Biology subjects

Fleming, E.

Publications and source records attributed to Fleming, E..

13 recordsLinked to original sources

bioRxiv2024

BioMapAI: Artificial Intelligence Multi-Omics Framework Modeling of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic illness with a multifactorial etiology and heterogeneous symptomatology, posing major challenges for diagnosis and treatment. Here, we present BioMapAI, a supervised deep neural network trained on a four-year, longitudinal, multi-omics dataset from 249 participants, which integrates gut metagenomics, plasma metabolomics, immune cell profiling, blood laboratory data, and detailed clinical symptoms. By simultaneously modeling these diverse data types to predict clinical severity, BioMapAI identifies disease- and symptom-specific biomarkers and robustly classifies ME/CFS in both held-out and independent external cohorts. Using an explainable AI approach, we construct the first connectivity map spanning the microbiome, immune system, and plasma metabolome in health and ME/CFS, adjusted for age, gender, and additional clinical factors. This map uncovers disrupted associations between microbial metabolism (e.g., short-chain fatty acids, branched-chain amino acids, tryptophan, benzoate), plasma lipids and bile acids, and heightened inflammatory responses in mucosal and inflammatory T cell subsets (MAIT, {gamma}{delta}T) secreting IFN{gamma} and GzA. Overall, BioMapAI provides unprecedented systems-level insights into ME/CFS, refining existing hypotheses and hypothesizing new pathways associated to the diseases heterogeneous symptoms.

bioinformatics↗

bioRxiv2023

Species- and strain-effects in interferon, innate immunity, and barrier transcriptional response of microbiome interactions of 3D respiratory epithelial cultures

The microbiome modulates the respiratory epitheliums immunomodulatory functions. To explore how the microbiomes biodiversity affects microbe-epithelial interactions, we screened 58 phylogenetically diverse microbes for their transcriptomic effect on tracheobronchial air-liquid interface cell (ALI) cultures. We found distinct species- and strain-level differences in host innate immunity, and epithelial barrier response. Strikingly, we found that host interferon, an antiviral immune response, was one of the most variable host processes, and this variability was not driven by microbial phylogenetic diversity, bioburden, nor by the microbes ability to stimulate other innate immunity pathways. Our study provides a foundation for understanding how the respiratory microbiomes biodiversity affects epithelial, and particularly, antiviral innate immunity.

microbiology↗

bioRxiv2023

High-Throughput Bioprinting of the Nasal Epithelium using Patient-derived Nasal Epithelial Cells

Human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models. However, the manual approach is slow, low-throughput and has limitations in terms of achieving the intricate 3D structure of the natural nasal epithelium in a uniform manner. 3D Bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of nasal progenitor cells ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4-week air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes such as disease modeling, immunological studies, and drug screening.

bioengineering↗

bioRxiv2023

Skin microbiome attributes associate with biophysical skin aging

Two major arms of skin aging are changes in the skins biophysical conditions and alterations in the skin microbiome. This work partitioned both arms to study their interaction in detail. Leveraging the resolution provided by shotgun metagenomics, we explored how skin microbial species, strains, and gene content interact with the biophysical traits of the skin during aging. With a dataset well-controlled for confounding factors, we found that skin biophysical traits, especially the collagen diffusion coefficient, are associated with the composition and the functional potential of the skin microbiome, including the abundance of bacterial strains found in nosocomial infections and the abundance of antibiotic resistance genes. Our findings reveal important associations between skin biophysical features and aging-related changes in the skin microbiome and generate testable hypotheses for the mechanisms of such associations.

genomics↗

bioRxiv2023

Assessing the impact of successive soil cultivation on Meloidogyne enterolobii infection and on soil bacterial assemblages

Soil cultivation may change the soil microbiome and alter interactions between plants and parasites. The objective of this work was to evaluate temporal changes in plant health, microbiome abundance, bacterial diversity and the plant-parasitic nematode, Meloidogyne enterolobii incidence in two soil fields with different agricultural uses. Soil samples were collected from a commercial tomato production field (agricultural soil) and a single-cultivation strawberry field (native soil). Samples for the second experiment were collected from the same fields the following year. Tomato plants cv. Yearly Girl were grown in a greenhouse and inoculated with M. enterolobii. After 45 days, plants were evaluated for the plant growth parameters, nematode reproduction, and soil bacterial assemblages were assessed using cultivation-independent sequencing methods (V3/V4 region of the rRNA 16S). Overall the average of fruit fresh weight in the second experiment was 2.4-fold to 14-fold higher than the first experiment. Moreover, there was a 80.5% decrease in eggs present per root system from the first experiment to the second. The relative abundance of bacterial assemblages from Experiment 1 to Experiment 2 changed for most of the top phyla (eg. Actinobacteria, Bacteroidetes, and Chloroflexi) and genera (eg. Bacillus, Streptomyces, and Flavisolibacter) and there was no change in microbial diversity between the two experiments. This study suggests that soil management can lead to an overall decrease in nematode reproduction and better crop yield, as well as a shift in the overall bacterial assemblages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/525929v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@2b76b2org.highwire.dtl.DTLVardef@17db6c5org.highwire.dtl.DTLVardef@6a6d69org.highwire.dtl.DTLVardef@1352fb0_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2023

Measuring prion propagation in single bacteria elucidates mechanism of loss

Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies in mammals, but prions can also constitute non-toxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes with distinct stability and inheritance characteristics. We find that the prion is lost through random partitioning of aggregates to one of the two daughter cells at division. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation across domains of life.

microbiology↗

bioRxiv2021

Multi-omics of host-microbiome interactions in short- and long-term Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex, multi-system, debilitating disability manifesting as severe fatigue and post-exertional malaise. The chronic dysfunctions in ME/CFS are increasingly recognized as significant health factors with potential parallels with long COVID. However, the etiology of ME/CFS remains elusive with limited high-resolution human studies. In addition, reliable biomarker-based diagnostics have not been well-established, but may assist in disease classification, particularly during different temporal phases of the disease. Here, we performed deep multi- omics (shotgun metagenomics of gut microbiota and plasma metabolomics) and clinical phenotyping of healthy controls (n=79) vs. two cohorts of ME/CFS patients - those with short-term disease (<4 years, n=75), and patients with long-term disease (>10y, n=79). Overall, ME/CFS was characterized by reduced gut microbiome diversity and richness with high heterogeneity, and depletion of sphingomyelins and short-chain fatty acids in the plasma. We found significant differences when stratifying by cohort; short-term ME/CFS was associated with more microbial dysbiosis, but long-term ME/CFS was associated with markedly more severe phenotypic and metabolic abnormalities. We identified a reduction in the gene-coding capacity (and relative abundance of butyrate producers) of microbial butyrate biosynthesis together with a reduction in the plasma concentration of butyrate, especially in the short-term group. Global co-association and detailed gene pathway correlation analyses linking the microbiome and metabolome identified additional potential biological mechanisms underlying host-microbiome interactions in ME/CFS, including bile acids and benzoate pathways. Finally, we built multiple state-of-the-art classifiers to identify microbes, microbial gene pathways, metabolites, and clinical features that individually or together, were most able to differentiate short or long-term MECFS, or MECFS vs. healthy controls. Taken together, our study presents the highest resolution, multi-cohort and multi- omics analysis to date, providing an important resource to facilitate mechanistic hypotheses of host-microbiome interactions in ME/CFS.

microbiology↗

bioRxiv2021

Mice with a humanized immune system are resilient to transplantation of human microbiota

Human gut microbiota has co-evolved with human, and plays important roles in regulating the development and functioning of the host immune system. To study the human-specific microbiome-immunune interaction in an animal model is challenging as the animal model needs to capture both the human-specific immune functions and the human-specific microbiome composition. Here we combined two widely-used humanization procedures to generate a humanized mouse model (HMA-huCD34) with functional human leukocytes developed from engrafted huCD34+ cells and human fecal microbes introduced through fecal microbiota transplantation, and investigated how the two introduced human components interact. We found that the engrafted human leukocytes are resilient to the transplanted human microbes, while reciprocally the transplanted microbial community in the huCD34 mice was significantly different from mice without a humanized immune system. By tracking the colonization of human fecal Bacteroides strains in the mouse gut, we found that the composition of the strain population changes over time, the trajectory of which depends upon the type of mouse. On the other hand, different from Bacteroides, Akkermansia muciniphila exhibited consistent and rapid fixation of a single donor strain in all tested mice, suggesting strong purifying selection common to all mouse types. Our prospect study illustrated the complex interactions between the transplanted microbiome and different host factors, and suggested that the humanized mouse model may not faithfully reproduce the human-specific microbiome-immune interaction.

microbiology↗

bioRxiv2021

Large-scale CRISPRi and transcriptomics of Staphylococcus epidermidis identify genetic factors implicated in commensal-pathogen lifestyle versatility

Staphylococcus (S.) epidermidis is a ubiquitous human commensal skin bacterium that is also one of the most prevalent nosocomial pathogens. The genetic factors underlying this remarkable lifestyle plasticity are incompletely understood, much due to the difficulties of genetic manipulation, precluding high-throughput functional profiling of this species. To probe S. epidermdis versatility to survive across a diversity of skin sites and infection niches, we developed a large-scale CRISPR interference (CRISPRi) screen complemented by transcriptional profiling (RNA-seq) across 24 diverse environmental conditions and piloted a droplet-based CRISPRi approach to enhance throughput and sensitivity. We identified putative essential genes, importantly, revealing amino acid metabolism as crucial to survival across diverse environments and demonstrated the importance of trace metal uptake for survival under multiple stress conditions. We identified pathways significantly enriched and repressed across our range of stress and nutrient limited conditions, demonstrating the considerable plasticity of S. epidermidis in responding to environmental stressors. We postulate a mechanism by which nitrogen metabolism is linked to lifestyle versatility in response to hyperosmotic challenges, such as those encountered on human skin. Finally, we examined S. epidermidis survival under acid stress and hypothesize a role for cell wall modification as a vital component of the survival response in acidic conditions. Taken together, this study integrates large scale CRISPRi and transcriptomics data across multiple environments to provide insights into a keystone member of the human skin microbiome. Our results additionally provide a valuable benchmarking analysis for CRISPRi screens and are rich resource for other staphylococcal researchers. Author summaryStaphylococcus epidermidis is an important bacteria of the skin microbiome. While it has an important role in skin health, it can also be a major infectious agent, especially in bloodstream and catheter infections. Understanding the underlying genes and pathways that contribute to S. epidermidis ability to have both health and disease-associated abilities will be important to promoting the former and targeting the latter. Yet the function of many S. epidermidis genes, particularly in skin and infection environments, remains unknown. We developed a CRISPRi platform to knock down the function of S. epidermidis genes to better understand to what degree they are essential for growth in these environments. We complemented this gene essentiality data with gene expression data in the same environments to understand how regulation of these genes contribute to S. epidermidis survival. These large-scale data generated numerous hypotheses for new genetic links to S. epidermidis growth versatility.

microbiology↗

bioRxiv2021

Species and strain cultivation of skin, oral, and gut microbiota

Genomics-driven discovery of microbial species have provided extraordinary insights into the biodiversity of human microbiota. High resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. Here, we describe and validate a streamlined workflow for cultivating microbes from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.

microbiology↗

bioRxiv2018

Serotonin regulates dynamics of cerebellar granule cell activity by modulating tonic inhibition

Understanding how afferent information is integrated by cortical structures requires identifying the factors shaping excitation and inhibition within their input layers. The input layer of the cerebellar cortex integrates diverse sensorimotor information to enable learned associations that refine the dynamics of movement. Specifically, mossy fiber afferents relay sensorimotor input into the cerebellum to excite granule cells, whose activity is regulated by inhibitory Golgi cells. To test how this integration can be modulated, we have used an acute brain slice preparation from young adult rats and found that encoding of mossy fiber input in the cerebellar granule cell layer can be regulated by serotonin (5-HT) via a specific action on Golgi cells. We find that 5-HT depolarizes Golgi cells, likely by activating 5-HT2A receptors, but does not directly act on either granule cells or mossy fibers. As a result of Golgi cell depolarization, 5-HT significantly increases tonic inhibition onto both granule cells and Golgi cells. 5-HT-mediated Golgi cell depolarization is not sufficient, however, to alter the probability or timing of mossy fiber-evoked feed-forward inhibition onto granule cells. Together, increased granule cell tonic inhibition paired with normal feed-forward inhibition acts to reduce granule cell spike probability without altering spike timing. These data hence provide a circuit mechanism by which 5-HT can reduce granule cell activity without altering temporal representations of mossy fiber input. Such changes in network integration could enable flexible, state-specific suppression of cerebellar sensorimotor input that should not be learned, or enable reversal learning for unwanted associations.\n\nNew and Noteworthy5-HT regulates synaptic integration at the input stage of cerebellar processing by increasing tonic inhibition of granule cells. This circuit mechanism reduces the probability of granule cell spiking without altering spike timing, thus suppressing cerebellar input without altering its temporal representation in the granule cell layer.

neuroscience↗

bioRxiv2017

A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

BackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.\n\nResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful--focusing on CRISPRi as a potential application--as demonstrated by essential gene knockdown in two staphylococcal species.\n\nConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

microbiology↗

bioRxiv2016

In situ estimates of iron-oxidation and accretion rates for iron-oxidizing bacterial mats at Loihi Seamount.

It is increasingly recognized that diffuse, hydrothermal venting is an important source of iron to the deep-sea that can influence oceanic iron dynamics and abundance. Lithotrophic Fe-oxidizing bacteria (FeOB) are dominant at diffuse hydrothermal vent sites, producing microbial iron mats that are often centimeters or more thick. At present, little is known about in situ Fe-oxidation rates, or accretion rates for iron mats. An in situ productivity chamber was developed that took advantage of the unique mineral morphotypes produced by FeOB to estimate rates of Fe-oxidation and accretion. Chambers were placed at two diffuse vents (1179 and 1300 mbsl) at L[o]ihi Seamount where they were colonized by FeOB for different amounts of time. From this analysis, it was estimated that Fe-oxidation rates could range from 8.2-51.9 x 10-6 mol {middle dot} hr-1, and that iron mats could accrete at around 2.2 cm {middle dot} yr-1. Molecular analysis indicated that the relative abundance of Zetaproteobacteria, a group of known FeOB, accounted for 80-90% of the bacteria colonizing the chambers. There was a distinct difference between populations at the 1179m site (Pohaku), and the 1300m site (North Hiolo Ridge). Microscope slides placed within the productivity chambers were colonized by different morphotypes of FeOB. The cells responsible for one common morphotype that produces a Y-shaped filament were identified as Zetaproteobacteria by use of a small subunit rRNA probe. This work confirms the importance of FeOB in the formation of chemosynthetic iron mats, and provides the first estimates for in situ Fe-oxidation rates and mat accretion rates.\n\nHighlightsO_LIAn in-situ productivity chamber was developed to estimate rates of Fe-oxidation and understand colonization patterns at chemosynthetic iron mats at L[o]ihi Seamount.\nC_LIO_LIFe-oxidation rates ranged from 8.2-51.9 x 10-6 mol {dot} hr-1, and it was estimated that the iron mats could accrete at around 2.2 cm {dot} yr-1.\nC_LIO_LIThe iron mat community was dominated by Zetaproteobacteria, whose relative abundance accounted for up to 89% of the microbial community.\nC_LIO_LIThe community membership that grew during short-term incubations reflected the community composition of nearby microbial mats.\nC_LI

microbiology↗