Biology subjects

Ismagilov, R. F.

Publications and source records attributed to Ismagilov, R. F..

12 recordsLinked to original sources

bioRxiv2026

Fungal microbial enrichment method enables fungal metagenomics directly from human clinical samples

Fungi play important roles in health and disease, but current methods such as culture, PCR, and amplicon sequencing cannot provide genome-level characterization directly from clinical samples. Although metagenomic sequencing could overcome these limitations, it remains impractical in clinical samples where fungal DNA is present at low abundance relative to human DNA. Here, we extend a recently described microbial enrichment method (MEM)(1) to fungi (fungal Microbial Enrichment Method; fMEM) and test the method in bronchoalveolar lavage (BAL) samples to demonstrate direct-from-sample fungal metagenomic analysis and metagenome-assembled genome (MAG) recovery. In BAL samples, fMEM depleted human DNA by more than 1000-fold while preserving fungal DNA within 10-fold, enabling shotgun sequencing from samples with fungal biomass as low as 10 pg fungal DNA per 200 {micro}L BAL. fMEM enabled de novo recovery of fungal MAGs from three of four sequenced BAL samples, including two near-complete MAGs (>90% BUSCO completeness) and one 82.1% complete MAG, with low BUSCO-estimated contamination ([≤]1.5%). Fungal MAGs recovered by fMEM also resolved potentially clinically-relevant genes, not fully predictable from taxonomy alone and revealed genomic content absent from currently-available same-species reference genomes. fMEM is compatible with a whole-genome amplification (including long-read sequencing workflows). Long reads from fMEM-processed samples provided high coverage (>10X) over fungal assemblies. fMEMs compatibility with long-read sequencing enables recovery of genes that would be difficult to assemble with short reads alone. fMEM may enable new insights into the role of human-associated fungi, impacting public health, clinical management, and research into complex diseases with suspected fungal roles. ImportanceFungi influence human health, infectious disease, and the microbiome, but direct genome analysis from clinical samples has remained impractical because fungal DNA is often overwhelmed by human DNA. We developed a fungal microbial enrichment method (fMEM) that enables direct-from-sample fungal metagenomic sequencing and genome recovery from bronchoalveolar lavage samples without requiring culture for genome assembly. fMEM recovers genome-level features not predicted by taxonomy or current same-species reference genomes and is compatible with long-read sequencing workflows that can recover loci missed by short-read sequencing. fMEM opens new opportunities for culture-independent fungal genomics, clinical microbiology, comparative genomics, and mechanistic studies of human-associated fungi.

bioengineering↗

bioRxiv2026

Ultra-low biomass sequencing workflow (LBV-Seq) enables de novo metagenomic reconstruction of DNA and RNA viral genomes

Genome-resolved virome analysis remains inaccessible for many samples, including those with clinical relevance, because viral nucleic acid recovered after enrichment is often too scarce to support de novo genome assembly. As a result, many analyses are limited to sparse read-level detection, which cannot recover divergent viruses, resolve strains, or interpret gene-level variation. Here, we developed Low Biomass Viral Sequencing (LBV-Seq), a workflow that couples low-input viral sample handling with modified primary template-directed amplification and short- or long-read sequencing to enable de novo reconstruction of DNA and RNA viral genomes from sub-femtogram to nanogram inputs. LBV-Seq reproducibly captures the same relative community composition, amplifies diverse viruses, and achieved broad genome coverage across nearly all targets regardless of viral genome structure, Baltimore class, abundance, or input mass. Short-read assemblies recovered near-complete genomes from femtogram-scale inputs. Long-read sequencing provided orthogonal support for genome structure, with PacBio HiFi reads spanning large portions of viral genomes and, in some cases, complete small viral genomes. Applied to virus-enriched human duodenal biopsy eluates, LBV-Seq provided proof-of-feasibility for recovering both bacteriophage and eukaryotic viral genomes from low-input biopsy-derived material. In the eluates tested, LBV-Seq recovered co-occurring Alphatorquevirus and Betatorquevirus genomes estimated to be present at roughly 10 copies/{micro}L and at viral masses below 0.1 fg/{micro}L. LBV-Seq enables genome-resolved virome analysis in samples previously limited to detection-based viromics, supporting viral discovery and strain-resolved analyses in settings where viral mass is low, including viruses enriched from human tissue biopsies.

genomics↗

bioRxiv2025

Faecalibacterium prausnitzii, depleted in the Parkinson's disease microbiome, improves motor deficits in α-synuclein overexpressing mice

Gut microbiome composition is altered in Parkinsons disease (PD), a neurodegenerative disorder characterized by motor dysfunction and frequently accompanied by gastrointestinal (GI) symptoms. Notably, microbial taxa with anti-inflammatory properties are consistently depleted in PD patients compared to controls. To explore whether specific gut bacteria may be disease-protective, we assembled a microbial consortium of 8 human-associated taxa that are reduced in individuals with PD across multiple cohorts and geographies. Treatment of -synuclein overexpressing (Thy1-ASO) mice, an animal model of PD, with this consortium improved motor and GI deficits. A single bacterial species from this consortium, Faecalibacterium prausnitzii, was sufficient to correct gut microbiome deviations in Thy1-ASO mice, induce anti-inflammatory immune responses, and promote protective colonic gene expression profiles. Accordingly, oral treatment with F. prausnitzii robustly ameliorated motor and GI symptoms and reduced -synuclein aggregates in the brain. These findings support the emerging hypothesis of functional contributions by the microbiome to PD and embolden development of potential probiotic therapies.

physiology↗

bioRxiv2024

Inducible, but not constitutive, pancreatic REG/Reg isoforms are regulated by intestinal microbiota and pancreatic diseases

The REG/Reg gene locus encodes for a conserved family of potent antimicrobial but also pancreatitis-associated proteins. Here we investigated whether REG/Reg family members differ in their baseline expression levels and abilities to be regulated in the pancreas and gut upon perturbations. We found, in human and mouse, pancreas and gut differed in REG/Reg isoform levels and preferences, with duodenum most resembling the pancreas. Pancreatic acinar cells and intestinal enterocytes were the dominant REG producers. Intestinal symbiotic microbes regulated the expression of the same, select Reg members in gut and pancreas. These Reg members had the most STAT3-binding sites close to the transcription start sites and were partially IL-22 dependent. We thus categorized them as "inducible" and others as "constitutive". Indeed, also in models of pancreatic-ductal adenocarcinoma and pancreatitis, only inducible Reg members were upregulated in pancreas. While intestinal Reg expression remained unchanged upon pancreatic perturbation, pancreatitis altered the microbial composition of the duodenum and feces shortly after disease onset. Our study reveals differential usage and regulation of REG/Reg isoforms as a mechanism for tissue-specific innate immunity, highlights the intimate connection of pancreas and duodenum, and implies a gut-to-pancreas communication axis resulting in a coordinated Reg response.

immunology↗

bioRxiv2022

Quantitative whole-tissue 3D imaging reveals bacteria in close association with mouse jejunum mucosa

BackgroundThe small intestine (SI) is the primary site of nutrient absorption, so its large surface area lacks the thick protective mucus that is characteristic of the large intestine. Because the SI epithelium is relatively exposed, any microbes that colonize the thin mucosa of the SI may exert a substantial effect on the host. Thus far, potential bacterial colonization of the SI mucosa has only been documented in disease states, suggesting mucosal colonization is a rare occurrence, likely requiring multiple perturbations. ResultsHere, we tested whether we could induce bacterial association with jejunum mucosa by a combination of malnutrition and oral co-gavage with a specific bacterial cocktail (E. coli and Bacteroides spp.) that has previously induced environmental enteropathy in mouse models. To overcome the current limitations in imaging and allow definite determination of whether bacterial colonization of the SI mucosa is occurring, we optimized our previously developed whole-tissue three-dimensional (3D) imaging tools with third-generation hybridization chain reaction (HCR v3.0) probes. Only in mice that were malnourished and gavaged with the bacterial cocktail did we detect dense bacterial clusters surrounding intestinal villi suggestive of colonization. Healthy mice gavaged with bacteria and malnourished mice not gavaged with bacteria showed no evidence of mucosal colonization. Furthermore, in malnourished mice gavaged with bacteria we detected villus loss, which may represent one possible consequence that bacterial colonization of the SI mucosa has on the host. ConclusionsOur results suggest that dense bacterial colonization of jejunum mucosa is possible in the presence of multiple perturbations and that villus loss may be one possible consequence to such colonization. Furthermore, our results demonstrate the utility of whole-tissue 3D imaging tools. Although 2D imaging of thin sections may have failed to detect and capture the full spatial complexity of such rare events, whole-tissue 3D imaging tools enabled their detection over large areas of intestinal mucosa and visualization of their spatial complexity in 3D.

microbiology↗

bioRxiv2021

GATA4 regionalizes intestinal metabolism and barrier function to prevent immunopathology

Different regions of the gastrointestinal tract have distinct digestive and absorptive functions, which may be locally disrupted by infection or autoimmune disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. Here, we used mouse models, transcriptomics, and immune profiling to show that regional epithelial expression of the transcription factor GATA4 prevented adherent bacterial colonization and inflammation in the proximal small intestine by regulating retinol metabolism and luminal IgA. Loss of epithelial GATA4 expression increased mortality in mice infected with Citrobacter rodentium. In active celiac patients with villous atrophy, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. This study reveals broad impacts of GATA4-regulated intestinal regionalization and highlights an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease. One-sentence summaryEpithelial GATA4 regulates intestinal regionalization of bacterial colonization, metabolic pathways, and tissue immunity.

immunology↗

bioRxiv2021

3D imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa

Improving our understanding of host-microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the 3D spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 days after recovery when overall bacterial loads returned to pre-antibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern was resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that bacterial taxa are distributed differently within crypts, with Lactobacilli laying closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different. Significance StatementMany human diseases are causally linked to the gut microbiota, yet the field still lacks mechanistic understanding of the underlying complex interactions because existing tools cannot simultaneously quantify microbial communities and their intact native context. In this work, we provide a new approach to tissue clearing and preservation that enables visualization, in 3D and at scales ranging from centimeters to micrometers, of the complete biogeography of the host-microbiota interface. We combine this new tool with sequencing and multiplexed labelling of the microbiota to provide the field with a platform on which to discover patterns in the spatial distribution of microbes. We validated this platform by quantifying the distribution of bacteria in the cecal mucosa at different stages of antibiotic exposure. This approach will enable researchers to formulate and test new hypotheses about host-microbe and microbe-microbe interactions.

microbiology↗

bioRxiv2020

A single-cell method to map higher-order 3D genome organization in thousands of individual cells reveals structural heterogeneity in mouse ES cells

In eukaryotes, the nucleus is organized into a three dimensional structure consisting of both local interactions such as those between enhancers and promoters, and long-range higher-order structures such as nuclear bodies. This organization is central to many aspects of nuclear function, including DNA replication, transcription, and cell cycle progression. Nuclear structure intrinsically occurs within single cells; however, measuring such a broad spectrum of 3D DNA interactions on a genome-wide scale and at the single cell level has been a great challenge. To address this, we developed single-cell split-pool recognition of interactions by tag extension (scSPRITE), a new method that enables measurements of genome-wide maps of 3D DNA structure in thousands of individual nuclei. scSPRITE maximizes the number of DNA contacts detected per cell enabling high-resolution genome structure maps within each cells and is easy-to-use and cost-effective. scSPRITE accurately detects chromosome territories, active and inactive compartments, topologically associating domains (TADs), and higher-order structures within single cells. In addition, scSPRITE measures cell-to-cell heterogeneity in genome structure at different levels of resolution and shows that TADs are dynamic units of genome organization that can vary between different cells within a population. scSPRITE will improve our understanding of nuclear architecture and its relationship to nuclear function within an individual nucleus from complex cell types and tissues containing a diverse population of cells.

molecular biology↗

bioRxiv2020

Metabolic multi-stability and hysteresis in a model aerobe-anaerobe microbiome community

Changes in the composition of the human microbiome are associated with health and disease. Some microbiome states persist in seemingly unfavorable conditions, e.g., the proliferation of aerobe-anaerobe communities in oxygen-exposed environments in wounds or small intestinal bacterial overgrowth. However, it remains unclear how different stable microbiome states can exist under the same conditions, or why some states persist under seemingly unfavorable conditions. Here, using two microbes relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and dynamical systems theory, to show that multi-stability and hysteresis (MSH) is a mechanism that can describe the shift from an aerobe-dominated state to a resilient, paradoxically persistent aerobe-anaerobe state. We examine the impact of changing oxygen and nutrient regimes and identify factors, including changes in metabolism and gene expression, that lead to MSH. When analyzing the transitions between the two states in this system, the familiar conceptual connection between causation and correlation is broken and MSH must be used to interpret the dynamics. Using MSH to analyze microbiome dynamics will improve our conceptual understanding of the stability of microbiome states and the transitions among microbiome states. One sentence summaryMulti-stability and hysteresis (MSH) is a potential mechanism to describe shifts to and persistence of aerobe-anaerobe communities in the microbiome.

ecology↗

bioRxiv2020

A Quantitative Sequencing Framework for Absolute Abundance Measurements of Mucosal and Lumenal Microbial Communities

A fundamental goal in microbiome studies is to determine which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative microbial abundances, which cannot capture absolute changes. Moreover, studies often focus on a single site (usually stool), although microbial demographics differ substantially among gastrointestinal (GI) locations. Here, we developed a quantitative framework to accurately measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compared microbial loads in lumenal and mucosal samples at several sites along the GI tract. Measurements of absolute (but not relative) abundances revealed decreases in total microbial loads on the ketogenic diet and enabled us to accurately determine the effect of the diet on each taxon at each GI location. Quantitative measurements also revealed different patterns in how the ketogenic diet affected each taxons abundance in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework applied to samples from relevant GI locations will enable mapping microbial biogeography of the mammalian GI tract and more accurately capture the changes of microbial taxa in experimental microbiome studies.

microbiology↗

bioRxiv2020

Quantitative microbiome profiling in lumenal and tissue samples with broad coverage and dynamic range via a single-step 16S rRNA gene DNA copy quantification and amplicon barcoding

Current methods for detecting, accurately quantifying, and profiling complex microbial communities based on the microbial 16S rRNA marker genes are limited by a number of factors, including inconsistent extraction of microbial nucleic acids, amplification interference from contaminants and host DNA, different coverage of PCR primers utilized for quantification and sequencing, and potentially biases in PCR amplification rates among microbial taxa during amplicon barcoding. Here, we describe a single-step method that enables the quantification of microbial 16S rRNA gene DNA copies with wide dynamic range and broad microbial diversity, and simultaneous amplicon barcoding for quantitative 16S rRNA gene amplicon profiling of microbiota. The method is suitable for a variety of sample types and is robust in samples with low microbial abundance, including samples containing high levels of host mammalian DNA, as is common in human clinical samples. We demonstrate that our modification to the Earth Microbiome Project (EMP) V4 16S rRNA gene primers expands their microbial coverage while dramatically reducing non-specific mammalian mitochondrial DNA amplification, thus achieving wide dynamic range in microbial quantification and broad coverage for capturing high microbial diversity in samples with or without high host DNA background. The approach relies only on broadly available hardware (real-time PCR instruments) and standard reagents utilized for conventional 16S rRNA gene amplicon library preparation both of which make it amenable for immediate and widespread adoption. Simultaneous 16S rRNA gene DNA copy quantification and amplicon barcoding for multiplexed next-generation sequencing from the same analyzed sample, performed in a combined workflow, reduces the amount of sample needed and reduces time and reagent costs. Additionally, we demonstrate that using our modified 16S rRNA gene primers in a digital PCR (dPCR) format enables precise and exact microbial quantification in samples with very high host DNA background levels without the need for quantification standards. Potential future applications of this approach include: (1) quantitative microbiome profiling in human and animal microbiome research; (2) detection of monoinfections and profiling of polymicrobial infections in tissues, stool, and bodily fluids in human and veterinary medicine; (3) environmental sample analyses (e.g., soil and water); and (4) broad-coverage detection of microbial food contamination in products high in mammalian DNA, such as meat products. We predict that utilization of this approach primarily for quantitative microbiome profiling will be invaluable to microbiome studies, which have historically been limited to analysis of relative abundances of microbes.

bioengineering↗

bioRxiv2018

High-molecular-weight polymers from dietary fiber drive aggregation of particulates in the murine small intestine

The lumen of the small intestine (SI) is filled with particulates: microbes, therapeutic particles, and food granules. The structure of this particulate suspension could impact uptake of drugs and nutrients and the function of microorganisms; however, little is understood about how this suspension is re-structured as it transits the gut. Here, we demonstrate that particles spontaneously aggregate in SI luminal fluid ex vivo. We find that mucins and immunoglobulins are not required for aggregation. Instead, aggregation can be controlled using polymers from dietary fiber in a manner that is qualitatively consistent with polymer-induced depletion interactions, which do not require specific chemical interactions. Furthermore, we find that aggregation is tunable; by feeding mice dietary fibers of different molecular weights, we can control aggregation in SI luminal fluid. This work suggests that the molecular weight and concentration of dietary polymers play an underappreciated role in shaping the physicochemical environment of the gut.

biophysics↗