scholarly journals The use of CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to reveal the eukaryotic microbiome of metazoans

2020 ◽  
Author(s):  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christophe M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle
Keyword(s):  
Amplicon Sequencing ◽  
Model Organisms ◽  
Phylogenetic Groups ◽  
Guide Rna ◽  
Rdna Sequences ◽  
Alternative Approach ◽  
18S Rdna Sequences ◽  
Health And Disease ◽  
Taxonomic Markers

AbstractCharacterization of the eukaryotic microbiome is required to understand the role of microbial communities in health and disease. Such investigation relies on sequencing 18S ribosomal RNA genes (rDNA), which serve as taxonomic markers; however, this is compromised by contaminating host rDNA sequences. To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rDNA sequences of the host. Validation shows that >96.5% of rDNA amplicons from ten model organisms were cleaved, while rDNA from protists and fungi were unaffected. In oyster spat, CCSAS resolved ∼8.5-fold more taxa, and several additional major phylogenetic groups when compared to the best available alternative approach. We designed taxon-specific sgRNA for ∼16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected because of methodological challenges.

scholarly journals Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

2021 ◽  
Author(s):  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christophe M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle
Keyword(s):  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Model Organisms ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Oyster Spat ◽  
Guide Rna ◽  
16S Rna ◽  
Eukaryotic Microbes

Abstract BackgroundThe microbiome affects the health of plants and animals, including humans, and has many biological, ecological and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. ResultsTo overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that >96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. ConclusionCCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Keywords: Eukaryotic microbiome, 18S rRNA gene, Microeukaryote, CRISPR-Cas, Taxon-specific single-guide RNA, gRNA-target-site, CasOligo, CCSAS

scholarly journals Wild black bears harbor simple gut microbial communities with little difference between the jejunum and colon

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sierra J. Gillman ◽  
Erin A. McKenney ◽  
Diana J. R. Lafferty
Keyword(s):  
Gastrointestinal Tract ◽  
Microbial Communities ◽  
Ursus Americanus ◽  
Black Bear ◽  
Amplicon Sequencing ◽  
Black Bears ◽  
Model Organisms ◽  
Rrna Gene ◽  
Number Of Bacteria

AbstractThe gut microbiome (GMB), comprising the commensal microbial communities located in the gastrointestinal tract, has co-evolved in mammals to perform countless micro-ecosystem services to facilitate physiological functions. Because of the complex inter-relationship between mammals and their gut microbes, the number of studies addressing the role of the GMB on mammalian health is almost exclusively limited to human studies and model organisms. Furthermore, much of our knowledge of wildlife–GMB relationships is based on studies of colonic GMB communities derived from the feces of captive specimens, leaving our understanding of the GMB in wildlife limited. To better understand wildlife–GMB relationships, we engaged hunters as citizen scientists to collect biological samples from legally harvested black bears (Ursus americanus) and used 16S rRNA gene amplicon sequencing to characterize wild black bear GMB communities in the colon and jejunum, two functionally distinct regions of the gastrointestinal tract. We determined that the jejunum and colon of black bears do not harbor significantly different GMB communities: both gastrointestinal sites were dominated by Firmicutes and Proteobacteria. However, a number of bacteria were differentially enriched in each site, with the colon harboring twice as many enriched taxa, primarily from closely related lineages.

scholarly journals Perturbations of the ileal mycobiota by necrotic enteritis in broiler chickens

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qing Yang ◽  
Jing Liu ◽  
Kelsy J. Robinson ◽  
Melanie A. Whitmore ◽  
Sydney N. Stewart ◽  
...  
Keyword(s):  
Broiler Chickens ◽  
Animal Health ◽  
Amplicon Sequencing ◽  
Significant Negative Correlation ◽  
Necrotic Enteritis ◽  
Fold Reduction ◽  
Poultry Disease ◽  
Health And Disease ◽  
Composition Changes

Abstract Background Intestinal microbiota is critical for maintaining animal health and homeostasis. However, involvement of the fungal community, also known as the mycobiota, in animal health and disease is poorly understood. This study was aimed to examine the association between the intestinal mycobiota and the severity of necrotic enteritis (NE), an economically significant poultry disease. Methods A total of 90 day-of-hatch Cobb broilers were infected with Eimeria maxima on d 10, followed by an oral challenge with C. perfringens on d 14 to induce NE, while another 10 broilers were served as mock-infected controls. On d 17, the lesions in the jejunum were scored, and the ileal digesta were subjected to DNA isolation and real-time PCR quantification of total bacterial and fungi populations. Internal transcribed spacer 2 (ITS2) amplicon sequencing was also performed to profile the ileal mycobiota composition. Changes in the ileal mycobiota in response to NE were investigated. Spearman correlation analysis was further conducted to identify the correlations between relative abundances of individual ileal fungi and the severity of NE. Results While the total bacterial population in the ileum was increased by 2- to 3-fold in NE chickens, the total fungal population was progressively declined in more exacerbated NE, with the most severely infected chickens showing a nearly 50-fold reduction relative to mock-infected controls. Richness of the ileal mycobiota also tended to reduce in chickens with NE (P = 0.06). Compositionally, among 30 most abundant fungal amplicon sequence variants (ASVs), 11 were diminished and 7 were enriched (P < 0.05), while 12 remained largely unchanged in NE-afflicted chickens (P > 0.05). Multiple Wallemia and Aspergillus species were markedly diminished in NE (P < 0.05) and also showed a significant negative correlation with NE severity (P < 0.05). Conclusions Dysbiosis of the ileal mycobiota is induced evidently by NE and the extent of the dysbiosis is positively correlated with disease severity. These findings suggest a possible role of the intestinal mycobiota in NE pathogenesis and highlight the mycobiota as a new potential target for NE mitigation in poultry.

scholarly journals Histone Variant H3.3 Mutations in Defining the Chromatin Function in Mammals

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2716
Author(s):  
Matteo Trovato ◽  
Vibha Patil ◽  
Maja Gehre ◽  
Kyung Min Noh
Keyword(s):  
Mammalian Cells ◽  
Functional Role ◽  
Histone Variant ◽  
Model Organisms ◽  
Post Translational Modification ◽  
Mammalian Development ◽  
Histone 3 ◽  
Oncogenic Mutations ◽  
Health And Disease

The systematic mutation of histone 3 (H3) genes in model organisms has proven to be a valuable tool to distinguish the functional role of histone residues. No system exists in mammalian cells to directly manipulate canonical histone H3 due to a large number of clustered and multi-loci histone genes. Over the years, oncogenic histone mutations in a subset of H3 have been identified in humans, and have advanced our understanding of the function of histone residues in health and disease. The oncogenic mutations are often found in one allele of the histone variant H3.3 genes, but they prompt severe changes in the epigenetic landscape of cells, and contribute to cancer development. Therefore, mutation approaches using H3.3 genes could be relevant to the determination of the functional role of histone residues in mammalian development without the replacement of canonical H3 genes. In this review, we describe the key findings from the H3 mutation studies in model organisms wherein the genetic replacement of canonical H3 is possible. We then turn our attention to H3.3 mutations in human cancers, and discuss H3.3 substitutions in the N-terminus, which were generated in order to explore the specific residue or associated post-translational modification.

scholarly journals The Good, the Bad, and the Ugly of ROS: New Insights on Aging and Aging-Related Diseases from Eukaryotic and Prokaryotic Model Organisms

2018 ◽  
Vol 2018 ◽  
pp. 1-23 ◽  
Author(s):  
Ana L. Santos ◽  
Sanchari Sinha ◽  
Ariel B. Lindner
Keyword(s):  
Cellular Damage ◽  
Model Organisms ◽  
Oxygen Species ◽  
Cellular Physiology ◽  
Age Related ◽  
Normal Metabolism ◽  
Health And Disease ◽  
Genetic Interventions ◽  
Mitochondrial Networks

Aging is associated with the accumulation of cellular damage over the course of a lifetime. This process is promoted in large part by reactive oxygen species (ROS) generated via cellular metabolic and respiratory pathways. Pharmacological, nonpharmacological, and genetic interventions have been used to target cellular and mitochondrial networks in an effort to decipher aging and age-related disorders. While ROS historically have been viewed as a detrimental byproduct of normal metabolism and associated with several pathologies, recent research has revealed a more complex and beneficial role of ROS in regulating metabolism, development, and lifespan. In this review, we summarize the recent advances in ROS research, focusing on both the beneficial and harmful roles of ROS, many of which are conserved across species from bacteria to humans, in various aspects of cellular physiology. These studies provide a new context for our understanding of the parts ROS play in health and disease. Moreover, we highlight the utility of bacterial models to elucidate the molecular pathways by which ROS mediate aging and aging-related diseases.

scholarly journals Chromosome Instability, Aging and Brain Diseases

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1256
Author(s):  
Ivan Y. Iourov ◽  
Yuri B. Yurov ◽  
Svetlana G. Vorsanova ◽  
Sergei I. Kutsev
Keyword(s):  
Brain Aging ◽  
Chromosome Instability ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Accelerated Aging ◽  
Brain Diseases ◽  
Aging Brain ◽  
Ontogenetic Changes ◽  
Health And Disease

Chromosome instability (CIN) has been repeatedly associated with aging and progeroid phenotypes. Moreover, brain-specific CIN seems to be an important element of pathogenic cascades leading to neurodegeneration in late adulthood. Alternatively, CIN and aneuploidy (chromosomal loss/gain) syndromes exhibit accelerated aging phenotypes. Molecularly, cellular senescence, which seems to be mediated by CIN and aneuploidy, is likely to contribute to brain aging in health and disease. However, there is no consensus about the occurrence of CIN in the aging brain. As a result, the role of CIN/somatic aneuploidy in normal and pathological brain aging is a matter of debate. Still, taking into account the effects of CIN on cellular homeostasis, the possibility of involvement in brain aging is highly likely. More importantly, the CIN contribution to neuronal cell death may be responsible for neurodegeneration and the aging-related deterioration of the brain. The loss of CIN-affected neurons probably underlies the contradiction between reports addressing ontogenetic changes of karyotypes within the aged brain. In future studies, the combination of single-cell visualization and whole-genome techniques with systems biology methods would certainly define the intrinsic role of CIN in the aging of the normal and diseased brain.

scholarly journals The Role of Cell Metabolism in Innate Immune Memory

2020 ◽  
pp. 1-9
Author(s):  
Anaisa Valido Ferreira ◽  
Jorge Domiguéz-Andrés ◽  
Mihai Gheorghe Netea
Keyword(s):  
Metabolic Pathways ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Innate Immune ◽  
Immune Memory ◽  
Functional Changes ◽  
Trained Immunity ◽  
Health And Disease

Immunological memory is classically attributed to adaptive immune responses, but recent studies have shown that challenged innate immune cells can display long-term functional changes that increase nonspecific responsiveness to subsequent infections. This phenomenon, coined <i>trained immunity</i> or <i>innate immune memory</i>, is based on the epigenetic reprogramming and the rewiring of intracellular metabolic pathways. Here, we review the different metabolic pathways that are modulated in trained immunity. Glycolysis, oxidative phosphorylation, the tricarboxylic acid cycle, amino acid, and lipid metabolism are interplaying pathways that are crucial for the establishment of innate immune memory. Unraveling this metabolic wiring allows for a better understanding of innate immune contribution to health and disease. These insights may open avenues for the development of future therapies that aim to harness or dampen the power of the innate immune response.

scholarly journals SARS-CoV-2 does not have a strong effect on the nasopharyngeal microbial composition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tzipi Braun ◽  
Shiraz Halevi ◽  
Rotem Hadar ◽  
Gilate Efroni ◽  
Efrat Glick Saar ◽  
...  
Keyword(s):  
Clinical Microbiology ◽  
Amplicon Sequencing ◽  
Viral Disease ◽  
Nasopharyngeal Swab ◽  
Microbial Composition ◽  
16S Amplicon Sequencing ◽  
The World ◽  
Health And Disease ◽  
Longitudinal Sampling ◽  
Potential Interactions

AbstractThe coronavirus disease 2019 (COVID-19) has rapidly spread around the world, impacting the lives of many individuals. Growing evidence suggests that the nasopharyngeal and respiratory tract microbiome are influenced by various health and disease conditions, including the presence and the severity of different viral disease. To evaluate the potential interactions between Severe Acute Respiratory Syndrome Corona 2 (SARS-CoV-2) and the nasopharyngeal microbiome. Microbial composition of nasopharyngeal swab samples submitted to the clinical microbiology lab for suspected SARS-CoV-2 infections was assessed using 16S amplicon sequencing. The study included a total of 55 nasopharyngeal samples from 33 subjects, with longitudinal sampling available for 12 out of the 33 subjects. 21 of the 33 subjects had at least one positive COVID-19 PCR results as determined by the clinical microbiology lab. Inter-personal variation was the strongest factor explaining > 75% of the microbial variation, irrespective of the SARS-CoV-2 status. No significant effect of SARS-CoV-2 on the nasopharyngeal microbial community was observed using multiple analysis methods. These results indicate that unlike some other viruses, for which an effect on the microbial composition was noted, SARS-CoV-2 does not have a strong effect on the nasopharynx microbial habitants.

Sign in / Sign up

Export Citation Format

Share Document