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Frontiers in Medicine 2022Most colorectal cancer (CRC) cases are sporadic and develop along the adenoma-carcinoma sequence. Intestinal microbial dysbiosis is involved in the development of...
BACKGROUND
Most colorectal cancer (CRC) cases are sporadic and develop along the adenoma-carcinoma sequence. Intestinal microbial dysbiosis is involved in the development of colorectal cancer. However, there are still no absolute markers predicting the progression from adenoma to carcinoma. This study aimed to investigate the characteristics of intestinal microbiota in patients with colorectal adenoma and carcinoma and its correlations with clinical characteristics.
METHODS
Fecal samples were collected from 154 patients with CRC, 20 patients with colorectal adenoma (AD) and 199 healthy controls. To analyze the differences in the intestinal microbiota, 16S rRNA gene sequencing was conducted.
RESULTS
At the genus level, there were four significantly different genera among the three groups, namely Acidaminococcus, Alloprevotella, Mycoplasma, and Sphingobacterium, while Acidaminococcus significantly decreased with the order of Control-AD-CRC ( < 0.05). In addition, Parvimonas, Peptostreptococcus, Prevotella, Butyricimonas, Alistipes, and Odoribacter were the key genera in the network of colorectal adenoma/carcinoma-associated bacteria. The top 10 most important species, including , , , , , , , , and , showed the best performance in distinguishing AD from CRC (AUC = 85.54%, 95% CI: 78.83-92.25%). The clinicopathologic features, including age, gender, tumor location, differentiation degree, and TNM stage, were identified to be closely linked to the intestinal microbiome in CRC.
CONCLUSION
Several intestinal bacteria changed along the adenoma-carcinoma sequence and might be the potential markers for the diagnosis and treatment of colorectal adenoma/carcinoma. Intestinal microbiota characteristics in CRC should account for the host factors.
PubMed: 35935780
DOI: 10.3389/fmed.2022.888340 -
Gut Microbes Dec 2023Despite improved cardiometabolic outcomes following bariatric surgery, its long-term impact on colorectal cancer (CRC) risk remains uncertain. In parallel, the influence...
Despite improved cardiometabolic outcomes following bariatric surgery, its long-term impact on colorectal cancer (CRC) risk remains uncertain. In parallel, the influence of bariatric surgery on the host microbiome and relationships with disease outcomes is beginning to be appreciated. Therefore, we investigated the impact of Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) on the patterns of sulfide-reducing and butyrate-producing bacteria, which are hypothesized to modulate CRC risk after bariatric surgery. In this single-center, cross-sectional study, we included 15 pre-surgery subjects with severe obesity and patients who are at a median (range) of 25.6 (9.9-46.5) months after RYGB ( = 16) or VSG ( = 10). The DNA abundance of fecal bacteria and enzymes involved in butyrate and sulfide metabolism were identified using metagenomic sequencing. Differences between pre-surgery and post-RYGB or post-VSG cohorts were quantified using the linear discriminant analysis (LDA) effect size (LEfSe) method. Our sample was predominantly female (87%) with a median (range) age of 46 (23-71) years. Post-RYGB and post-VSG patients had a higher DNA abundance of fecal sulfide-reducing bacteria than pre-surgery controls (LDA = 1.3-4.4, < .05). The most significant enrichments were for fecal , and after RYGB, and for , after VSG. As for butyrate-producing bacteria, was more abundant, whereas and were lower post-RYGB vs. pre-surgery. was also lower in post-VSG vs. pre-surgery. Consistent with these findings, our analysis showed a greater enrichment of sulfide-reducing enzymes after bariatric surgery, especially RYGB, vs. pre-surgery. The DNA abundance of butyrate-producing enzymes was lower post-RYGB. In conclusion, the two most used bariatric surgeries, RYGB and VSG, are associated with microbiome patterns that are potentially implicated in CRC risk. Future studies are needed to validate and understand the impact of these microbiome changes on CRC risk after bariatric surgery.
Topics: Humans; Female; Middle Aged; Aged; Male; Butyrates; Cross-Sectional Studies; Escherichia coli; Gastrointestinal Microbiome; Bariatric Surgery; Bacteria; Colorectal Neoplasms
PubMed: 37702461
DOI: 10.1080/19490976.2023.2255345 -
Plant Biotechnology Journal Aug 2017CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high-throughput tools... (Review)
Review
CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high-throughput tools for genetic modification by the specific targeting of double-strand breaks in the genome of hosts. Plant biotechnologists have extensively used the S. pyogenes Cas9-based system since its inception in 2013. However, there are still some limitations to its even broader usage in plants. Major restrictions, especially in agricultural biotechnology, are the currently unclear regulatory status of plants modified with CRISPR/Cas9 and the lack of suitable delivery methods for some plant species. Solutions to these limitations could come in the form of new variants of genome editing enzymes that have recently been discovered and have already proved comparable to or even better in performance than S. pyogenes CRISPR/Cas9 in terms of precision and ease of delivery in mammal cells. Although some of them have already been tested in plants, most of them are less well known in the plant science community. In this review, we describe the following new enzyme systems engineered for genome editing, transcriptional regulation and cellular imaging-C2c2 from L. shahii; Cas9 from F. novicida, S. aureus, S. thermophiles, N. meningitidis; Cpf1 from F. novicida, Acidaminococcus and Lachnospiraceae; nickase, split, enhanced and other Cas9 variants from S. pyogenes; catalytically inactive SpCas9 linked to various nuclease or gene-regulating domains-with an emphasis on their advantages in comparison with the broadly used SpCas9. In addition, we discuss new possibilities they offer in plant biotechnology.
Topics: Biotechnology; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Models, Biological; Plants, Genetically Modified; Streptococcus pyogenes
PubMed: 28371222
DOI: 10.1111/pbi.12736 -
Microbiome May 2024Left ventricular diastolic dysfunction (LVDD) is an important precursor of heart failure (HF), but little is known about its relationship with gut dysbiosis and...
BACKGROUND
Left ventricular diastolic dysfunction (LVDD) is an important precursor of heart failure (HF), but little is known about its relationship with gut dysbiosis and microbial-related metabolites. By leveraging the multi-omics data from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), a study with population at high burden of LVDD, we aimed to characterize gut microbiota associated with LVDD and identify metabolite signatures of gut dysbiosis and incident LVDD.
RESULTS
We included up to 1996 Hispanic/Latino adults (mean age: 59.4 years; 67.1% female) with comprehensive echocardiography assessments, gut microbiome, and blood metabolome data. LVDD was defined through a composite criterion involving tissue Doppler assessment and left atrial volume index measurements. Among 1996 participants, 916 (45.9%) had prevalent LVDD, and 212 out of 594 participants without LVDD at baseline developed incident LVDD over a median 4.3 years of follow-up. Using multivariable-adjusted analysis of compositions of microbiomes (ANCOM-II) method, we identified 7 out of 512 dominant gut bacterial species (prevalence > 20%) associated with prevalent LVDD (FDR-q < 0.1), with inverse associations being found for Intestinimonas_massiliensis, Clostridium_phoceensis, and Bacteroide_coprocola and positive associations for Gardnerella_vaginali, Acidaminococcus_fermentans, Pseudomonas_aeruginosa, and Necropsobacter_massiliensis. Using multivariable adjusted linear regression, 220 out of 669 circulating metabolites with detection rate > 75% were associated with the identified LVDD-related bacterial species (FDR-q < 0.1), with the majority being linked to Intestinimonas_massiliensis, Clostridium_phoceensis, and Acidaminococcus_fermentans. Furthermore, 46 of these bacteria-associated metabolites, mostly glycerophospholipids, secondary bile acids, and amino acids, were associated with prevalent LVDD (FDR-q < 0.1), 21 of which were associated with incident LVDD (relative risk ranging from 0.81 [p = 0.001, for guanidinoacetate] to 1.25 [p = 9 × 10, for 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4)]). The inclusion of these 21 bacterial-related metabolites significantly improved the prediction of incident LVDD compared with a traditional risk factor model (the area under the receiver operating characteristic curve [AUC] = 0.73 vs 0.70, p = 0.001). Metabolite-based proxy association analyses revealed the inverse associations of Intestinimonas_massilliensis and Clostridium_phoceensis and the positive association of Acidaminococcus_fermentans with incident LVDD.
CONCLUSION
In this study of US Hispanics/Latinos, we identified multiple gut bacteria and related metabolites linked to LVDD, suggesting their potential roles in this preclinical HF entity. Video Abstract.
Topics: Humans; Gastrointestinal Microbiome; Female; Middle Aged; Male; Hispanic or Latino; Ventricular Dysfunction, Left; United States; Dysbiosis; Aged; Bacteria; Metabolome; Echocardiography
PubMed: 38725043
DOI: 10.1186/s40168-024-01797-x -
Scientific Reports Jul 2023The fecal microbiome of 55 obese children and adolescents (BMI-SDS 3.2 ± 0.7) and of 25 normal-weight subjects, matched both for age and sex (BMI-SDS...
The fecal microbiome of 55 obese children and adolescents (BMI-SDS 3.2 ± 0.7) and of 25 normal-weight subjects, matched both for age and sex (BMI-SDS - 0.3 ± 1.1) was analysed. Streptococcus, Acidaminococcus, Sutterella, Prevotella, Sutterella wadsworthensis, Streptococcus thermophilus, and Prevotella copri positively correlated with obesity. The inferred pathways strongly associated with obesity concern the biosynthesis pathways of tyrosine, phenylalanine, tryptophan and methionine pathways. Furthermore, polyamine biosynthesis virulence factors and pro-inflammatory lipopolysaccharide biosynthesis pathway showed higher abundances in obese samples, while the butanediol biosynthesis showed low abundance in obese subjects. Different taxa strongly linked with obesity have been related to an increased risk of multiple diseases involving metabolic pathways related to inflammation (polyamine and lipopolysaccharide biosynthesis). Cholesterol, LDL, and CRP positively correlated with specific clusters of microbial in obese patients. The Firmicutes/Bacteroidetes-ratio was lower in obese samples than in controls and differently from the literature we state that this ratio could not be a biomarker for obesity.
Topics: Child; Adolescent; Humans; Pediatric Obesity; Lipopolysaccharides; Gastrointestinal Microbiome; Microbiota; Algorithms
PubMed: 37438382
DOI: 10.1038/s41598-023-36533-2 -
FEMS Microbiology Letters Apr 2019The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a...
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.
Topics: Acidaminococcus; Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; Catalytic Domain; DNA Cleavage; Endodeoxyribonucleases; Endonucleases; Escherichia coli; Gene Editing; HEK293 Cells; Humans; Nucleotide Motifs; Plasmids
PubMed: 31004485
DOI: 10.1093/femsle/fnz085 -
Frontiers in Cellular and Infection... 2022This is the first study on gut microbiota (GM) in children affected by coronavirus disease 2019 (COVID-19). Stool samples from 88 patients with suspected severe acute...
This is the first study on gut microbiota (GM) in children affected by coronavirus disease 2019 (COVID-19). Stool samples from 88 patients with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 95 healthy subjects were collected (admission: 3-7 days, discharge) to study GM profile by 16S rRNA gene sequencing and relationship to disease severity. The study group was divided in COVID-19 (68), Non-COVID-19 (16), and MIS-C (multisystem inflammatory syndrome in children) (4). Correlations among GM ecology, predicted functions, multiple machine learning (ML) models, and inflammatory response were provided for COVID-19 and Non-COVID-19 cohorts. The GM of COVID-19 cohort resulted as dysbiotic, with the lowest α-diversity compared with Non-COVID-19 and CTRLs and by a specific β-diversity. Its profile appeared enriched in , , and and reduced in , , , , , , and , compared with CTRLs ( 0.05). All GM paired-comparisons disclosed comparable results through all time points. The comparison between COVID-19 and Non-COVID-19 cohorts highlighted a reduction of in the COVID-19 cohort ( < 0.05). The GM of MIS-C cohort was characterized by an increase of , , , , and and a decrease of , , , and , compared with CTRLs. Stratifying for disease severity, the GM associated to "moderate" COVID-19 was characterized by lower α-diversity compared with "mild" and "asymptomatic" and by a GM profile deprived in , , , and and enriched in , , , , , , and The ML models identified , , , , , , , , , , , , , , , , and as microbial markers of COVID-19. The KEGG ortholog (KO)-based prediction of GM functional profile highlighted 28 and 39 KO-associated pathways to COVID-19 and CTRLs, respectively. Finally, and correlated with proinflammatory cytokines regardless disease severity. Unlike adult GM profiles, was a specific marker of pediatric COVID-19 GM. The durable modification of patients' GM profile suggested a prompt GM quenching response to SARS-CoV-2 infection since the first symptoms. and reduced fatty acid and amino acid degradation were proposed as specific COVID-19 disease traits, possibly associated to restrained severity of SARS-CoV-2-infected children. Altogether, this evidence provides a characterization of the pediatric COVID-19-related GM.
Topics: Adult; Bacteroides; Bifidobacterium; COVID-19; Child; Clostridium; Feces; Gastrointestinal Microbiome; Humans; RNA, Ribosomal, 16S; SARS-CoV-2; Systemic Inflammatory Response Syndrome
PubMed: 35873161
DOI: 10.3389/fcimb.2022.908492 -
Biomolecules Apr 2024In recent years, CRISPR-Cas toolboxes for editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes...
In recent years, CRISPR-Cas toolboxes for editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of strains. This study explored the first use of sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.
Topics: Streptomyces; Gene Editing; CRISPR-Cas Systems; Acidaminococcus; CRISPR-Associated Protein 9; Multigene Family; Bacterial Proteins; CRISPR-Associated Proteins; Genome, Bacterial
PubMed: 38672502
DOI: 10.3390/biom14040486 -
The Journal of Biological Chemistry 2021Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron...
Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.
Topics: Acidaminococcus; Bacterial Proteins; Catalysis; Electron Transport; Electrons; Energy Metabolism; Flavin-Adenine Dinucleotide; Kinetics; Oxidation-Reduction
PubMed: 33755021
DOI: 10.1016/j.jbc.2021.100294 -
International Journal of Molecular... Jan 2024The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand...
The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a marker gene into the safe harbor site and a super-exon into in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
Topics: Humans; CRISPR-Cas Systems; Pluripotent Stem Cells; Induced Pluripotent Stem Cells; Hematopoietic Stem Cells; Alleles
PubMed: 38256061
DOI: 10.3390/ijms25020985