-
Nature Biomedical Engineering May 2017Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and...
Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered Cpf1 (AsCpf1) CRISPR RNAs (crRNAs) and 5 types of AsCpf1 mRNAs, and show that the top-performing modified crRNA (cr3'5F, containing five 2'-fluoro ribose at the 3' termini) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3'5F and ψ-modified AsCpf1 or Cpf1 (LbCpf1) mRNAs augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologs enabled genome editing in the presence of AsCpf1. Engineered CRISPR-Cpf1 systems should facilitate a broad range of genome editing applications.
PubMed: 28840077
DOI: 10.1038/s41551-017-0066 -
Standards in Genomic Sciences Jul 2010Acidaminococcus fermentans (Rogosa 1969) is the type species of the genus Acidaminococcus, and is of phylogenetic interest because of its isolated placement in a...
Acidaminococcus fermentans (Rogosa 1969) is the type species of the genus Acidaminococcus, and is of phylogenetic interest because of its isolated placement in a genomically little characterized region of the Firmicutes. A. fermentans is known for its habitation of the gastrointestinal tract and its ability to oxidize trans-aconitate. Its anaerobic fermentation of glutamate has been intensively studied and will now be complemented by the genomic basis. The strain described in this report is a nonsporulating, nonmotile, Gram-negative coccus, originally isolated from a pig alimentary tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Acidaminococcaceae, and the 2,329,769 bp long genome with its 2,101 protein-coding and 81 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
PubMed: 21304687
DOI: 10.4056/sigs.1002553 -
Food & Nutrition Research 2023The relationship between fruit, whole grain, and total energy consumption and the gut microbiome in the Chinese population remains unclear.
BACKGROUND
The relationship between fruit, whole grain, and total energy consumption and the gut microbiome in the Chinese population remains unclear.
OBJECTIVE
We investigated the relationship between intakes of fruits, whole grains, and energy, and the diversity and composition of gut microbiota.
DESIGN
This cross-sectional study included 167 subjects aged 40-75 years who underwent colonoscopy at Nankai Hospital in Tianjin, China. Each of the participants completed a personal history questionnaire, a 7-day dietary record, and donated a fecal sample. The V3-V4 hypervariable region of the bacterial 16S rRNAgene was amplified and sequenced using Illumina Novaseq. The relationship between diet and gut microbiota was evaluated in terms of both the overall composition and the abundance of specific taxon.
RESULTS
Fruits intake was positively related to the abundance of Bacilli, Porphyromonadaceae, Streptococcaceae, , and Bilophila in fecal samples. Higher whole grains intake was associated with higher microbial diversity, as measured by Shannon, Simpson, and Chao1 indices. Specifically, there was a significant increase inthe relative abundance of Lachnospiraceae and a decrease in Actinobacteria with increased whole grains intake. Moreover, higher intake of total energy was associated with a lower abundance of and a higher abundance of Lactobacillales and .
CONCLUSION
Whole grains intake was positively associated with gut microbial diversity. Fruits and total energy intake were related to the abundance of specifictaxon (e.g., Bacilli and Acidaminococcus). These findings highlight the potential importance of dietary interventions for modulating gut microbiota composition and promoting overall health.
PubMed: 38084154
DOI: 10.29219/fnr.v67.9725 -
Hepatology (Baltimore, Md.) Mar 2021Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), caused by autoimmune regulator (AIRE) mutations, manifests with chronic mucocutaneous...
BACKGROUND AND AIMS
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), caused by autoimmune regulator (AIRE) mutations, manifests with chronic mucocutaneous candidiasis (CMC) and multisystem autoimmunity, most often hypoparathyroidism (HP) and adrenal insufficiency (AI). European cohorts previously reported a ~10% prevalence of APECED-associated hepatitis (APAH) with presentations ranging from asymptomatic laboratory derangements to fatal fulminant hepatic failure. Herein, we characterized APAH in a large APECED cohort from the Americas.
APPROACH AND RESULTS
Forty-five consecutive patients with APECED were evaluated (2013-2015) at the National Institutes of Health (NIH; NCT01386437). Hepatology consultation assessed hepatic and autoimmune biomarkers and liver ultrasound in all patients. Liver biopsies evaluated autoimmune features and fibrosis. The 16S ribosomal RNA (rRNA) sequencing was performed in 35 patients' stools (12 with and 23 without APAH). Among 43 evaluable patients, 18 (42%) had APAH; in 33.3% of those with APAH, APAH occurred before developing classic APECED diagnostic criteria. At APAH diagnosis, the median age was 7.8 years, and patients manifested with aminotransferase elevation and/or hyperbilirubinemia. All patients with APAH were in clinical remission during their NIH evaluation while receiving immunomodulatory treatment. We found no difference in age, sex, or prevalence of CMC, AI, or HP between patients with or without APAH. Autoantibody positivity against aromatic L-amino acid decarboxylase, cytochrome P450 family 1 subfamily A member 2, histidine decarboxylase (HDC), bactericidal/permeability-increasing fold-containing B1, tryptophan hydroxlase, and 21-hydroxylase (21-OH), and the homozygous c.967_979del13 AIRE mutation were associated with APAH development. Classical serological biomarkers of autoimmune hepatitis (AIH) were only sporadically positive. AIH-like lymphoplasmacytic inflammation with mild fibrosis was the predominant histological feature. Stool microbiome analysis found Slackia and Acidaminococcus in greater abundance in patients with APAH.
CONCLUSIONS
APAH is more common than previously described, may present early before classic APECED manifestations, and most often manifests with milder, treatment-responsive disease. Several APECED-associated autoantibodies, but not standard AIH-associated biomarkers, correlate with APAH.
Topics: Adolescent; Adult; Americas; Autoantibodies; Biomarkers; Biopsy; Female; Gene Deletion; Hepatitis, Autoimmune; Humans; Immunotherapy; Liver; Liver Cirrhosis; Male; Polyendocrinopathies, Autoimmune; Young Adult
PubMed: 32557834
DOI: 10.1002/hep.31421 -
European Journal of Biochemistry Aug 19811. Glutaconate CoA-transferase catalyses the transfer of CoAS- from acetyl-CoA preferentially to (E)-glutaconate, but glutarate, (R)-2-hydroxyglutarate, acrylate and...
1. Glutaconate CoA-transferase catalyses the transfer of CoAS- from acetyl-CoA preferentially to (E)-glutaconate, but glutarate, (R)-2-hydroxyglutarate, acrylate and propionate are also good acceptors. No reaction was observed with (Z)-glutaconate and C4-dicarboxylic acids. 2. The product of the reaction of acetyl-CoA with (E)-glutaconate is the 1-isomer of glutaconyl-CoA, i.e. the thiol ester is conjugated with the double bond. Other results indicate, however, that with (R)-2-hydroxyglutarate as substrate both possible isomers are generated. 3. Glutaconate CoA-transferase was purified from cell-free extracts of Acidaminococcus fermentans to apparent homogeneity and crystallized. The relative molecular mass of the enzyme is approximately 275000. It consists of two different polypeptide chains (M, 32000 and 34000). On the catalytic pathway a thiolester is formed between CoASH and a carboxylate of the smaller polypeptide chain. 4. The structural and functional relationships between glutaconate CoA-transferase and other CoA-transferases are discussed. 5. Glutaconate CoA-transferase is also present in other bacteria fermenting glutamate via hydroxyglutarate. Experiments with an antiserum against the enzyme indicate that the transferase is necessary for the decarboxylation of glutaconate but not for the dehydration of (R)-2-hydroxyglutarate.
Topics: Acyl Coenzyme A; Coenzyme A; Coenzyme A-Transferases; Crystallization; Glutarates; Gram-Negative Anaerobic Bacteria; Kinetics; Molecular Weight; Substrate Specificity; Sulfhydryl Reagents; Sulfurtransferases
PubMed: 6945182
DOI: 10.1111/j.1432-1033.1981.tb06404.x -
The Journal of Biological Chemistry 2021Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. β-FAD of the electron transfer flavoprotein (EtfAB) from the...
Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. β-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD). The resultant α-FAD hydroquinone (α-FADH) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD to monitor formation of α-FAD or α-FADH, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to β-FAD at a rate of 920 s, yielding the charge-transfer complex NAD:β-FADH with an absorbance maximum at 650 nm. β-FADH bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD. With α-FAD, the reduction of β-FAD with NADH was 1500 times slower. Reduction of β-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.
Topics: Acidaminococcus; Bacterial Proteins; Electron Transport; Electron-Transferring Flavoproteins; Flavins; Kinetics; Oxidation-Reduction; Phylogeny
PubMed: 33239361
DOI: 10.1074/jbc.RA120.016017 -
Journal of Bacteriology May 1969Acidaminococcus gen. n. and the type species Acidaminococcus fermentans sp. n. were described. Amino acids, of which glutamic acid is the most important, could serve as...
Acidaminococcus gen. n. and the type species Acidaminococcus fermentans sp. n. were described. Amino acids, of which glutamic acid is the most important, could serve as the sole energy source for growth. Acetic and butyric acids and CO(2) were produced; propionic acid and hydrogen were not produced. Amino acid media supporting growth and the amino acid and vitamin requirements were described. Glucose was frequently not fermented or was weakly catabolized. Derivative products from glucose autoclaved in media, but not glucose itself, stimulated or were required for growth in amino acid media. A wide range of polyols and carbohydrates were not attacked. Lactate, fumarate, malate, succinate, citrate, and pyruvate were not used as energy sources for growth. Pyruvate completely suppressed growth. Cytochrome oxidase and benzidine reactions were negative; catalase, indole, acetyl methyl carbinol, and H(2)S were not produced; nitrate and sulfonthalein indicators were not reduced; ammonia was produced; gelatin liquefaction was negative or slow and partial; vancomycin (7.5 mug/ml) was resisted. Acidaminococcus was different from Veillonella in morphology, serology, nutrition, utilization of substrates, and accumulation of products in media supporting growth; Acidaminococcus resembled Peptococcus in utilization of glutamic acid and accumulation of similar products, but the two genera differed in morphology, gram reaction, serology, guanine plus cytosine content of deoxyribonucleic acid, and nutrition.
Topics: Aldehydes; Amino Acids; Bacteria; Culture Media; DNA, Bacterial; Glucose; Glutamates; Veillonella; Vitamins
PubMed: 5784223
DOI: 10.1128/jb.98.2.756-766.1969 -
Frontiers in Psychiatry 2024Schizophrenia is a complex psychiatric disorder, of which molecular pathogenesis remains largely unknown. Accumulating evidence suggest that gut microbiota may affect...
INTRODUCTION
Schizophrenia is a complex psychiatric disorder, of which molecular pathogenesis remains largely unknown. Accumulating evidence suggest that gut microbiota may affect brain function via the complex gut-brain axis, which may be a potential contributor to schizophrenia. However, the alteration of gut microbiota showed high heterogeneity across different studies. Therefore, this study aims to identify the consistently altered gut microbial taxa associated with schizophrenia.
METHODS
We conducted a systematic search and synthesis of the up-to-date human gut microbiome studies on schizophrenia, and performed vote counting analyses to identify consistently changed microbiota. Further, we investigated the effects of potential confounders on the alteration of gut microbiota.
RESULTS
We obtained 30 available clinical studies, and found that there was no strong evidence to support significant differences in α-diversity and β-diversity between schizophrenic patients and healthy controls. Among 428 differential gut microbial taxa collected from original studies, we found that 8 gut microbial taxa were consistently up-regulated in schizophrenic patients, including Proteobacteria, Gammaproteobacteria, , , , , and . While 5 taxa were consistently down-regulated in schizophrenia, including , , , and .
DISCUSSION
These findings suggested that gut microbial changes in patients with schizophrenia were characterized by the depletion of anti-inflammatory butyrate-producing genera, and the enrichment of certain opportunistic bacteria genera and probiotics. This study contributes to further understanding the role of gut microbiota in schizophrenia, and developing microbiota-based diagnosis and therapy for schizophrenia.
PubMed: 38596637
DOI: 10.3389/fpsyt.2024.1366311 -
FEMS Microbiology Reviews Oct 2004Several clostridia and fusobacteria ferment alpha-amino acids via (R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination. This reaction is of great... (Review)
Review
Several clostridia and fusobacteria ferment alpha-amino acids via (R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination. This reaction is of great mechanistic interest, since the beta-hydrogen, to be eliminated as proton, is not activated (pK 40-50). A mechanism has been proposed, in which one high-energy electron acts as cofactor and transiently reduces the electrophilic thiol ester carbonyl to a nucleophilic ketyl radical anion. The 2-hydroxyacyl-CoA dehydratases are two-component systems composed of an extremely oxygen-sensitive component A, an activator, and component D, the actual dehydratase. Component A, a homodimer with one [4Fe-4S]cluster, transfers an electron to component D, a heterodimer with 1-2 [4Fe-4S]clusters and FMN, concomitant with hydrolysis of two ATP. From component D the electron is further transferred to the substrate, where it facilitates elimination of the hydroxyl group. In the resulting enoxyradical the beta-hydrogen is activated (pK14). After elimination the electron is handed-over to the next incoming substrate without further hydrolysis of ATP. The helix-cluster-helix architecture of component A forms an angle of 105 degrees, which probably opens to 180 degrees upon binding of ATP resembling an archer shooting arrows. Therefore we designated component A as 'Archerase'. Here, we describe 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, 2-phenyllactate dehydratase from Clostridium sporogenes, 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, and lactyl-CoA dehydratase from Clostridium propionicum. A relative of the 2-hydroxyacyl-CoA dehydratases is benzoyl-CoA reductase from Thauera aromatica. Analogous but unrelated archerases are the iron proteins of nitrogenase and bacterial protochlorophyllide reductase. In anaerobic organisms, which do not oxidize 2-oxo acids, a second energy-driven electron transfer from NADH to ferredoxin, the electron donor of component A, has been established. The transfer is catalysed by a membrane-bound NADH-ferredoxin oxidoreductase driven by an electrochemical Na(+)-gradient. This enzyme is related to the Rnf proteins involved in Rhodobacter capsulatus nitrogen fixation.
Topics: Acidaminococcus; Acyl Coenzyme A; Amino Acids; Bacteria, Anaerobic; Clostridium; Fermentation; Ferredoxin-NADP Reductase; Fusobacterium nucleatum; Hydro-Lyases; Nitrogenase; Rhodobacter capsulatus; Thauera
PubMed: 15374661
DOI: 10.1016/j.femsre.2004.03.001 -
Plant Biotechnology Journal Oct 2019The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in...
The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.
Topics: Arabidopsis; CRISPR-Cas Systems; Endonucleases; Gene Editing; Genome, Plant; Solanum lycopersicum; Mutagenesis; Sequence Deletion; Nicotiana
PubMed: 30950179
DOI: 10.1111/pbi.13113