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1.
Biomed Res Int ; 2021: 9995225, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34258290

RESUMO

The aim of the study was to compare the microbiota composition and bacterial diversity of subgingival plaque in chronic periodontitis patients with and without gingival erosive oral lichen planus. The subgingival plaque samples of 20 chronic periodontitis patients with gingival erosive oral lichen planus (CP-OLP group) and 19 chronic periodontitis patients without gingival erosive oral lichen planus (CP group) were analyzed by 16S rRNA gene high-throughput sequencing. Compared with the CP group, the richness and diversity of subgingival plaque microflora in the CP-OLP group decreased significantly. There were some differences between the two groups in the composition of microflora on the levels of phylum and genus. Distributions of Prevotella and Leptotrichia in the CP-OLP group were significantly lower than those in the CP group. The dominant genera in CP-OLP group were Pseudomonas and Granulicatella. These results indicated that gingival erosive oral lichen planus may influence the structure and proportion of subgingival plaque microflora.


Assuntos
Periodontite Crônica/microbiologia , Genes de RNAr , Gengiva/microbiologia , Líquen Plano Bucal/genética , Microbiota , RNA Ribossômico 16S/genética , Adulto , Bactérias/genética , Biologia Computacional , Estudos Transversais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leptotrichia , Masculino , Pessoa de Meia-Idade , Prevotella , Pseudomonas
2.
Toxins (Basel) ; 13(4)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916905

RESUMO

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Córtex Cerebral/enzimologia , Leptotrichia/enzimologia , Crescimento Neuronal , Neurônios/enzimologia , Animais , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Córtex Cerebral/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leptotrichia/genética , Camundongos , Neurônios/patologia
3.
J Infect Chemother ; 27(8): 1265-1269, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33867268

RESUMO

The in vitro antibacterial spectra and activities of five antimicrobial agents, including lascufloxacin (LSFX) and two quinolones, were investigated against 69 species of anaerobes in 31 genera and 188 strains in 9 genera, respectively. In this study, minimum inhibitory concentrations (MICs) of lascufloxacin against the reference strains associated with respiratory and head and neck infections. LSFX inhibited the growth of 33 gram-positive and gram-negative reference strains at ≤0.015-2 µg/mL, except for Leptotrichia buccalis. MICs ranges of LSFX against the clinical isolates of 44 Porphyromonas spp., 45 Prevotella spp., 25 Fusobacterium spp., 7 Leptotrichia spp., 25 Parvimonas micra, 25 other gram-positive anaerobic cocci, and 17 Veillonella spp., were ≤0.015-4, 0.125-4, 0.06-0.5, 2, 0.25-16, ≤0.015-2, ≤0.015-16 µg/mL, respectively. LSFX demonstrated potent antibacterial efficacy against a wide range of species isolated from specimens involved in respiratory as well as head and neck infections.


Assuntos
Fluoroquinolonas , Leptotrichia , Antibacterianos/farmacologia , Bactérias Anaeróbias , Firmicutes , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana
4.
PLoS One ; 16(3): e0242396, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33720954

RESUMO

The objective of this pilot study was to describe the microbial profiles present in the plaque and saliva of children who continued to develop new carious lesions following treatment with silver diamine fluoride ("nonresponders") compared to caries active, caries-free, and children immediately receiving SDF treatment for untreated caries in order to identify potential microbial differences that may relate to a re-incidence of caries. Saliva and plaque samples from infected and contralateral sites were obtained from twenty children who were either caries free, had active carious lesions, were caries active and received SDF treatment immediately before sampling, or had previously received SDF treatment and developed new caries. In total, 8,057,899 Illumina-generated sequence reads from 60 samples were obtained. Reads were processed using the Quantitative Insights Into Microbial Ecology pipeline. Group differences were assessed using Analysis of Variance Models and Tukey Honest Significant Differences. To identify significant taxa between treatment groups, Linear discriminant analysis Effect Size (LefSe) and Analysis of Differential Abundance Taking Sample Variation Into Account were used. Differential abundant analysis indicated that members of the Lachnospiraceae family were significantly enriched in non-responders and the genus Tannerella and species Granulicatella adiances were also highly abundant in this group. LefSe analysis between non-responders and SDF-treated groups revealed that genera Leptotrichia and Granulicatella were enriched in non-responders. We observed the highest abundance of phosphotransferase system and lowest abundance of lipopolysaccharide synthesis in non-responders. The microbiome in dental biofilms is responsible for initiation and progression of dental caries. SDF has been shown to be effective in arresting the progression carious lesions, in part due to its antimicrobial properties. Findings suggest that the differential abundance of select microbiota and specific pathway functioning in individuals that present with recurrent decay after SDF treatment may contribute to a potential failure of silver diamine fluoride to arrest dental caries. However, the short duration of sample collection following SDF application and the small sample size emphasize the need for further data and additional analysis.


Assuntos
Cárie Dentária/tratamento farmacológico , Microbiota , Compostos de Amônio Quaternário/uso terapêutico , Compostos de Prata/uso terapêutico , Carnobacteriaceae/genética , Carnobacteriaceae/isolamento & purificação , Criança , Estudos Transversais , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Cárie Dentária/patologia , Placa Dentária/microbiologia , Análise Discriminante , Fluoretos Tópicos/uso terapêutico , Humanos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Projetos Piloto , Análise de Componente Principal , Saliva/microbiologia , Análise de Sequência de DNA , Falha de Tratamento
5.
Mol Cell ; 81(5): 1100-1115.e5, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33472057

RESUMO

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Leptotrichia/genética , RNA Guia/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Clivagem do RNA , RNA Guia/genética , RNA Guia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
6.
Genomics ; 113(1 Pt 2): 664-676, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33010388

RESUMO

Although the prevalence of inflammatory bowel disease (IBD) has been increasing worldwide, the etiology remains elusive. Investigating oral microbiota dysbiosis is essential to understanding IBD pathogenesis. Our study evaluated variations in salivary microbiota and identified potential associations with IBD. The saliva microbiota of 22 IBD patients and 8 healthy controls (HCs) was determined using 16S ribosomal RNA (rRNA) gene sequencing and analyzed using QIIME2. A distinct saliva microbiota dysbiosis in IBD, characterized by alterations in microbiota biodiversity and composition, was identified. Saccharibacteria (TM7), Absconditabacteria (SR1), Leptotrichia, Prevotella, Bulleidia, and Atopobium, some of which are oral biofilm-forming bacteria, were significantly increased. Moreover, levels of inflammatory cytokines associated with IBD were elevated and positively correlated with TM7 and SR1. Functional variations include down-regulation of genetic information processing, while up-regulation of carbohydrate metabolism and protein processing in the endoplasmic reticulum in IBD. Our data implicate salivary microbiota dysbiosis involving in IBD pathogenesis.


Assuntos
Disbiose/microbiologia , Doenças Inflamatórias Intestinais/microbiologia , Metagenoma , Boca/microbiologia , Adulto , Disbiose/complicações , Disbiose/epidemiologia , Feminino , Microbioma Gastrointestinal , Humanos , Doenças Inflamatórias Intestinais/complicações , Leptotrichia/genética , Leptotrichia/patogenicidade , Masculino , Prevotella/genética , Prevotella/patogenicidade
7.
Clin Oral Investig ; 25(5): 3033-3042, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33057825

RESUMO

OBJECTIVES: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. MATERIALS AND METHODS: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). RESULTS: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/µg dentin and 3.45 log10 EU/mL/µg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). CONCLUSION: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. CLINICAL RELEVANCE: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment.


Assuntos
Cárie Dentária , Pulpite , Bactérias , Capnocytophaga , Dentina , Endotoxinas , Humanos , Leptotrichia
8.
J Dent ; 104: 103539, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33248211

RESUMO

OBJECTIVE: Microbiota comparisons between healthy and diseased dental tissues have accentuated the importance of cultivating and identifying bacterial species that play a role in the initiation and progression of dental caries. The objective of this study was to evaluate the bacterial community composition in caries-active and caries-free children. METHODS: Supragingival plaque samples were collected from 64 caries-active and 64 caries-free Middle Eastern children. The hypervariable V3-V4 of the bacterial 16S rRNA gene was sequenced with Human Oral Microbe Identification using Next Generation Sequencing. Microbial community structure and composition analyses were performed by processing operational taxonomic units. Bioinformatic analyses, including analysis of similarity, alpha and beta diversities, and principal coordinate analysis, were carried out. RESULTS: Diversity indices did not find differences between the caries-active and caries-free groups (p > 0.05). Similarity analysis demonstrated that the microbiota composition did not differ between the two groups. Comparative analysis at the species level revealed a significantly higher relative abundance of Leptotrichia shahii, Prevotella melaninogenica, Veillonella dispar, Leptotrichia HOT 498, and Streptococcus mutans in caries-active children (p < 0.05). Corynebacterium matruchotii, Lautropia mirabilis, Neisseria elongata, and Corynebacterium durum were relatively more abundant in the caries-free group (p < 0.05). Species belonging to the Leptotrichia, Prevotella, and Veillonella genera were significantly predominant in the caries-active subjects. CONCLUSION: In view of the lack of a clear association between Corynebacterium spp. and dental caries status in the literature, the predominance of these species in caries-free children warrants further research to understand their possible role in a health-associated microbial community. CLINICAL SIGNIFICANCE: Understanding the relationship between specific bacteria present in dental biofilms and health and disease is essential for preventing and combating dental caries. Using advanced next generation sequencing techniques, the present study demonstrated the complexity of the caries microbiome and identified species/genera whose virulence or protective properties should be further explored.


Assuntos
Cárie Dentária , Placa Dentária , Microbiota , Burkholderiaceae , Criança , Corynebacterium , Suscetibilidade à Cárie Dentária , Dentição , Humanos , Leptotrichia , Microbiota/genética , RNA Ribossômico 16S/genética , Veillonella
9.
Artigo em Inglês | MEDLINE | ID: mdl-33288524

RESUMO

The oral aerotolerant anaerobe Leptotrichia goodfellowii is an unusual cause of endocarditis and is amenable to treatment with ß-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case of culture-negative, aortic and mitral valve endocarditis in a 66-yr-old woman with a history of cardiomyopathy, atrial fibrillation with intracardiac pacer, poor dentition, and recent tooth infection. In this case, 16S rDNA amplicon Sanger sequencing was not sufficient for pathogen identification because of interfering DNA, but deconvolution of the clinical sample using reflexive next-generation amplicon sequencing enabled confident identification of a single pathogenic organism, L. goodfellowii The patient developed a sigmoid colon perforation and died despite additional surgical treatment. Most Leptotrichia endocarditis cases have been subacute and have been successfully treated with antibiotics, with or without valve replacement. This case highlights both an unusual etiologic agent of endocarditis, as well as the rational utilization of advanced molecular diagnostics tools for characterizing serious infections.


Assuntos
Endocardite Bacteriana/diagnóstico , Endocardite/diagnóstico , Infecções por Fusobacteriaceae/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Idoso , DNA Ribossômico/genética , Feminino , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
10.
Nucleic Acids Res ; 48(17): e101, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32797156

RESUMO

Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Ensaios Enzimáticos/métodos , Inibidores da Síntese de Ácido Nucleico/análise , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Corantes Fluorescentes/química , Leptotrichia , Ligantes , Inibidores da Síntese de Ácido Nucleico/farmacologia , Riboswitch , Rifampina/análise , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
11.
Nat Biomed Eng ; 4(12): 1140-1149, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32848209

RESUMO

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.


Assuntos
COVID-19/diagnóstico , Proteínas Associadas a CRISPR/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/virologia , Humanos , Leptotrichia/enzimologia , Pandemias/prevenção & controle
12.
Int J Syst Evol Microbiol ; 70(3): 2084-2088, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32228774

RESUMO

The reclassification of Leptotrichia goodfellowii as Pseudoleptotrichia goodfellowii gen. nov., comb. nov. is proposed because of the separate phylogenetic position on the basis of the results of 16S rRNA gene sequence analysis, the genomic differences from all other Leptotrichia species and phenotypic differences from Leptotrichia species. The species Pseudoleptotrichia goodfellowii is the type species of the genus. The type strain is LB 57T, CCUG 32286 T, DSM 19756T.


Assuntos
Leptotrichia/classificação , Filogenia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
13.
Mol Cell ; 78(5): 850-861.e5, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348779

RESUMO

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.


Assuntos
Proteínas Associadas a CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas/fisiologia , Edição de RNA/fisiologia , Animais , Bactérias/genética , Bacteriófagos/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Células HEK293 , Humanos , Leptotrichia/genética , Leptotrichia/metabolismo , RNA/genética , Edição de RNA/genética
17.
PLoS One ; 14(11): e0225636, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31770392

RESUMO

OBJECTIVE: To investigate age-associated changes in airway microbiome composition and their relationships with lung function and arterial stiffness among genetically matched young and elderly pairs. METHODS: Twenty-four genetically linked family pairs comprised of younger (≤40 years) and older (≥60 years) healthy participants were recruited (Total n = 48). Lung function and arterial stiffness (carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx)) were assessed. Sputum samples were collected for targeted 16S rRNA gene amplicon sequencing and correlations between microbiome composition, lung function and arterial stiffness were investigated. RESULTS: Elderly participants exhibited reductions in lung function (FEV1 (p<0.001), FVC (p<0.001) and percentage FEV1/FVC (p = 0.003)) and a 1.3-3.9-fold increase in arterial stiffness (p<0.001) relative to genetically related younger adults. Elderly adults had a higher relative abundance of Firmicutes (p = 0.035) and lower relative abundance of Proteobacteria (p = 0.014), including specific genera Haemophilus (p = 0.024) and Lautropia (p = 0.020) which were enriched in the younger adults. Alpha diversity was comparable between young and elderly pairs (p>0.05) but was inversely associated with lung function (FEV1%Predicted and FVC %Predicted) in the young (p = 0.006 and p = 0.003) though not the elderly (p = 0.481 and p = 0.696). Conversely, alpha diversity was negatively associated with PWV in the elderly (p = 0.01) but not the young (p = 0.569). Specifically, phylum Firmicutes including the genus Gemella were correlated with lung function (FVC %Predicted) in the young group (p = 0.047 and p = 0.040), while Fusobacteria and Leptotrichia were associated with arterial stiffness (PWV) in the elderly (both p = 0.004). CONCLUSION: Ageing is associated with increased Firmicutes and decreased Proteobacteria representation in the airway microbiome among a healthy Asian cohort. The diversity and composition of the airway microbiome is independently associated with lung function and arterial stiffness in the young and elderly groups respectively. This suggests differential microbial associations with these phenotypes at specific stages of life with potential prognostic implications.


Assuntos
Pulmão/fisiologia , Microbiota , Rigidez Vascular/fisiologia , Adulto , Fatores Etários , Idoso , Família , Firmicutes/genética , Firmicutes/isolamento & purificação , Haemophilus/genética , Haemophilus/isolamento & purificação , Voluntários Saudáveis , Humanos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Pessoa de Meia-Idade , Análise de Onda de Pulso , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Testes de Função Respiratória , Escarro/microbiologia , Adulto Jovem
18.
Nat Protoc ; 14(10): 2986-3012, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548639

RESUMO

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
19.
Emerg Infect Dis ; 25(8): 1614-1616, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31310211

RESUMO

We report a case of Sneathia amnii as the causative agent of maternal chorioamnionitis and congenital pneumonia resulting in a late fetal death in Mozambique, with strong supportive postmortem molecular and histopathologic confirmation. This rare, fastidious gram-negative coccobacillus has been reported to infrequently cause abortions, stillbirths, and neonatal infections.


Assuntos
Corioamnionite/diagnóstico , Corioamnionite/microbiologia , Infecções por Fusobacteriaceae/diagnóstico , Infecções por Fusobacteriaceae/microbiologia , Leptotrichia , Complicações Infecciosas na Gravidez/diagnóstico , Complicações Infecciosas na Gravidez/microbiologia , Natimorto , Adulto , Autopsia , Corioamnionite/epidemiologia , Feminino , Infecções por Fusobacteriaceae/epidemiologia , Humanos , Imuno-Histoquímica , Pulmão/microbiologia , Pulmão/patologia , Moçambique/epidemiologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia
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