RESUMO
OBJECTIVES: The relevance of spatial composition in the microbial changes associated with UC is unclear. We coupled luminal brush samples, mucosal biopsies and laser capture microdissection with deep sequencing of the gut microbiota to develop an integrated spatial assessment of the microbial community in controls and UC. DESIGN: A total of 98 samples were sequenced to a mean depth of 31,642 reads from nine individuals, four control volunteers undergoing routine colonoscopy and five patients undergoing surgical colectomy for medically-refractory UC. Samples were retrieved at four colorectal locations, incorporating the luminal microbiota, mucus gel layer and whole mucosal biopsies. RESULTS: Interpersonal variability accounted for approximately half of the total variance. Surprisingly, within individuals, asymmetric Eigenvector map analysis demonstrated differentiation between the luminal and mucus gel microbiota, in both controls and UC, with no differentiation between colorectal regions. At a taxonomic level, differentiation was evident between both cohorts, as well as between the luminal and mucosal compartments, with a small group of taxa uniquely discriminating the luminal and mucosal microbiota in colitis. There was no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity. CONCLUSIONS: Our study demonstrates a conserved spatial structure to the colonic microbiota, differentiating the luminal and mucosal communities, within the context of marked interpersonal variability. While elements of this structure overlap between UC and control volunteers, there are differences between the two groups, both in terms of the overall taxonomic composition and how spatial structure is ascribable to distinct taxa.
Assuntos
Bactérias/isolamento & purificação , Colite Ulcerativa/microbiologia , Colo/microbiologia , Microbiota/fisiologia , Adulto , Bactérias/genética , Biópsia , Colite Ulcerativa/patologia , Colo/patologia , Colonoscopia , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , RNA Bacteriano/análise , Voluntários , Adulto JovemRESUMO
AIM: The colonic mucus gel layer is composed of mucins that may be sulphated or sialyated. Sulphated mucins predominate in health while in ulcerative colitis (UC) sulphation is reduced. These differences result directly from inflammatory events. It may also be hypothesized that they arise in part from alterations in the colonic microbiota, particularly changes in the burden of sulphated mucin-metabolizing species, such as Desulfovibrio (DSV) bacteria. The aim of this study was to correlate colonic mucin chemotypes and inflammatory scores in health and UC and relate these changes to changes in the colonization of colonic crypts by DSV. METHOD: Paired colonic biopsies from 34 healthy controls (HC) and 19 patients with active UC were collected for the purpose of parallel histological and microbiological assessment. High-iron diamine and Alcian blue staining and haematoxylin and eosin of mucosal biopsy specimens were used to assess histological changes within the clinical spectrum of UC. Quantitative real-time polymerase chain reaction analysis was employed to determine the total and DSV copy number within the colonic crypts. RESULTS: Compared with HC, the mucin chemotype in UC was less sulphated and inversely correlated with the degree of mucosal inflammation. A weak but significant negative correlation was found between the abundance of sulphated mucins and DSV burden. CONCLUSION: Mucin composition strongly correlates with the degree of mucosal inflammation, and to a lesser extent with DSV burden. These data suggest that mucin chemotype and DSV burden are linked phenomena and highlight the need to consider changes in mucin chemotype in the setting of microbial dysbiosis occurring within the colitic colon. What does this paper add to the literature? Decreased sulphation of mucins has been associated with inflammation in ulcerative colitis. Currently there are few data describing the relationship between microbial species and changes in mucin chemotype. This study validates previous findings and presents evidence of changes in mucin chemotype occurring in tandem with coherent changes in the microbiota within crypt niches.
Assuntos
Colite Ulcerativa/metabolismo , Colo/química , DNA Bacteriano/análise , Desulfovibrio/isolamento & purificação , Mucosa Intestinal/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Colo/microbiologia , Colo/patologia , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Mucinas/análise , Sialomucinas/análise , Adulto JovemRESUMO
This study assesses the impact of permitting unrestricted access to requests for soluble transferrin receptor (sTfR) analysis in screening for iron deficiency (ID). Biochemical data including sTfR, serum ferritin (sFn), transferrin saturation, zinc protoporphyrins (ZPP) and also erythrocyte indices are used to highlight the differences between hospital (H) and general practitioner (GP) patient groups. A significantly higher number of abnormal sFn values (40%) over abnormal sTfR values (25%) occurred in GP patients. This trend was reversed in the H patient group where high sTfR values predominated. Consequently, screening with sFn, exclusively, missed ID (sTfR > 28.1 nmol/l) in 5% of GP patients and in 20% of H patients. Some 40% of H patients had elevated CRP values (CRP > 10 mg/l) indicating inflammatory disease, however, ZPP was more efficient than CRP at screening the validity of normal sFn values in the group. Unrestricted access to sTfR, sFn and ZPP analyses should expedite diagnosis in all patients, particularly H patients, but may be costly. The high specificity (>90%) of the mean cell haemoglobin for ID may be under-utilized diagnostically.
Assuntos
Anemia Ferropriva/diagnóstico , Receptores da Transferrina/sangue , Anemia Ferropriva/sangue , Biomarcadores/sangue , Diagnóstico Diferencial , Serviços de Diagnóstico/normas , Serviços de Diagnóstico/estatística & dados numéricos , Índices de Eritrócitos , Feminino , Ferritinas/sangue , Humanos , Irlanda , Masculino , Valor Preditivo dos Testes , Estudos Prospectivos , Protoporfirinas/sangue , Sensibilidade e EspecificidadeRESUMO
A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Carcinoma Hepatocelular/enzimologia , Enoil-CoA Hidratase/genética , Isomerases/genética , Neoplasias Hepáticas/enzimologia , Microcorpos/enzimologia , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Hepatocelular/genética , Linhagem Celular , Clonagem Molecular , Humanos , Fígado/enzimologia , Neoplasias Hepáticas/genética , Dados de Sequência Molecular , Enzima Bifuncional do Peroxissomo , Biossíntese de Proteínas , Ratos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Fibroblasts from patients with the inherited disorder Zellweger syndrome have few or no peroxisomes; multiple biochemical processes that normally occur in this organelle are defective. Rhizomelic chondrodysplasia punctata (RCDP) is another inherited disorder in which two unrelated peroxisomal metabolic processes, plasmalogen synthesis and phytanic acid oxidation, are impaired despite the normal appearance of peroxisomal structure. It was previously reported that one of the enzymes of peroxisomal fatty acid beta-oxidation, 3-ketoacyl-CoA thiolase (beta-keto-thiolase), was present in precursor rather than mature form in both of these diseases. Immunofluorescent staining for peroxisomal beta-ketothiolase showed the immunoreactivity to be localized in subcellular particles in fibroblasts from both Zellweger syndrome and RCDP patients, even though the former lack normal peroxisomes. Immunoblot studies were performed to determine the subcellular location of the thiolase precursor in fractionated fibroblasts from Zellweger and RCDP patients. In both disorders, thiolase immunoreactivity was detected in subcellular fractions having a lower density than normal peroxisomes and mitochondria, and was resistant to digestion by proteinase K. The density of the thiolase precursor-containing fractions was similar to that of peroxisomal membrane "ghost" fractions recently described by Santos et al. (J Biol Chem 263:10502-10509, 1988). Our results suggest that these are not empty membrane vesicles but contain at least one peroxisomal matrix protein. Furthermore, they exist not only in cells in which normal peroxisomes fail to form (Zellweger syndrome), but also in some cells which have catalase-containing peroxisomes (RCDP).