RESUMEN
The human intestinal microbiota is a homeostatic ecosystem with a remarkable impact on human health and the disruption of this equilibrium leads to an increased susceptibility to infection by numerous pathogens. In this study, we used shotgun metagenomic sequencing and two different bioinformatic approaches, based on mapping of the reads onto databases and on the reconstruction of putative draft genomes, to investigate possible changes in the composition of the intestinal microbiota in samples from patients with Shiga Toxin-producing E. coli (STEC) infection compared to healthy and healed controls, collected during an outbreak caused by a STEC O26:H11 infection. Both the bioinformatic procedures used, produced similar result with a good resolution of the taxonomic profiles of the specimens. The stool samples collected from the STEC infected patients showed a lower abundance of the members of Bifidobacteriales and Clostridiales orders in comparison to controls where those microorganisms predominated. These differences seemed to correlate with the STEC infection although a flexion in the relative abundance of the Bifidobacterium genus, part of the Bifidobacteriales order, was observed also in samples from Crohn's disease patients, displaying a STEC-unrelated dysbiosis. The metagenomics also allowed to identify in the STEC positive samples, all the virulence traits present in the genomes of the STEC O26 that caused the outbreak as assessed through isolation of the epidemic strain and whole genome sequencing. The results shown represent a first evidence of the changes occurring in the intestinal microbiota of children in the course of STEC infection and indicate that metagenomics may be a promising tool for the culture-independent clinical diagnosis of the infection.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Heces/microbiología , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Metagenoma , Metagenómica , Escherichia coli Shiga-Toxigénica , Biodiversidad , Preescolar , Biología Computacional/métodos , Humanos , Lactante , Recién Nacido , Metagenómica/métodos , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , Virulencia/genéticaRESUMEN
BACKGROUND: An enriched environment for residents with dementia may have a positive effect on the rest-activity rhythm. A small scaled homelike special care unit might be such an enriched environment. The present study shows whether the rest-activity rhythm of residents with moderate to severe dementia responds positively to a transfer from a regular Special Care Unit (SCU) to a small scaled homelike SCU. METHODS: Initially, a group of 145 residents living in a regular SCU participated. Out of this group, 77 residents moved to a small scaled homelike SCU. This group was compared to the group of 68 residents that remained at the regular SCU. Rest-activity rhythm was assessed by means of actigraphy and observation scales before and after relocation. RESULTS: No significant main effects nor significant interaction effects in intradaily and interdaily activity were found for the data of 38 residents in the small scaled homelike SCU and 20 residents of the regular SCU. The effect sizes, however, ranged from small to large. CONCLUSIONS: Considering the effect sizes, a new study with a larger number of participants is necessary before firm conclusions can be drawn. TRIAL REGISTRATION: Current Controlled Trials ISRCTN11151241 . registration date: 21-06-2017. Retrospectively registered.
Asunto(s)
Actigrafía/tendencias , Demencia/fisiopatología , Hogares para Ancianos/tendencias , Actividad Motora/fisiología , Casas de Salud/tendencias , Descanso/fisiología , Actigrafía/métodos , Anciano , Anciano de 80 o más Años , Demencia/epidemiología , Demencia/terapia , Planificación Ambiental/tendencias , Femenino , Humanos , Masculino , Países Bajos/epidemiologíaRESUMEN
AIM: The aim of the study was to evaluate the effect of full-mouth tooth extraction on the oral microflora, with emphasis on the presence and load of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. MATERIAL AND METHODS: Adult patients (n = 30), with moderate to advanced periodontitis and scheduled for full-mouth tooth extraction, were consecutively selected. Prior to and 1 and 3 months after full-mouth tooth extraction saliva, tongue, buccal and gingival mucosa and subgingival plaque/prosthesis samples were obtained. Aerobic and anaerobic culture techniques and quantitative real-time polymerase chain reaction (qPCR) were employed for the detection of oral pathogens. RESULTS: Full-mouth tooth extraction resulted in reduction below detection level of A. actinomycetemcomitans and P. gingivalis in 15 of 16 and 8 of 16 previously positive patients using culture techniques and qPCR, respectively. Those patients remaining qPCR positive showed a significant reduction in load of these bacteria. CONCLUSION: Full-mouth tooth extraction significantly changes the oral microflora. These changes include reduction of A. actinomycetemcomitans and P. gingivalis, frequently to levels below detection threshold. In some patients, A. actinomycetemcomitans and P. gingivalis can persist in the edentulous oral cavity up to 3 months after full-mouth tooth extraction.
Asunto(s)
Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Carga Bacteriana , Boca Edéntula/microbiología , Boca/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Extracción Dental/métodos , Adulto , Técnicas Bacteriológicas , Estudios de Cohortes , Placa Dental/microbiología , Prótesis Dental/microbiología , Femenino , Estudios de Seguimiento , Encía/microbiología , Humanos , Masculino , Persona de Mediana Edad , Mucosa Bucal/microbiología , Pérdida de la Inserción Periodontal/cirugía , Bolsa Periodontal/cirugía , Periodontitis/cirugía , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Saliva/microbiología , Lengua/microbiologíaRESUMEN
Clostridia dominate the rodent intestinal bacterial community and play an important role in physiological functions of the host. However, their ecology and diversity are still unclear. In our previous report, we showed that phylogenetically novel groups of clostridia inhabit the mouse intestine and contribute to the normalization of germfree mice. In this study, five new oligonucleotide probes were designed and applied to detect these clostridial groups that are essential for the normalization of germfree mice. Faecal microbiota of conventional mouse strains and specific pathogen-free mice from different breeding colonies were analysed by fluorescence in situ hybridization using these five probes. Our results showed that the composition of clostridia differed among mouse strains and also among mouse groups of the same inbred strain from different breeding colonies. These five new probes for mouse clostridia were able to detect the difference in clostridial diversity in each mouse group. In addition to Clostridium, we also analysed Bacteroides and Lactobacillus using previously described probes and the number or the frequency of occurrence of Bacteroides was shown to be different among mouse groups analysed. The oligonucleotide probe set including our newly developed and previously described probes used in this study can be applied to monitoring of significant groups of mouse intestinal microbiota.
Asunto(s)
Técnicas Bacteriológicas/métodos , Clostridium/genética , Intestinos/microbiología , Sondas de Oligonucleótidos/genética , Animales , Bacteroides/genética , Clostridium/aislamiento & purificación , Heces/microbiología , Hibridación Fluorescente in Situ , Lactobacillus/genética , Ratones , Especificidad de la Especie , Organismos Libres de Patógenos EspecíficosRESUMEN
Verbal descriptors of dyspnea have been suggested as being useful in providing information on the underlying pathophysiology. However, little is known about the reliability of these descriptors. The present study examined the reliability of a German language list of respiratory symptom descriptors and studied the association of these descriptors with the intensity and unpleasantness of perceived dyspnea. Fourteen healthy volunteers performed cycle-ergometer exercise and voluntary breath-holding during which they rated the perceived intensity (VAS-I) and unpleasantness (VAS-U) of dyspnea on visual analog scales. Following this, they judged their sensations of dyspnea using the list of symptom descriptors. Both conditions were repeated in reverse order on a subsequent occasion 10 days apart. Ventilatory measures, heart rate, blood lactate, VAS-I and VAS-U during cycle-exercise as well as breath-holding time, VAS-I and VAS-U during breath-holding showed no differences between both occasions. Separate hierarchical cluster analyses identified four clusters of verbal descriptors of dyspnea which were widely comparable between both occasions: effort, speed, obstruction and suffocation. Separate multidimensional scaling analyses (MDS) confirmed these four clusters for each occasion. On both days, perceived unpleasantness of dyspnea was correlated with all four clusters during cycle-exercise, while perceived intensity showed only correlations with effort or speed, respectively. No such correlations were obtained for breath-holding. The results suggest that separable clusters of German language descriptors of dyspnea are reliably used by healthy volunteers. The obtained clusters are widely comparable to previously described clusters in other languages and are differently related to the intensity and unpleasantness of perceived dyspnea.
Asunto(s)
Disnea/diagnóstico , Terminología como Asunto , Adulto , Femenino , Humanos , MasculinoRESUMEN
For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.
Asunto(s)
Bacterias/genética , ADN Bacteriano/análisis , Heces/microbiología , ARN Ribosómico 16S/genética , Bacterias/clasificación , Humanos , Sondas de Oligonucleótidos/metabolismo , ARN Ribosómico 16S/análisisRESUMEN
Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.
Asunto(s)
Antígenos de Superficie/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/análisis , Unión Competitiva/fisiología , Química Encefálica/fisiología , Proteínas Portadoras/análisis , Células Cultivadas , ADN Complementario , Hipocampo/citología , Humanos , Riñón/citología , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Plasticidad Neuronal/fisiología , Neuronas/química , Neuronas/citología , Proteínas R-SNARE , ARN Mensajero/análisis , Ratas , Proteínas SNARE , Transmisión Sináptica/fisiología , Proteína 25 Asociada a Sinaptosomas , Sinaptosomas/química , Sinaptosomas/metabolismo , Sintaxina 1 , Linfocitos T/citología , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Previous studies from this laboratory have demonstrated that extracellular calcium entry through the NMDA subtype of glutamate receptors in hippocampal neurons selectively down-regulated ligatin gene expression in a rapid and long-lasting manner. Here we investigated the molecular mechanism that underlies this phenomenon. We demonstrate that glutamate receptor activation transiently increased the transcriptional activity of the ligatin gene and simultaneously shortened the half-life of its message. Using nuclear run-on assays and northern analyses of total RNA from alpha-amanitin-treated cells, we measured the effects of glutamate on the transcriptional activity and mRNA stability of the ligatin gene. The transcriptional activity of ligatin was found to be transiently increased (1.4-fold) 20 min after the addition of glutamate, with a return to basal levels by 60 min. Thus, the glutamate-dependent decrease in ligatin message could not be explained by a decline in its synthesis. Instead, concurrent with transcriptional up-regulation, glutamate shortened the half-life of the ligatin message from 10 h to 58 min, leading to a net decrease (0.7-fold) in its steady-state levels by 60 min. This posttranscriptional destablization of ligatin mRNA was mimicked by the translation inhibitor, cycloheximide, but not by puromycin. This finding indicated that the stability of ligatin mRNA was translation independent and distinguished this posttranscriptional regulatory mechanism from those previously described. Moreover, using in situ hybridization and confocal microscopy, we showed that control of message stability occurred both in the cell body and in the dendritic regions distant from the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Regulación de la Expresión Génica , Hipocampo/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Glutamato/fisiología , Transcripción Genética , Actinas/genética , Amanitinas/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Hibridación in Situ , Cinética , Proteínas de la Membrana/genética , Ratas , Transcripción Genética/efectos de los fármacosRESUMEN
Previous work from this laboratory has documented that glutamate receptor activation and extracellular calcium entry into hippocampal neurons caused a long-lasting down-regulation of ligatin mRNA and protein. Here, we investigated whether glutamate reduced ligatin mRNA levels by decreasing the transcriptional activity of the gene and/or by regulating post-transcriptional RNA processing steps including mRNA stability. Using nuclear run-on assays, it was demonstrated that transcriptional activity of the ligatin gene was not significantly decreased after glutamate receptor activation. Further, Northern analysis of RNA from neurons maintained in the presence of the transcription inhibitor, alpha-amanitin, showed that glutamate shortened the half life of the ligatin message from 10 h to 58 min. This post-transcriptional destabilization of ligatin mRNA was mimicked by NMDA, dependent on Ca2+, blocked by MK801, and not affected by AMPA and kainic acid, indicating that message stability was governed by changes in intracellular calcium. Moreover, using in situ hybridization and confocal microscopy, we showed that glutamate and NMDA decreased ligatin message within dendritic and somal regions without increasing nuclear levels. These findings demonstrated that glutamate receptor activation altered neuronal gene expression posttranscriptionally by destabilizing mRNA. Our data suggest that post-transcriptional regulation of gene expression may be part of the normal receptor mediated regulatory program of plasticity and provides the first description of a glutamate receptor-modulated, calcium-dependent mechanism which rapidly destabilizes mRNA in neurons.
Asunto(s)
Hipocampo/citología , Neuronas/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Receptores de Glutamato/genética , Animales , Calcio/fisiología , Células Cultivadas/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores AMPA/fisiología , Receptores de Ácido Kaínico/fisiología , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
To test the hypothesis that repeated subconvulsive stimulations required to induce kindling can permanently alter gene expression of hippocampal neurons, we used Northern and in situ hybridization analyses to measure steady-state mRNA levels encoding several phenotypic proteins. mRNA encoding a membrane-bound protein, ligatin, was significantly reduced in kindled brains relative to naive and sham control animals. This change in gene expression persisted for over 4 months after kindling, was associated with a decrease in ligatin protein expression, was not produced by single or multiple seizures that did not induce the kindling phenomena, and was blocked by MK801. These results provide direct evidence that kindling can cause persistent changes in the expression of specific genes in hippocampal neurons and suggest that N-methyl-D-aspartate receptor-activated changes in gene expression may be a basic molecular mechanism underlying some of the long-lasting plasticity changes seen in kindling or models of long-term memory.
Asunto(s)
Hipocampo/fisiología , Excitación Neurológica/fisiología , Animales , Expresión Génica , Hibridación in Situ , Masculino , Proteínas de la Membrana/genética , Peso Molecular , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal , Proteínas Quinasas/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Factores de TiempoRESUMEN
Activation of excitatory amino acid (EAA) receptors in cultured hippocampal neurons causes down-regulation of the protein ligatin, a receptor for phosphoglycoproteins and a marker protein for membrane-vesicle transport systems. This reduction occurs at both physiologic and excitotoxic levels of glutamate stimulation and is accompanied by a significant decrease in steady state levels of ligatin mRNA. Reduction in ligatin mRNA occurs within 60 min and persists 24 h later. Steady state levels of mRNAs encoding cyclophilin, an ubiquitous cytosolic protein, and neuron specific-enolase (N-SE) are not diminished by glutamate receptor activation, demonstrating that down-regulation of ligatin mRNA was not a result of general catabolism. Further, this reduction in ligatin mRNA occurred without induction of HSP 70. Pharmacological studies using selective antagonists and agonists indicate that this down-regulation of ligatin gene expression is predominantly mediated by the N-methyl-D-aspartate (NMDA) subclass of EAA receptors and that Ca2+ is required. This is the first report that EAA receptor activation in hippocampal neurons can pretranslationally down-regulate gene expression in a rapid and long-lasting manner under physiologic, as well as cytotoxic conditions. The data support the hypothesis that modulation of neuronal gene expression may represent a molecular mechanism mediating some of the long-lasting functional and pathophysiological effects of EAA on cell function.