Subgingival host-microbiome metatranscriptomic changes following scaling and root planing in grade II/III periodontitis.

AIM: To assess the effects of scaling and root planing (SRP) on the dynamics of gene expression by the host and the microbiome in subgingival plaque samples. MATERIALS AND METHODS: Fourteen periodontitis patients were closely monitored in the absence of periodontal treatment for 12 months. During this period, comprehensive periodontal examination and subgingival biofilm sample collection were performed bi-monthly. After 12 months, clinical attachment level (CAL) data were compiled and analysed using linear mixed models (LMM) fitted to longitudinal CAL measurements for each tooth site. LMM classified the sites as stable (S), progressing (P), or fluctuating (F). After the 12-month visit, subjects received SRP, and at 15 months they received comprehensive examination and supportive periodontal therapy. Those procedures were repeated at the 18-month visit, when patients were also sampled. Each patient contributed with one S, one P, and one F site collected at the 12- and 18-month visits. Samples were analysed using Dual RNA-Sequencing to capture host and bacterial transcriptomes simultaneously. RESULTS: Microbiome and host response behaviour were specific to the site's progression classification (i.e., S, P, or F). Microbial profiles of pre- and post-treatment samples exhibited specific microbiome changes, with progressing sites showing the most significant changes. Among them, Porphyromonas gingivalis was reduced after treatment, while Fusobacterium nucleatum showed an increase in proportion. Transcriptome analysis of the host response showed that interleukin (IL)-17, TNF signalling pathways, and neutrophil extracellular trap formation were the primary immune response activities impacted by periodontal treatment. CONCLUSIONS: SRP resulted in a significant "rewiring" of host and microbial activities in the progressing sites, while restructuring of the microbiome was minor in stable and fluctuating sites.

Long-term dynamics of the human oral microbiome during clinical disease progression.

BACKGROUND: Oral microbiome dysbiosis is linked to overt inflammation of tooth-supporting tissues, leading to periodontitis, an oral condition that can cause tooth and bone loss. Microbiome dysbiosis has been described as a disruption in the symbiotic microbiota composition's stability that could adversely affect the host's health status. However, the precise microbiome dynamics that lead to dysbiosis and the progression of the disease are largely unknown. The objective of our study was to investigate the long-term dynamics of periodontitis progression and its connection to dysbiosis. RESULTS: We studied three different teeth groups: sites that showed disease progression, sites that remained stable during the study, and sites that exhibited a cyclic deepening followed by spontaneous recovery. Time-series analysis revealed that communities followed a characteristic succession of bacteria clusters. Stable and fluctuating sites showed high asynchrony in the communities (i.e., different species responding dissimilarly through time) and a reordering of the communities where directional changes dominated (i.e., sample distance increases over time) in the stable sites but not in the fluctuating sites. Progressing sites exhibited low asynchrony and convergence (i.e., samples distance decreases over time). Moreover, new species were more likely to be recruited in stable samples if a close relative was not recruited previously. In contrast, progressing and fluctuating sites followed a neutral recruitment model, indicating that competition between closely related species is a significant component of species-species interactions in stable samples. Finally, periodontal treatment did not select similar communities but stabilized α-diversity, centered the abundance of different clusters to the mean, and increased community rearrangement. CONCLUSIONS: Here, we show that ecological principles can define dysbiosis and explain the evolution and outcomes of specific microbial communities of the oral microbiome in periodontitis progression. All sites showed an ecological succession in community composition. Stable sites were characterized by high asynchrony, a reordering of the communities where directional changes dominated, and new species were more likely to be recruited if a close relative was not recruited previously. Progressing sites were characterized by low asynchrony, community convergence, and a neutral model of recruitment. Finally, fluctuating sites were characterized by high asynchrony, community convergence, and a neutral recruitment model.

Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has four Na+/H+ antiporters that interconvert and balance Na+ and H+ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na+/H+ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na+ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na+ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na+/H+ antiporters was extremely sensitive to the Na+ concentration and showed essentially no Na+(Li+)/H+ antiporter activity, but it retained most K+/H+ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has four Na+/H+ antiporters that connect and interconvert its Na+ and H+ gradients. We have constructed four deletion mutants, each of which has only one of the four Na+/H+ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na+ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H+ and Na+ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Impact of Mediterranean Diet prior to Stroke on the Prognosis of Patients Undergoing Endovascular Treatment.

INTRODUCTION: Mediterranean diet (MeDiet) has been associated with lower risk of stroke. Additionally, animal models suggested that some components of MeDiet are associated with better outcomes after ischemic stroke (IS). We aimed to evaluate the association between global adherence to the MeDiet and the consumption of particular components of the MeDiet with stroke outcomes. MATERIAL AND METHODS: Multicenter observational study of consecutive IS patients treated with endovascular therapy. Inclusion criteria were large anterior circulation vessel occlusion and pre-stroke modified Rankin scale (mRS) <2. Adherence to MeDiet prior to stroke was evaluated using MEDAS 14-item scale. We evaluated the total score and also individual components of the scale. Clinical, radiological, and prognostic variables were collected. Good functional prognosis was considered as mRS ≤2 and complete recanalization as thrombolysis in cerebral infarction 3. RESULTS: From January 1 to October 30, 2018, 239 patients were included (mean age 71 years, 48% women, median baseline NIHSS 16). Median MEDAS scale was 8 points (7-10). Patients with a higher adherence to MeDiet had significantly lower total and LDL-cholesterol levels. Total adherence score was not associated with stroke outcomes. In multivariate analyses, consumption of olive oil as the principal source of fat was independently associated with good functional outcome at 3 months, OR 3.2 (1.1-10.1) and daily consumption of wine was independently associated with complete recanalization, OR 2.0 (1.1-3.8). CONCLUSIONS: Our study suggests that some components of MeDiet, such as olive oil and wine consumption, are related to better prognosis after stroke. More studies are needed to confirm these findings.

Metatranscriptomic analyses of the oral microbiome.

Although the composition of the oral human microbiome is now well studied, regulation of genes within oral microbial communities remains mostly uncharacterized. Current concepts of periodontal disease and caries highlight the importance of oral biofilms and their role as etiological agents of those diseases. Currently, there is increased interest in exploring and characterizing changes in the composition and gene-expression profiles of oral microbial communities. These efforts aim to identify changes in functional activities that could explain the transition from health to disease and the reason for the chronicity of those infections. It is now clear that the functions of distinct species within the subgingival microbiota are intimately intertwined with the rest of the microbial community. This point highlights the relevance of examining the expression profile of specific species within the subgingival microbiota in the case of periodontal disease or caries lesions, in the context of the other members of the biofilm in vivo. Metatranscriptomic analysis of the oral community is the starting point for identifying environmental signals that modulate the shift in metabolism of the community from commensal to dysbiotic. These studies give a snapshot of the expression patterns of microbial communities and also allow us to determine triggers to diseases. For example, in the case of caries, studies have unveiled a potential new pathway of sugar metabolism, namely the use of sorbitol as an additional source of carbon by Streptococcus mutans; and in the case of periodontal disease, high levels of extracellular potassium could be a signal of disease. Longitudinal studies are needed to identify the real markers of the initial stages of caries and periodontal disease. More information on the gene-expression profiles of the host, along with the patterns from the microbiome, will lead to a clearer understanding of the modulation of health and disease. This review presents a summary of these initial studies, which have opened the door to a new understanding of the dynamics of the oral community during the dysbiotic process in the oral cavity.

Potassium is a key signal in host-microbiome dysbiosis in periodontitis.

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human ß-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression.

The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease.

The periodontal pathogen Porphyromonas gingivalis induces expression of transposases and cell death of Streptococcus mitis in a biofilm model.

Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage.

A Porphyromonas gingivalis hypothetical protein controlled by the type I-C CRISPR-Cas system is a novel adhesin important in virulence.

The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.

Effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model.

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (Actinomyces naeslundii, Lactobacillus casei, Streptococcus mitis, Veillonella parvula, and Fusobacterium nucleatum) and the same biofilm plus the periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.

[Factors associated with survival at one year of life in neonates with severe congenital cardiopathy in A National Hospital in Peru]. / Factores asociados a la supervivencia al año de vida en neonatos con cardiopatía congénita severa en un Hospital Nacional de Perú.

OBJECTIVES.: To determine factors associated with survival in the first year of life in neonates with severe congenital heart disease treated in a national hospital in Peru. MATERIALS AND METHODS.: 160 children born between 2012 and 2015 with a diagnosis of severe congenital cardiopathy were studied and admitted to the Neonatology Service of the Edgardo Rebagliati Martins National Hospital of the Peruvian Social Security. The Kaplan-Meier method and the Log-Rank test were used in the survival analysis. Crude and adjusted analyses were performed using Cox regression models. RESULTS.: Fifty-two, point 5 percent (52.5%) of patients were male and the most frequent severe congenital cardiopathy was pulmonary atresia (26.3%). Thirty-three, point seven percent (33.7%) of patients died, with a 66.3% (IC95% 58.4-73.0) one-year survival. Prenatal diagnosis improved survival (HRa 0.54, 95% CI 0.30-0.98) while cyanotic cardiopathies (HRa 2.93, 95% CI 1.36-6.34) and the presence of another congenital anomaly (HRa 3.28, 95% CI 1.79-6.01) decreased it; these factors were also significant in a second model stratified by surgical treatment with the exception of the stratified model by complications where a prenatal diagnosis ceased to be significant. CONCLUSIONS.: Prenatal diagnosis increases survival from severe congenital heart disease. However, cyanotic heart diseases and other congenital anomalies, which decrease this chance, should be considered, if surgery is performed or complications occur.

OBJETIVOS.: Determinar los factores asociados a la supervivencia en el primer año de vida en neonatos con cardiopatía congénita severa atendidos en un hospital nacional de Perú. MATERIALES Y MÉTODOS.: Se estudiaron 160 niños nacidos entre el 2012 y 2015 con diagnóstico de alguna cardiopatía congénita severa que ingresaron al Servicio de Neonatología del Hospital Nacional Edgardo Rebagliati Martins del Seguro Social del Perú. En el análisis de supervivencia se utilizó el método de Kaplan-Meier y la prueba Log-Rank. Se realizaron análisis crudos y ajustados mediante modelos de regresión de Cox. RESULTADOS.: El 52,5% de los pacientes fueron de sexo masculino y la cardiopatía congénita severa más frecuente fue la atresia pulmonar (26,3%). El 33,7% de los pacientes fallecieron, siendo la supervivencia al año del 66,3% (IC95% 58,4-73,0). El diagnóstico prenatal mejoró la supervivencia (HRa 0,54, IC95%: 0,30-0,98) mientras que las cardiopatías de tipo cianóticas (HRa 2,93, IC95%: 1,36-6,34) y la presencia de otra anomalía congénita (HRa 3,28, IC95%: 1,79-6,01) la disminuyeron, estos factores fueron también significativos en un segundo modelo estratificado por tratamiento quirúrgico con excepción del modelo estratificado por complicaciones donde un diagnóstico prenatal dejó de ser significativo. CONCLUSIONES.: El diagnóstico prenatal incrementa la supervivencia ante una cardiopatía congénita severa y permitiría un tratamiento quirúrgico oportuno; sin embargo, se debe considerar que las cardiopatías de tipo cianóticas y la presencia de otras anomalías congénitas extracardíacas disminuyen la supervivencia si se realiza una intervención quirúrgica o se presentan complicaciones.

The contribution of Tannerella forsythia dipeptidyl aminopeptidase IV in the breakdown of collagen.

In this study, we characterized a serine protease from Tannerella forsythia that degrades gelatin, type I, and III collagen. Tannerella forsythia is associated with periodontitis progression and severity. The primary goal of this research was to understand the mechanisms by which T. forsythia contributes to periodontitis progression. One of our previous metatranscriptomic analysis revealed that during periodontitis progression T. forsythia highly expressed the bfor_1659 ORF. The N-terminal end is homologous to dipeptidyl aminopeptidase IV (DPP IV). DPP IV is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal end of proteins. Collagen type I is rich in X-Pro and X-Ala sequences, and it is the primary constituent of the periodontium. This work assessed the collagenolytic and gelatinolytic properties of BFOR_1659. To that end, the complete BFOR_1659 and its domains were purified as His-tagged recombinant proteins, and their collagenolytic activity was tested on collagen-like substrates, collagen type I and III combined, and on the extracellular matrix (ECM) formed on human gingival fibroblasts culture HGF-1. BFOR_1659 was only found in T. forsythia supernatants, highlighting its potential role on the pathogenicity of T. forsythia. We also found that BFOR_1659 efficiently degrades all tested substrates but the individual domains were inactive. Given that BFOR_1659 is highly expressed in the periodontal pocket, its clinical relevance is suggested to periodontitis progression.

The effect of the stress hormone cortisol on the metatranscriptome of the oral microbiome.

Imbalances of the microbiome, also referred to as microbial dysbiosis, could lead to a series of different diseases. One factor that has been shown to lead to dysbiosis of the microbiome is exposure to psychological stressors. Throughout evolution microorganisms of the human microbiome have developed systems for sensing host-associated signals such as hormones associated with those stressors, enabling them to recognize essential changes in their environment, thus changing their expression gene profile to fit the needs of the new environment. The most widely accepted theory explaining the ability of hormones to affect the outcome of an infection involves the suppression of the immune system. Commensal microbiota is involved in stressor-induced immunomodulation, but other biological effects are not yet known. Here we present the impact that cortisol had on the community-wide transcriptome of the oral community. We used a metatranscriptomic approach to obtain first insights into the metabolic changes induced by this stress hormone as well as which members of the oral microbiome respond to the presence of cortisol in the environment. Our findings show that the stress hormone cortisol directly induces shifts in the gene expression profiles of the oral microbiome that reproduce results found in the profiles of expression of periodontal disease and its progression.

Analysis of the reasons for exclusion from tPA therapy after early arrival in acute stroke patients.

OBJECTIVE: Only a small percentage of patients with acute stroke receive thrombolytic therapy, mainly due to late hospital arrival. Factors excluding those who arrive within 3h after stroke onset are less well known. PATIENTS AND METHODS: During the first year after implementing a protocol for stroke thrombolysis, we prospectively evaluated all patients with stroke admitted to our center within 3h from onset. Within-hospital time intervals were calculated and the reasons for exclusion from thrombolysis were analyzed. RESULTS: Ninety-six patients (representing 16% of all stroke patients admitted) arrived in less than 3h, and 25 of them (representing 7.5% of all patients with ischemic stroke) received thrombolytic therapy, with a door-to-needle interval of 51 min (range, 33-121). The reasons that accounted for 75% of therapy exclusions were non-modifiable (a too mild or improving deficit, and intracranial hemorrhage), except for a time window exceeded, which would probably require increasing public awareness about stroke. CONCLUSIONS: Most reasons for not applying thrombolysis to patients who arrive early enough are non-modifiable. Minimizing the door-to-needle time could compensate for late hospital arrival, which continues to be the main reason for not applying this therapy to stroke patients throughout the world.

Laterality does not influence early mortality in MCA ischemic stroke.

OBJECTIVES: There is clinical and experimental evidence involving the insula in the control of the autonomic system and cardiac function, with differential participation of right and left brain hemispheres, and these differences could influence mortality in the acute phase of brain hemisphere ischemic infarcts. METHODS: We have analyzed retrospectively the mortality during initial hospitalization in a series of 504 consecutive, unselected patients with middle cerebral artery (MCA) ischemic infarcts to detect potential differences between right (220 patients) and left (284 patients) lesions. RESULTS: Factors associated with a higher mortality were the infarct size, the occurrence of nosocomial respiratory infection, age and a history of diabetes mellitus or chronic obstructive pulmonary disease. CONCLUSIONS: The laterality of the infarct did not have a significant influence on stroke mortality during the admission period for the acute stage.

Beyond microbial community composition: functional activities of the oral microbiome in health and disease.

The oral microbiome plays a relevant role in the health status of the host and is a key element in a variety of oral and non-oral diseases. Despite advances in our knowledge of changes in microbial composition associated with different health conditions the functional aspects of the oral microbiome that lead to dysbiosis remain for the most part unknown. In this review, we discuss the progress made towards understanding the functional role of the oral microbiome in health and disease and how novel technologies are expanding our knowledge on this subject.

Functional signatures of oral dysbiosis during periodontitis progression revealed by microbial metatranscriptome analysis.

BACKGROUND: Periodontitis is a polymicrobial biofilm-induced inflammatory disease that affects 743 million people worldwide. The current model to explain periodontitis progression proposes that changes in the relative abundance of members of the oral microbiome lead to dysbiosis in the host-microbiome crosstalk and then to inflammation and bone loss. Using combined metagenome/metatranscriptome analysis of the subgingival microbiome in progressing and non-progressing sites, we have characterized the distinct molecular signatures of periodontitis progression. METHODS: Metatranscriptome analysis was conducted on samples from subgingival biofilms from progressing and stable sites from periodontitis patients. Community-wide expression profiles were obtained using Next Generation Sequencing (Illumina). Sequences were aligned using 'bowtie2' against a constructed oral microbiome database. Differential expression analysis was performed using the non-parametric algorithm implemented on the R package 'NOISeqBio'. We summarized global functional activities of the oral microbial community by set enrichment analysis based on the Gene Ontology (GO) orthology. RESULTS: Gene ontology enrichment analysis showed an over-representation in the baseline of active sites of terms related to cell motility, lipid A and peptidoglycan biosynthesis, and transport of iron, potassium, and amino acids. Periodontal pathogens (Tannerella forsythia and Porphyromonas gingivalis) upregulated different TonB-dependent receptors, peptidases, proteases, aerotolerance genes, iron transport genes, hemolysins, and CRISPR-associated genes. Surprisingly, organisms that have not been usually associated with the disease (Streptococcus oralis, Streptococcus mutans, Streptococcus intermedius, Streptococcus mitis, Veillonella parvula, and Pseudomonas fluorenscens) were highly active transcribing putative virulence factors. We detected patterns of activities associated with progression of clinical traits. Among those we found that the profiles of expression of cobalamin biosynthesis, proteolysis, and potassium transport were associated with the evolution towards disease. CONCLUSIONS: We identified metabolic changes in the microbial community associated with the initial stages of dysbiosis. Regardless of the overall composition of the community, certain metabolic signatures are consistent with disease progression. Our results suggest that the whole community, and not just a handful of oral pathogens, is responsible for an increase in virulence that leads to progression. TRIAL REGISTRATION: NCT01489839, 6 December 2011.

Community-wide transcriptome of the oral microbiome in subjects with and without periodontitis.

Despite increasing knowledge on phylogenetic composition of the human microbiome, our understanding of the in situ activities of the organisms in the community and their interactions with each other and with the environment remains limited. Characterizing gene expression profiles of the human microbiome is essential for linking the role of different members of the bacterial communities in health and disease. The oral microbiome is one of the most complex microbial communities in the human body and under certain circumstances, not completely understood, the healthy microbial community undergoes a transformation toward a pathogenic state that gives rise to periodontitis, a polymicrobial inflammatory disease. We report here the in situ genome-wide transcriptome of the subgingival microbiome in six periodontally healthy individuals and seven individuals with periodontitis. The overall picture of metabolic activities showed that iron acquisition, lipopolysaccharide synthesis and flagellar synthesis were major activities defining disease. Unexpectedly, the vast majority of virulence factors upregulated in subjects with periodontitis came from organisms that are not considered major periodontal pathogens. One of the organisms whose gene expression profile was characterized was the uncultured candidate division TM7, showing an upregulation of putative virulence factors in the diseased community. These data enhance understanding of the core activities that are characteristic of periodontal disease as well as the role that individual organisms in the subgingival community play in periodontitis.