Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Dysbiosis in the oral microbiomes of anti-CCP positive individuals at risk of developing rheumatoid arthritis.

OBJECTIVES: An increased prevalence of periodontitis and perturbation of the oral microbiome has been identified in patients with rheumatoid arthritis (RA). The periodontal pathogen Porphyromonas gingivalis may cause local citrullination of proteins, potentially triggering anti-citrullinated protein antibody production. However, it is not known if oral dysbiosis precedes the onset of clinical arthritis. This study comprehensively characterised the oral microbiome in anti-cyclic citrullinated peptide (anti-CCP) positive at-risk individuals without clinical synovitis (CCP+at risk). METHODS: Subgingival plaque was collected from periodontally healthy and diseased sites in 48 CCP+at risk, 26 early RA and 32 asymptomatic healthy control (HC) individuals. DNA libraries were sequenced on the Illumina HiSeq 3000 platform. Taxonomic profile and functional capability of the subgingival microbiome were compared between groups. RESULTS: At periodontally healthy sites, CCP+at risk individuals had significantly lower microbial richness compared with HC and early RA groups (p=0.004 and 0.021). Microbial community alterations were found at phylum, genus and species levels. A large proportion of the community differed significantly in membership (523 species; 35.6%) and structure (575 species; 39.1%) comparing CCP+at risk and HC groups. Certain core species, including P. gingivalis, had higher relative abundance in the CCP+at risk group. Seventeen clusters of orthologous gene functional units were significantly over-represented in the CCP+at risk group compared with HC (adjusted p value <0.05). CONCLUSION: Anti-CCP positive at-risk individuals have dysbiotic subgingival microbiomes and increased abundance of P. gingivalis compared with controls. This supports the hypothesis that the oral microbiome and specifically P. gingivalis are important in RA initiation.

Influence of saliva on the oral microbiota.

Saliva plays a major role in determining the composition and activity of the oral microbiota, via a variety of mechanisms. Molecules, mainly from saliva, form a conditioning film on oral surfaces, thus providing receptors for bacterial attachment. The attached cells use saliva components, such as glycoproteins, as their main source of nutrients for growth. Oral bacteria work sequentially and in a concerted manner to catabolize these structurally complex molecules. Saliva also buffers the pH in the biofilm to around neutrality, creating an environment which is conducive to the growth of many oral bacteria that provide important benefits to the host. Components of the adaptive and innate host defences are delivered by saliva, and these often function synergistically, and at sublethal concentrations, so a complex relationship develops between the host and the resident microbiota. Dysbiosis can occur rapidly if the flow of saliva is perturbed.

Ecological approaches to oral biofilms: control without killing.

Humans have co-evolved with micro-organisms and have a symbiotic or mutualistic relationship with their resident microbiome. As at other body surfaces, the mouth has a diverse microbiota that grows on oral surfaces as structurally and functionally organised biofilms. The oral microbiota is natural and provides important benefits to the host, including immunological priming, down-regulation of excessive pro-inflammatory responses, regulation of gastrointestinal and cardiovascular systems, and colonisation by exogenous microbes. On occasions, this symbiotic relationship breaks down, and previously minor components of the microbiota outcompete beneficial bacteria, thereby increasing the risk of disease. Antimicrobial agents have been formulated into many oral care products to augment mechanical plaque control. A delicate balance is needed, however, to control the oral microbiota at levels compatible with health, without killing beneficial bacteria and losing the key benefits delivered by these resident microbes. These antimicrobial agents may achieve this by virtue of their recommended twice daily topical use, which results in pharmacokinetic profiles indicating that they are retained in the mouth for relatively long periods at sublethal levels. At these concentrations they are still able to inhibit bacterial traits implicated in disease (e.g. sugar transport/acid production; protease activity) and retard growth without eliminating beneficial species. In silico modelling studies have been performed which support the concept that either reducing the frequency of acid challenge and/or the terminal pH, or by merely slowing bacterial growth, results in maintaining a community of beneficial bacteria under conditions that might otherwise lead to disease (control without killing).

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

Molecular basis for avirulence of spontaneous variants of Porphyromonas gingivalis: Genomic analysis of strains W50, BE1 and BR1.

The periodontal pathogen Porphyromonas gingivalis is genetically heterogeneous. However, the spontaneous generation of phenotypically different sub-strains has also been reported. McKee et al. (1988) cultured P. gingivalis W50 in a chemostat during investigations into the growth and properties of this bacterium. Cell viability on blood agar plates revealed two types of non-pigmenting variants, W50 beige (BE1), and W50 brown (BR1), in samples grown in a high-hemin medium after day 7, and the population of these variants increased to approximately 25% of the total counts by day 21. W50, BE1 and BR1 had phenotypic alterations in pigmentation, reduced protease activity and haemagglutination and susceptibility to complement killing. Furthermore, the variants exhibited significant attenuation in a mouse model of virulence. Other investigators showed that in BE1, the predominant extracellular Arg-gingipain was RgpB, and no reaction with an A-lipopolysaccharide-specific MAb 1B5 (Collinson et al., 1998; Slaney et al., 2006). In order to determine the genetic basis for these phenotypic properties, we performed hybrid DNA sequence long reads using Oxford Nanopore and the short paired-end DNA sequence reads of Illumina HiSeq platforms to generate closed circular genomes of the parent and variants. Comparative analysis indicated loss of intact kgp in the 20 kb region of the hagA-kgp locus in the two variants BE1 and BR1. Deletions in hagA led to smaller open reading frames in the variants, and BR1 had incurred a major chromosomal DNA inversion. Additional minor changes to the genomes of both variants were also observed. Given the importance of Kgp and HagA to protease activity and haemagglutination, respectively, in this bacterium, genomic changes at this locus may account for most of the phenotypic alterations of the variants. The homologous and repetitive nature of hagA and kgp and the features at the inverted junctions are indicative of specific and stable homologous recombination events, which may underlie the genetic heterogeneity of this species.

Atomic-scale interactions between quorum sensing autoinducer molecules and the mucoid P. aeruginosa exopolysaccharide matrix.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung coloniser whose chronicity is associated with the formation of cation cross-linked exopolysaccharide (EPS) matrices, which form a biofilm that acts as a diffusion barrier, sequestering cationic and neutral antimicrobials, and making it extremely resistant to pharmacological challenge. Biofilm chronicity and virulence of the colony is regulated by quorum sensing autoinducers (QSAIs), small signalling metabolites that pass between bacteria, through the biofilm matrix, regulating genetic responses on a population-wide scale. The nature of how these molecules interact with the EPS is poorly understood, despite the fact that they must pass through EPS matrix to reach neighbouring bacteria. Interactions at the atomic-scale between two QSAI molecules, C4-HSL and PQS-both utilised by mucoid P. aeruginosa in the CF lung-and the EPS, have been studied for the first time using a combined molecular dynamics (MD) and density functional theory (DFT) approach. A large-scale, calcium cross-linked, multi-chain EPS molecular model was developed and MD used to sample modes of interaction between QSAI molecules and the EPS that occur at physiological equilibrium. The thermodynamic stability of the QSAI-EPS adducts were calculated using DFT. These simulations provide a thermodynamic rationale for the apparent free movement of C4-HSL, highlight key molecular functionality responsible for EPS binding and, based on its significantly reduced mobility, suggest PQS as a viable target for quorum quenching.

Residual Bacteriome after Chemomechanical Preparation of Root Canals in Primary and Secondary Infections.

INTRODUCTION: Secondary infections may be linked to the presence of residual microorganisms within dental root canals. The purpose of this study was to investigate the bacterial composition of primary and secondary root canal infections before and after chemomechanical treatment. METHODS: Samples were collected before chemomechanical preparation (S1) and before obturation (S2) from 19 subjects (10 primary and 9 secondary infections). DNA was extracted, and the V3/V4 region of the 16S ribosomal RNA gene was amplified using the 347 F/803R primers and paired-end sequenced using the MiSeq (Illumina, San Diego, CA) instrument. RESULTS: Sequencing analysis yielded partial 16S ribosomal RNA gene sequences that were taxonomically classified into 10 phyla and 143 genera. The most prevalent phyla in the S1 and S2 samples were Firmicutes and Bacteroides; however, when comparing between sample groups, Proteobacteria seem to have been enriched in secondary infections. The dominant genera in the primary S1 samples were Bacillus, Streptococcus, and Prevotella, whereas Bacillus, Streptococcus, and Selenomonas dominated the secondary infection S1 samples. Bacillus and Marinilactibacillus were the most dominant genera in the primary and secondary S2 samples. The mean number of operational taxonomic units per sample was 32,656 (±12,124 SD) and 37,113 (±16,994 SD) in the S1 and S2 samples, respectively. Alpha and beta diversities presented the same pattern within samples from both groups. CONCLUSIONS: Great interindividual variations in the bacterial composition of the root canal biofilms were observed. There was no difference in the bacterial composition before and after treatment, although some genera survived and seem to be part of a residual microbiome. Our findings revealed a high diversity of the bacterial communities present in root canal infections after chemomechanical treatment, although the majority of the taxa detected were in low abundance.

Optimization of conditions for in vitro modeling of subgingival normobiosis and dysbiosis.

Modeling subgingival microbiome in health and disease is key to identifying the drivers of dysbiosis and to studying microbiome modulation. Here, we optimize growth conditions of our previously described in vitro subgingival microbiome model. Subgingival plaque samples from healthy and periodontitis subjects were used as inocula to grow normobiotic and dysbiotic microbiomes in MBEC assay plates. Saliva supplemented with 1%, 2%, 3.5%, or 5% (v/v) heat-inactivated human serum was used as a growth medium under shaking or non-shaking conditions. The microbiomes were harvested at 4, 7, 10 or 13 days of growth (384 microbiomes in total) and analyzed by 16S rRNA gene sequencing. Biomass significantly increased as a function of serum concentration and incubation period. Independent of growth conditions, the health- and periodontitis-derived microbiomes clustered separately with their respective inocula. Species richness/diversity slightly increased with time but was adversely affected by higher serum concentrations especially in the periodontitis-derived microbiomes. Microbial dysbiosis increased with time and serum concentration. Porphyromonas and Alloprevotella were substantially enriched in higher serum concentrations at the expense of Streptococcus, Fusobacterium and Prevotella. An increase in Porphyromonas, Bacteroides and Mogibacterium accompanied by a decrease in Prevotella, Catonella, and Gemella were the most prominent changes over time. Shaking had only minor effects. Overall, the health-derived microbiomes grown for 4 days in 1% serum, and periodontitis-derived microbiomes grown for 7 days in 3.5%-5% serum were the most similar to the respective inocula. In conclusion, normobiotic and dysbiostic subgingival microbiomes can be grown reproducibly in saliva supplemented with serum, but time and serum concentration need to be adjusted differently for the health and periodontitis-derived microbiomes to maximize similarity to in vivo inocula. The optimized model could be used to identify drivers of dysbiosis, and to evaluate interventions such as microbiome modulators.

Temperature-dependent modulation of Porphyromonas gingivalis lipid A structure and interaction with the innate host defenses.

Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalis lipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalis grown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by ß-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

How is the development of dental biofilms influenced by the host?

BACKGROUND: The host provides environmental conditions that support diverse communities of microorganisms on all environmentally-exposed surfaces of the body. MATERIALS AND METHODS: To review the literature to determine which properties of the host substantially influence the development of dental biofilms. RESULTS: The mouth facilitates the growth of a characteristic resident microbiota. The composition of the oral microbiota is influenced by temperature, pH, and atmosphere, as well as by the host defences and host genetics. In addition, the host supplies endogenous nutrients and a variety of surfaces for biofilm formation. In health, the resident oral microbiota forms a symbiotic relationship with the host, regulated by active host-microbe cross talk. This resident microbiota is sensitive to perturbations in the host environment, especially to changes in nutrient supply and pH, so that previously minor components of the microbiota can become more competitive (and vice versa), resulting in reorganization of biofilm community structure. CONCLUSION: The host environment dictates the composition and gene expression of the resident microbiota. Changes in oral environmental conditions can disrupt the normal symbiotic relationship between the host and its resident microbes, and increase the risk of disease.

Cation complexation by mucoid Pseudomonas aeruginosa extracellular polysaccharide.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung colonizer, producing an extracellular matrix (ECM) composed predominantly of the extracellular polysaccharide (EPS) alginate. The ECM limits antimicrobial penetration and, consequently, CF sufferers are prone to chronic mucoid P. aeruginosa lung infections. Interactions between cations with elevated concentrations in the CF lung and the anionic EPS, enhance the structural rigidity of the biofilm and exacerbates virulence. In this work, two large mucoid P. aeruginosa EPS models, based on ß-D-mannuronate (M) and ß-D-mannuronate-α-L-guluronate systems (M-G), and encompassing thermodynamically stable acetylation configurations-a structural motif unique to mucoid P. aeruginosa-were created. Using highly accurate first principles calculations, stable coordination environments adopted by the cations have been identified and thermodynamic stability quantified. These models show the weak cross-linking capability of Na+ and Mg2+ ions relative to Ca2+ ions and indicate a preference for cation binding within M-G blocks due to the smaller torsional rearrangements needed to reveal stable binding sites. The geometry of the chelation site influences the stability of the resulting complexes more than electrostatic interactions, and the results show nuanced chemical insight into previous experimental observations.

Immunomodulatory streptococci that inhibit CXCL8 secretion and NFκB activation are common members of the oral microbiota.

Introduction. Oral tissues are generally homeostatic despite exposure to many potential inflammatory agents including the resident microbiota. This requires the balancing of inflammation by regulatory mechanisms and/or anti-inflammatory commensal bacteria. Thus, the levels of anti-inflammatory commensal bacteria in resident populations may be critical in maintaining this homeostatic balance.Hypothesis/Gap Statement. The incidence of immunosuppressive streptococci in the oral cavity is not well established. Determining the proportion of these organisms and the mechanisms involved may help to understand host-microbe homeostasis and inform development of probiotics or prebiotics in the maintenance of oral health.Aim. To determine the incidence and potential modes of action of immunosuppressive capacity in resident oral streptococci.Methodology. Supragingival plaque was collected from five healthy participants and supragingival and subgingival plaque from five with gingivitis. Twenty streptococci from each sample were co-cultured with epithelial cells±flagellin or LL-37. CXCL8 secretion was detected by ELISA, induction of cytotoxicity in human epithelial cells by lactate dehydrogenase release and NFκB-activation using a reporter cell line. Bacterial identification was achieved through partial 16S rRNA gene sequencing and next-generation sequencing.Results. CXCL8 secretion was inhibited by 94/300 isolates. Immunosuppressive isolates were detected in supragingival plaque from healthy (4/5) and gingivitis (4/5) samples, and in 2/5 subgingival (gingivitis) plaque samples. Most were Streptococcus mitis/oralis. Seventeen representative immunosuppressive isolates all inhibited NFκB activation. The immunosuppressive mechanism was strain specific, often mediated by ultra-violet light-labile factors, whilst bacterial viability was essential in certain species.Conclusion. Many streptococci isolated from plaque suppressed epithelial cell CXCL8 secretion, via inhibition of NFκB. This phenomenon may play an important role in oral host-microbe homeostasis.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries.

A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Enrichment of periodontal pathogens from the biofilms of healthy adults.

Periodontitis is associated with shifts in the balance of the subgingival microbiome. Many species that predominate in disease have not been isolated from healthy sites, raising questions as to the origin of these putative pathogens. The study aim was to determine whether periodontal pathogens could be enriched from pooled saliva, plaque and tongue samples from dentally-healthy adult volunteers using growth media that simulate nutritional aspects of the inflamed subgingival environment. The microbiome was characterised before and after enrichment using established metagenomic approaches, and the data analysed bioinformatically to identify major functional changes. After three weeks, there was a shift from an inoculum in which Streptococcus, Haemophilus, Neisseria, Veillonella and Prevotella species predominated to biofilms comprising an increased abundance of taxa implicated in periodontitis, including Porphyromonas gingivalis, Fretibacterium fastidiosum, Filifactor alocis, Tannerella forsythia, and several Peptostreptococcus and Treponema spp., with concomitant decreases in health-associated species. Sixty-four species were present after enrichment that were undetectable in the inoculum, including Jonquetella anthropi, Desulfovibrio desulfuricans and Dialister invisus. These studies support the Ecological Plaque Hypothesis, providing evidence that putative periodontopathogens are present in health at low levels, but changes to the subgingival nutritional environment increase their competitiveness and drive deleterious changes to biofilm composition.

Prevalence of Periodontal Disease and Periodontopathic Bacteria in Anti-Cyclic Citrullinated Protein Antibody-Positive At-Risk Adults Without Arthritis.

Importance: The prevalence of periodontitis is increased in patients with rheumatoid arthritis (RA) and periodontopathic bacteria can citrullinate proteins. Periodontitis may, therefore, be an initiator of RA and a target for prevention. Periodontal disease and periodontal bacteria have not been investigated in at-risk individuals with RA autoimmunity but no arthritis. Objective: To examine periodontal disease and periodontopathic bacteria in anti-cyclic citrullinated protein (anti-CCP) antibody-positive at-risk individuals without arthritis. Design, Setting, and Participants: This cross-sectional study took place at a teaching hospital from April 27, 2015, to May 8, 2017. Forty-eight anti-CCP-positive individuals without arthritis (CCP+ at-risk) were recruited nationally. Twenty-six patients with early RA (ERA) and 32 healthy control individuals were recruited locally. Data were analyzed between June 1, 2017, and December 1, 2017. Interventions: Periodontal assessment and examination of joints using ultrasonography. Main Outcomes and Measures: Prevalence of diseased periodontal sites, clinical periodontitis, and periodontal inflamed surface area in CCP+ at-risk individuals compared with patients with ERA and healthy individuals matched for age and smoking. Paired-end sequencing of DNA from subgingival plaque from diseased and healthy periodontal sites was performed and DNA was profiled and analyzed. Results: A total of 48 CCP+ at-risk individuals (mean [SD] age, 51.9 [11.4] years; 31 [65%] female), 26 patients with ERA (mean [SD] age, 54.4 [16.7] years; 14 [54%] female), and 32 healthy individuals (mean [SD] age, 49.4 [15.3] years; 19 [59%] female) were recruited. Of 48 CCP+ at-risk individuals, 46 had no joint inflammation on ultrasonography. Thirty-five CCP+ at-risk individuals (73%), 12 healthy individuals (38%), and 14 patients with ERA (54%) had clinical periodontitis. The median (interquartile range) percentage of periodontal sites with disease was greater in CCP+ at-risk individuals compared with healthy individuals (3.3% [0%-11.3%] vs 0% [0%-0.7%]) and similar to patients with ERA (1.1% [0%-13.1%]). Median (interquartile range) periodontal inflamed surface area was higher in CCP+ at-risk individuals compared with healthy individuals (221 mm2 [81-504 mm2] vs 40 mm2 [12-205 mm2]). Patients with CCP+ at-risk had increased relative abundance of Porphyromonas gingivalis (but not Aggregatibacter actinomycetemcomitans) at healthy periodontal sites compared with healthy individuals (effect size, 3.00; 95% CI, 1.71-4.29) and patients with ERA (effect size, 2.14; 95% CI, 0.77-3.52). Conclusions and Relevance: This study found increased prevalence of periodontitis and P gingivalis in CCP+ at-risk individuals. This suggests periodontitis and P gingivalis are associated with disease initiation and could be targets for preventive interventions in RA.

The commensal Streptococcus salivarius K12 downregulates the innate immune responses of human epithelial cells and promotes host-microbe homeostasis.

Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and strain K12 has reported probiotic effects. An emerging paradigm indicates that commensal bacteria downregulate immune responses through the action on NF-kappaB signaling pathways, but additional mechanisms underlying probiotic actions are not well understood. Our objective here was to identify host genes specifically targeted by K12 by comparing their responses with responses elicited by pathogens and to determine if S. salivarius modulates epithelial cell immune responses. RNA was extracted from human bronchial epithelial cells (16HBE14O- cells) cocultured with K12 or bacterial pathogens. cDNA was hybridized to a human 21K oligonucleotide-based array. Data were analyzed using ArrayPipe, InnateDB, PANTHER, and oPOSSUM. Interleukin 8 (IL-8) and growth-regulated oncogene alpha (Groalpha) secretion were determined by enzyme-linked immunosorbent assay. It was demonstrated that S. salivarius K12 specifically altered the expression of 565 host genes, particularly those involved in multiple innate defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. It inhibited baseline IL-8 secretion and IL-8 responses to LL-37, Pseudomonas aeruginosa, and flagellin in epithelial cells and attenuated Groalpha secretion in response to flagellin. Immunosuppression was coincident with the inhibition of activation of the NF-kappaB pathway. Thus, the commensal and probiotic behaviors of S. salivarius K12 are proposed to be due to the organism (i) eliciting no proinflammatory response, (ii) stimulating an anti-inflammatory response, and (iii) modulating genes associated with adhesion to the epithelial layer and homeostasis. S. salivarius K12 might thereby ensure that it is tolerated by the host and maintained on the epithelial surface while actively protecting the host from inflammation and apoptosis induced by pathogens.

Gene expression of bacterial collagenolytic proteases in root caries.

Objective: It is unknown whether bacteria play a role in the collagen matrix degradation that occurs during caries progression. Our aim was to characterize the expression level of genes involved in bacterial collagenolytic proteases in root biofilms with and without caries. Method: we collected samples from active cavitated root caries lesions (RC, n = 30) and from sound root surfaces (SRS, n = 10). Total microbial RNA was isolated and cDNA sequenced on the Illumina Hi-Seq2500. Reads were mapped to 162 oral bacterial reference genomes. Genes encoding putative bacterial collagenolytic proteases were identified. Normalization and differential expression analysis was performed on all metatranscriptomes (FDR<10-3). Result: Genes encoding collagenases were identified in 113 bacterial species the majority were peptidase U32. In RC, Streptococcus mutans and Veillonella parvula expressed the most collagenases. Organisms that overexpressed collagenolytic protease genes in RC (Log2FoldChange>8) but none in SRS were Pseudoramibacter alactolyticus [HMPREF0721_RS02020; HMPREF0721_RS04640], Scardovia inopinata [SCIP_RS02440] and Olsenella uli DSM7084 [OLSU_RS02990]. Conclusion: Our findings suggest that the U32 proteases may be related to carious dentine. The contribution of a small number of species to dentine degradation should be further investigated. These proteases may have potential in future biotechnological and medical applications, serving as targets for the development of therapeutic agents.

Insulin-Like Growth Factor Axis Expression in Dental Pulp Cells Derived From Carious Teeth.

The insulin-like growth factor (IGF) axis plays an important role in dental tissue regeneration and most components of this axis are expressed in human dental pulp cells (DPCs). In our previous study, we analyzed IGF axis gene expression in DPCs and demonstrated a novel role of IGF binding protein (IGFBP)-2 and -3 in coordinating mineralized matrix formation in differentiating DPCs. A more recent study from our laboratory partially characterized dental pulp stem cells from teeth with superficial caries (cDPCs) and showed that their potential to differentiate odontoblasts and/or into osteoblasts is enhanced by exposure to the mild inflammatory conditions characteristic of superficial caries. In the present study, we examine whether changes apparent in IGF axis expression during osteogenic differentiation of healthy DPCs are also apparent in DPCs derived from carious affected teeth.

Hyaluronan-Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes.

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.