Qian Zhang

Salivary exosomal PSMA7: a promising biomarker of inflammatory bowel disease

ABSTRACTInflammatory bowel disease (IBD) is an intestinal immune-dysfunctional disease worldwide whose prevalence increasing in Asia including China. It is a chronic disease of the gastrointestinal tract with unknown cause. Exosomes are small vesicles in various body fluids. They have diameters of 40–120xa0nm, and one of their functions is long-distance transfer of various substances. In this study, we investigated the contents of salivary exosomes in patients with IBD and in healthy controls to explore a new biomarker in patients with IBD. In this study, whole saliva was obtained from patients with IBD (ulcerative colitis (UC), nxa0=xa037; Crohn’s disease (CD), nxa0=xa011) and apparently healthy individuals (HC, nxa0=xa010). Salivary exosomes were extracted from samples, and the proteins within the exosomes were identified by liquid chromatograph-mass spectrometer (LC-MS/MS). The results showed that more than 2000 proteins were detected in salivary exosomes from patients with IBD. Through gene ontology analysis, we found that proteasome subunit alpha type 7 (PSMA7) showed especially marked differences between patients with IBD and the healthy controls, in that its expression level was much higher in the CD and UC groups. This exosomal protein is related to proteasome activity and inflammatory responses. So we conclude that in this research, salivary exosomal PSMA7 was present at high levels in salivary exosomes from subjects with IBD. It can be a very promising biomarker to release the patients from the pain of colonoscopy.

Identification of Lactobacillus from the Saliva of Adult Patients with Caries Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification.

Analysis of the Microbiota of Black Stain in the Primary Dentition

Black tooth stain is a characteristic extrinsic discoloration commonly seen on the cervical enamel following the contour of the gingiva. To investigate the relationship between black tooth stain and the oral microbiota, we used 16S rRNA gene sequencing to compare the microbial composition of dental plaque and saliva among caries-free children with and without black stain. Dental plaque and saliva, as well as black stain, were sampled from 10 children with and 15 children without black stain. Data were analyzed using the pipeline tool MOTHUR. Student’s t-test was used to compare alpha diversities and the Mann-Whitney U test to compare the relative abundances of the microbial taxa. A total of 10 phyla, 19 classes, 32 orders, 61 families and 102 genera were detected in these samples. Shannon and Simpson diversity were found to be significantly lower in saliva samples of children with black stain. Microbial diversity was reduced in the black stain compared to the plaque samples. Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Tannerella and Treponema were more abundant and Campylobacter less abundant in plaque samples of children with black stain. Principal component analysis demonstrated clustering among the dental plaque samples from the control group, while the plaque samples from the black stain group were not and appeared to cluster into two subgroups. Alterations in oral microbiota may be associated with the formation of black stain.

Exploring the Genomic Diversity and Cariogenic Differences of Streptococcus mutans Strains Through Pan-Genome and Comparative Genome Analysis

Pan-genome refers to the sum of genes that can be found in a given bacterial species, including the core-genome and the dispensable genome. In this study, the genomes from 183 Streptococcus mutans (S. mutans) isolates were analyzed from the pan-genome perspective. This analysis revealed that S. mutans has an “open” pan-genome, implying that there are plenty of new genes to be found as more genomes are sequenced. Additionally, S. mutans has a limited core-genome, which is composed of genes related to vital activities within the bacterium, such as metabolism and hereditary information storage or processing, occupying 35.6 and 26.6% of the core genes, respectively. We estimate the theoretical core-genome size to be about 1083 genes, which are fewer than other Streptococcus species. In addition, core genes suffer larger selection pressures in comparison to those that are less widely distributed. Not surprisingly, the distribution of putative virulence genes in S. mutans strains does not correlate with caries status, indicating that other factors are also responsible for cariogenesis. These results contribute to a more understanding of the evolutionary characteristics and dynamic changes within the genome components of the species. This also helps to form a new theoretical foundation for preventing dental caries. Furthermore, this study sets an example for analyzing large genomic datasets of pathogens from the pan-genome perspective.

DNA methylation profile is associated with the osteogenic potential of three distinct human odontogenic stem cells

Among the various sources of human autologous stem cells, stem cells isolated from dental tissues exhibit excellent properties in tissue engineering and regenerative medicine. However, the distinct potential of these odontogenic cell lines remains unclear. In this study, we analyzed DNA methylation patterns to determine whether specific differences existed among three different odontogenic cell types. Using the HumanMethylation450 Beadchip, the whole genomes of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) were compared. Then, the osteogenic potential of these cells was evaluated both in vitro and in vivo, and the methylation levels of certain genes related to bone formation differed among the three cell lines. P values less than 0.05 were considered to indicate statistical significance. The three cell types showed highly similar DNA methylation patterns, although specific differences were identified. Gene ontology analysis revealed that one of the most significantly different gene categories was related to bone formation. Thus, expression of cell surface epitopes and osteogenic-related transcription factors as well as the bone formation capacity were compared. The results showed that compared with DFPCs and DPSCs, PDLSCs had higher transcription levels of osteogenic-related factors, a higher in vitro osteogenic potential, and an increased new bone formation capacity in vivo. In conclusion, the results of this study suggested that the differential DNA methylation profiles could be related to the osteogenic potential of these human odontogenic cell populations. Additionally, the increased osteogenic potential of PDLSCs might aid researchers or clinicians in making better choices regarding tissue regeneration and clinical therapies.Stem cells: Differences in bone-forming potential of dental stem cellsStem cells isolated from the periodontal ligament are better at making new bone tissue than other kinds of dental stem cells. Feng Chen and colleagues from Peking University, China, analyzed the profile of DNA methylation that affect gene expression in stem cells isolated from human dental pulp (found in the center of the tooth) or periodontal ligaments (which attach tooth roots to bone), as well as from dental follicle progenitor cells that eventually form periodontal ligament cells. They saw significant differences in DNA methylation among genes related to bone production. They thus tested the bone-forming ability of each cell type, both in cell culture experiments and mice. The periodontal ligament stem cells proved the best, suggesting they could be useful for tissue engineering and regenerative medicine.

Population-Genomic Insights into Variation in Prevotella intermedia and Prevotella nigrescens Isolates and Its Association with Periodontal Disease

High-throughput sequencing has helped to reveal the close relationship between Prevotella and periodontal disease, but the roles of subspecies diversity and genomic variation within this genus in periodontal diseases still need to be investigated. We performed a comparative genome analysis of 48 Prevotella intermedia and Prevotella nigrescens isolates that from the same cohort of subjects to identify the main drivers of their pathogenicity and adaptation to different environments. The comparisons were done between two species and between disease and health based on pooled sequences. The results showed that both P. intermedia and P. nigrescens have highly dynamic genomes and can take up various exogenous factors through horizontal gene transfer. The major differences between disease-derived and health-derived samples of P. intermedia and P. nigrescens were factors related to genome modification and recombination, indicating that the Prevotella isolates from disease sites may be more capable of genomic reconstruction. We also identified genetic elements specific to each sample, and found that disease groups had more unique virulence factors related to capsule and lipopolysaccharide synthesis, secretion systems, proteinases, and toxins, suggesting that strains from disease sites may have more specific virulence, particularly for P. intermedia. The differentially represented pathways between samples from disease and health were related to energy metabolism, carbohydrate and lipid metabolism, and amino acid metabolism, consistent with data from the whole subgingival microbiome in periodontal disease and health. Disease-derived samples had gained or lost several metabolic genes compared to healthy-derived samples, which could be linked with the difference in virulence performance between diseased and healthy sample groups. Our findings suggest that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, which may reflect changes in microbial and environmental dynamics in subgingival microbial ecosystems. This provides insight into the potential of P. intermedia and P. nigrescens as new predictive biomarkers and targets for effective interventions in periodontal disease.

Investigating Oral Microbiome Profiles in Children with Cleft Lip and Palate for Prognosis of Alveolar Bone Grafting

In this study, we sought to investigate the oral microbiota structure of children with cleft lip and palate (CLP) and explore the pre-operative oral bacterial composition related to the prognosis of alveolar bone grafting. In total, 28 patients (19 boys, 9 girls) with CLP who were scheduled to undergo alveolar bone grafting for the first time were recruited. According to the clinical examination of operative sites at the third month after the operation, the individuals were divided into a non-inflammation group (n = 15) and an inflammation group (n = 13). In all, 56 unstimulated saliva samples were collected before and after the operation. The v3-v4 hypervariable regions of the 16S rRNA gene were sequenced using an Illumina MiSeq sequencing platform. Based on the beta diversity of the operational taxonomic units (OTUs) in the inflammation and non-inflammation samples, the microbial variation in the oral cavity differed significantly between the two groups before and after the operation (P < 0.05). Analysis of the relative abundances of pre-operative OTUs revealed 26 OTUs with a relative abundance higher than 0.01%, reflecting a significant difference of the relative abundance between groups (P < 0.05). According to a principal component analysis of the pre-operative samples, the inflammation-related OTUs included Tannerella sp., Porphyromonas sp., Gemella sp., Moraxella sp., Prevotella nigrescens, and Prevotella intermedia, most of which were enriched in the inflammation group and showed a significant positive correlation. A cross-validated random forest model based on the 26 different OTUs before the operation was able to fit the post-operative status of grafted sites and yielded a good classification result. The sensitivity and specificity of this classified model were 76.9% and 86.7%, respectively. These findings show that the oral microbiota profile before alveolar bone grafting may be related to the risk of post-operative inflammation at grafted sites.

A computational protocol to characterize elusive Candidate Phyla Radiation bacteria in oral environments using metagenomic data

Several studies have documented the diversity and potential pathogenic associations of organisms in the human oral cavity. Although much progress has been made in understanding the complex bacterial community inhabiting the human oral cavity, our understanding of some microorganisms is less resolved due to a variety of reasons. One such little-understood group is the candidate phyla radiation (CPR), which is a recently identified, but highly abundant group of ultrasmall bacteria with reduced genomes and unusual ribosomes. Here, we present a computational protocol for the detection of CPR organisms from metagenomic data. Our approach relies on a self-constructed dataset comprising published CPR genomic sequences as a filter to identify CPR sequences from metagenomic sequencing data. After assembly and functional prediction, the taxonomic affiliation of CPR contigs can be identified through phylogenetic analysis with publically available 16S rRNA gene and ribosomal proteins, in addition to sequence similarity analyses (e.g., average nucleotide identity calculations and contig mapping). Using this protocol, we reconstructed two draft genomes of organisms within the TM7 superphylum, that had genome sizes of 0.594 Mb and 0.678 Mb. Among the predicted functional genes of the constructed genomes, a high percentage were related to signal transduction, cell motility, and cell envelope biogenesis, which could contribute to cellular morphological changes in response to environmental cues. Importance Candidate phyla radiation (CPR) bacterial group is a recently identified, but highly diverse and abundant group of ultrasmall bacteria exhibiting reduced genomes and limited metabolic capacities. A number of studies have reported their potential pathogenic associations in multiple mucosal diseases including periodontitis, halitosis, and inflammatory bowel disease. However, CPR organisms are difficult to cultivate and are difficult to detect with PCR-based methods due to divergent genetic sequences. Thus, our understanding of CPR has lagged behind that of other bacterial component. Here, we used metagenomic approaches to overcome these previous barriers to CPR identification, and established a computational protocol for detection of CPR organisms from metagenomic samples. The protocol describe herein holds great promise for better understanding the potential biological functioning of CPR. Moreover, the pipeline could be applied to other organisms that are difficult to cultivate.

Nell-1-ΔE, a novel transcript of Nell-1, inhibits cell migration by interacting with enolase-1

NELL‐1 is a secreted protein that was originally found to be upregulated in pathologically fusing and fused sutures in non‐syndromic unilateral coronal synostosis patients. Apart from the ability of NELL‐1 to promote osteogenesis in long and craniofacial bones, NELL‐1 reportedly inhibits the formation of several benign and malignant tumors. We previously identified a novel transcript of Nell‐1 that lacked a calcium‐binding epidermal growth factor (EGF)‐like domain compared with full‐length Nell‐1; this new transcript was named Nell‐1‐ΔE. Three obvious structural differences between these two isoforms were revealed by homology modeling. Furthermore, the recombinant Nell‐1‐ΔE protein, but not the full‐length Nell‐1 protein, inhibited cell migration in vitro. However, full‐length Nell‐1 and Nell‐1‐ΔE proteins were present in similar subcellular locations and displayed similar expression patterns in both the intracellular and extracellular spaces. The results from the co‐immunoprecipitation and liquid chromatography/tandem mass spectrometry analyses using two cell lines demonstrated that Nell‐1‐ΔE but not full‐length Nell‐1 interacted with enolase‐1 in the extracellular spaces of both cell lines. The results of wound healing assays using ENO‐1‐overexpressing cells treated with full‐length Nell‐1/Nell‐1‐ΔE suggested that Nell‐1‐ΔE inhibited cell migration by interacting with ENO‐1. Our study indicated that the novel transcript Nell‐1‐ΔE, but not full‐length Nell‐1, might be a candidate tumor suppressor factor for basic research and clinical practice.

Dysbiosis and Ecotypes of the Salivary Microbiome Associated With Inflammatory Bowel Diseases and the Assistance in Diagnosis of Diseases Using Oral Bacterial Profiles

Inflammatory bowel diseases (IBDs) are chronic, idiopathic, relapsing disorders of unclear etiology affecting millions of people worldwide. Aberrant interactions between the human microbiota and immune system in genetically susceptible populations underlie IBD pathogenesis. Despite extensive studies examining the involvement of the gut microbiota in IBD using culture-independent techniques, information is lacking regarding other human microbiome components relevant to IBD. Since accumulated knowledge has underscored the role of the oral microbiota in various systemic diseases, we hypothesized that dissonant oral microbial structure, composition, and function, and different community ecotypes are associated with IBD; and we explored potentially available oral indicators for predicting diseases. We examined the 16S rRNA V3–V4 region of salivary bacterial DNA from 54 ulcerative colitis (UC), 13 Crohn’s disease (CD), and 25 healthy individuals using Illumina sequencing. Distinctive sample clusters were driven by disease or health based on principal coordinate analysis (PCoA) of both the Operational Taxonomic Unit profile and Kyoto Encyclopedia of Genes and Genomes pathways. Comparisons of taxa abundances revealed enrichment of Streptococcaceae (Streptococcus) and Enterobacteriaceae in UC and Veillonellaceae (Veillonella) in CD, accompanied by depletion of Lachnospiraceae and [Prevotella] in UC and Neisseriaceae (Neisseria) and Haemophilus in CD, most of which have been demonstrated to exhibit the same variation tendencies in the gut of IBD patients. IBD-related oral microorganisms were associated with white blood cells, reduced basic metabolic processes, and increased biosynthesis and transport of substances facilitating oxidative stress and virulence. Furthermore, UC and CD communities showed robust sub-ecotypes that were not demographic or severity-specific, suggesting their value for future applications in precision medicine. Additionally, indicator species analysis revealed several genera indicative of UC and CD, which were confirmed in a longitudinal cohort. Collectively, this study demonstrates evident salivary dysbiosis and different ecotypes in IBD communities and provides an option for identifying at-risk populations, not only enhancing our understanding of the IBD microbiome apart from the gut but also offering a clinically useful strategy to track IBD as saliva can be sampled conveniently and non-invasively.