Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus.

BACKGROUND: Emerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them. METHODS: We collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB. RESULTS: OLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased. CONCLUSIONS: A lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed.

Explore variation of salivary bacteria across time and geolocations.

Saliva is an informative body fluid that can be found at various crime scenes, and the salivary bacterial community has been revealed it is a potential auxiliary target for forensic identification. However, the variation of salivary bacterial community composition across time and geolocation needs to be explored. The study was designed to be carried out during the winter vacation that was across about 50 days and eight geographic locations. The high throughput sequencing was performed with the V3-V4 region of the16S rRNA gene to explore salivary bacterial community composition. An overall slight fluctuation of the salivary bacteria was observed, which primarily occurred in the relative abundance of the salivary bacterial taxa. The results of principal coordinate analysis and hierarchical clustering showed samples were clustered by the individuals. All individuals could be correctly identified with the random forest model. In summation, although the relative abundance of salivary bacteria varied across the changes of time and geolocation, the individualized characteristic of salivary bacteria remained steady, which is beneficial for the salivary bacterial application in personal identification.

Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

Microbiome of periodontitis and peri-implantitis before and after therapy: Long-read 16S rRNA gene amplicon sequencing.

AIMS: The microbial profiles of peri-implantitis and periodontitis (PT) are inconclusive. The controversies mainly arise from the differences in sampling sites, targeted gene fragment, and microbiome analysis techniques. The objective of this study was to explore the microbiomes of peri-implantitis (PI), control implants (CI), PT and control teeth (CT), and the microbial change of PI after nonsurgical treatment (PIAT). METHODS: Twenty-two patients diagnosed with both PT and peri-implantitis were recruited. Clinical periodontal parameters and radiographic bone levels were recorded. In each patient, the subgingival and submucosal plaque samples were collected from sites with PI, CI, PT, CT, and PIAT. Microbiome diversity was analyzed by high-throughput amplicon sequencing using full-length of 16S rRNA gene by next generation sequencing. RESULTS: The 16S rRNA gene sequencing analysis revealed 512 OTUs in oral microbiome and 377 OTUs reached strain levels. The PI and PT groups possessed their own unique core microbiome. Treponema denticola was predominant in PI with probing depth of 8-10 mm. Interestingly, Thermovirga lienii DSM 17291 and Dialister invisus DSM 15470 were found to associate with PI. Nonsurgical treatment for peri-implantitis did not significantly alter the microbiome, except Rothia aeria. CONCLUSION: Our study suggests Treponemas species may play a pivotal role in peri-implantitis. Nonsurgical treatment did not exert a major influence on the peri-implantitis microbiome in short-term follow-up. PT and peri-implantitis possess the unique microbiome profiles, and different therapeutic strategies may be suggested in the future.

Differential expression of circRNAs during osteogenic/odontogenic differentiation of stem cells from apical papilla promoted by blue light-emitting diode.

BACKGROUND: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED. MATERIALS AND METHODS: SCAPs were divided into the irradiation group (4 J/cm2) and the control group (0 J/cm2), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed. RESULTS: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed. CONCLUSION: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.

Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Impact of root canal disinfection on the bacteriome present in primary endodontic infection: A next generation sequencing study.

AIM: To investigate the bacteriome present in teeth with primary endodontic infection (PEI) and apical periodontitis (AP) and to determine quantitatively and qualitatively the impact of chemomechanical preparation (CMP) using 2.5% sodium hypochlorite NAOCl on the bacteriome found in PEI with AP using the Illumina MiSeq platform. METHODOLOGY: Thirty-six paired samples from 18 patients were successfully sequenced and analysed. Samples were collected at two sampling times: before (s1) and after (s2) CMP using 2.5% NaOCl. The DNA was extracted from s1 and s2 samples and quantified using quantitative PCR (qPCR). All 36 samples were sequenced using the Illumina MiSeq platform. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants (ASVs). Alpha diversity metrics representing abundance (Chao1) and diversity and evenness (Shannon, Simpson) were computed. The paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The PERMANOVA method (with 999 permutations) was applied to compare community composition between sample types (s1 versus s2) and between patient IDs. ALDEx2 (ANOVA-like differential expression tool for high-throughput sequencing data) to investigate differentially abundant taxa between s1 and s2. A paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. RESULTS: The qPCR counts were significantly higher in s1 compared to s2 (p = .0007). The Chao1 index indicated no difference in alpha diversity (p < .7019); whereas Shannon (p = .0056) and Simpson (p = .02685) indexes showed higher values in s2. The PERMANOVA test using Adonis2 showed a significant effect of sample time on community composition (R2 = .0630, p = .012). Patient ID also showed a significant effect on community composition (R2 = .6961, p = .001). At the genus level, Dialister, Mogibacterium, Prevotella, and Olsenella were differentially enriched at s1, while Actinomyces, Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified were differentially enriched in s2. CONCLUSION: The bacteriome present in teeth with PEI with AP is complex and diverse. CMP using 2.5% NaOCl showed a high quantitatively and qualitatively disinfectant impact on the bacteriome present in PEI with AP.

Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

AIM: The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. METHODOLOGY: Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. RESULTS: Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. CONCLUSIONS: The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

High throughput miRNA sequencing and bioinformatics analysis identify the mesenchymal cell proliferation and apoptosis related miRNAs during fetal mice palate development.

BACKGROUND: Palatogenesis requires a precise spatiotemporal regulation of gene expression. Recent studies indicate that microRNAs (miRNAs) are key factors in normal palatogenesis. The present study aimed to explain the regulatory mechanisms of miRNAs during palate development. METHODS: Pregnant ICR mice were choose at embryonic day 10.5 (E10.5). Hemotoxylin and eosin (H&E) staining was used to observe the morphological changes during the development of palatal process at embryonic day (E)13.5, E14.0, E14.5, E15.0 and E15.5. The fetal palatal tissues were collected at E13.5, E14.0, E14.5 and E15.0 to explore miRNA expression and function by high throughput sequencing and bioinformatic analysis. Mfuzz cluster analysis was used to look for miRNAs related to the fetal mice palate formation. The target genes of miRNAs were predicted by miRWalk. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed base on target genes. The mesenchymal cell proliferation and apoptosis related miRNAs-genes networks were predicted and constructed using miRWalk and Cytoscape software. The expression of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was detected by a quantitative real-time PCR (RT-qPCR) assay. RESULTS: H&E staining found that the palatal process grows vertically along the sides of the tongue at E13.5, the position of the tongue begins to descend and the bilateral palatal processes rise above the tongue at E14.0, the palatal process grows horizontally at E14.5, there is palatal contact fusion at E15.0, and the palatal suture disappeared at E15.5. Nine clusters of miRNA expression changes were identified in the fetal mice palate formation progression, including two reducing trends, two rising trends and five disordered trends. Next, the heatmap showed the miRNA expression from Clusters 4, 6, 9, 12 in the E13.5, E14.0, E14.5 and E15.0 groups. GO functional and KEGG pathway enrichment analysis found target genes of miRNAs in clusters involved in regulation of mesenchymal phenotype and the mitogen-activated protein kinase (MAPK) signaling pathway. Next, mesenchymal phenotype related miRNA-genes networks were constructed. The heatmap showing that the mesenchymal phenotype related miRNA expression of Clusters 4, 6, 9 and 12 at E13.5, E14.0, E14.5 and E15.0. Furthermore, the mesenchymal cell proliferation and apoptosis related miRNA-gene networks were identified in Clusters 6 and 12, including mmu-miR-504-3p-Hnf1b, etc. The expression level of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was verified by a RT-qPCR assay. CONCLUSIONS: For the first time, we identified that clear dynamic miRNA expression during palate development. Furthermore, we demonstrated that mesenchymal cell proliferation and apoptosis related miRNAs, genes and the MAPK signaling pathway are important during fetal mice palate development.

Community dynamics of subgingival microbiome in periodontitis and targets for microbiome modulation therapy.

The microbial aetiology for periodontitis has been widely studied and deciphered for more than a century. The evolving and changing concepts about periodontal microbiology can be attributed to continuously developing laboratory techniques. The current sequencing platforms have not only expanded the catalog of periodontal pathogens but have also facilitated the understanding of functional interactions of the ecological framework. However, the translation of this new knowledge to advance periodontal therapeutics is minimal. We contend that novel clinical interventions directed beyond conventional therapies need to be emphasized. A clear understanding of the structural and functional dynamics of subgingival microbiota is a pre-requisite for developing any microbiome-based interventions for applications in periodontal health care. In this review, we discuss the 16 s-rRNA gene sequencing-based knowledge of the subgingival microbial community structure, its interactions and functions, and our perspective on the potential to engineer it for periodontal therapeutics. Harnessing this next-generation sequencing-based knowledge, microbiome modulation therapies are poised to change microbiome therapeutics' face.

Influence of Environmental Factors on Salivary Microbiota and Their Metabolic Pathway: Next-Generation Sequencing Approach.

The current study aimed to investigate the effect of periodontitis and long-term heavy metal (HM) exposure on the salivary microbiome. The patients were divided into four groups as Wu Wei control (WWC) group involved healthy individuals, Wu Wei periodontitis (WWP) patients having periodontitis, Jing Chang with metal pollution periodontally healthy individuals (JCP), and Kuang periodontitis (KP). The most abundant bacteria identified at the phylum level in the WWC group were Bacteroides, Firmicutes, and Fusobacteria. Firmicutes were observed in a significantly higher proportion in the KP group than in the WWC, WWP, and JCP. At the genus level, the WWC has major dominating bacterial genera (such as Leptotrichia, Neisseria, and Fusobacterium) which were similar to WWP and KP group. The significant difference (p < 0.05) was found in alpha diversity while in beta diversity, the significant (p = 0.005) results were found among the four groups. The correlation of oral microbiota revealed that HMs present in the soil (Cr, Ni, and Cu) are associated with the growth of Capnocytophaga, Selenomonas, Aggregatibacter, and Campylobacter. The bacterial functions in the KP group were higher in translation and nucleotide metabolism than in the WWP group. This demonstrated that long-term exposure to HMs can influence the salivary microbiota which can alter the functioning, and diversity of bacteria.

Case report: isolated prevotella intermedia causing intracranial infection detected using metagenomic next generation sequencing.

BACKGROUND: Isolated Prevotella intermedia, a rare gram-negative, rod-shaped, anaerobic bacterium, is rarely detected in clinical practice. It has been associated with infections of the oral cavity and female genital tract, but has never been detected in cerebrospinal fluid (CSF) of patients in China. Accurate detection of causative pathogens is still an arduous task owing to the difficult conditions of anaerobic bacterial culture. Isolated Prevotella intermedia can be detected by metagenomic next generation sequencing (mNGS) of the CSF. Correct diagnosis and antibiotic treatment can help patients avoid life-threatening events. CASE PRESENTATION: Herein, we describe the case of a 64-year-old Chinese woman who presented with typical features of meningoencephalitis. Routine CSF culture failed to identify the causative pathogen. Isolated Prevotella intermedia was detected by mNGS, and the patient was treated with antibacterial agents including ceftriaxone, vancomycin, moxifloxacin, meropenem, metronidazole, and linezolid. The patient underwent surgical treatment for abscess of left frontal parietal lobe, which was observed on magnetic resonance imaging (MRI) and was suspected to be caused by Prevotella intermedia. It was further confirmed that it was a secondary infection from the oral cavity, and the possible etiology might have been dental surgery. Treatment was rendered to the patient based on metagenomic test result, and her condition improved after two months. CONCLUSIONS: This case highlights the role of mNGS in accurate diagnosis of patients with central nervous system infection. In particular, mNGS can be used to identify rare pathogens and confirm the diagnosis in patients with unknown etiology.

Meningitis caused by oral anaerobes detected using mNGS tool: a case report and review of literature.

BACKGROUND: Bacterial meningitis is a central nervous system (CNS) infection disease of the meninges and brain parenchyma caused by the bacteria. Few cases of meningitis related to oral anaerobes have been reported in the literature. Here, we report a case of meningitis in a middle-aged woman, caused by oral anaerobes. CASE PRESENTATION: A 58-year-old woman was admitted to hospital with fever, headache for 21 days and left limb weakness for 2 days. The blood cell counts (11.73 × 109/L), neutrophil counts (9.22 × 109/L) and high-sensitivity C-reactive protein levels (> 5.00 mg/L) were elevated. The brain computerized tomography (CT) scanning indicated the new right thalamus infarct. The brain cranial-enhanced magnetic resonance imaging (MRI) showed the right lateral paraventricular and right thalamic infarct, and abnormal signal in occipital horns of bilateral lateral ventricles were increased. In addition, the brain enhanced nuclear magnetic resonance (NMR) scanning suggested that meninges were thickened and enhanced at the base of the brain, with meningitis changes. The neck CT angiography (CTA) revealed arteriosclerotic changes. The metagenomic next-generation sequencing (mNGS) revealed Eubacterium brachy, Porphyromonas gingivalis, Fusobacterium nucleatum and Torque teno virus in her cerebrospinal fluid (CSF). The patient was diagnosed with purulent meningitis caused by infection of oral anaerobes, and treated with mannitol, ceftriaxone and vancomycin. Her symptoms alleviated. Subsequently, she was transferred to the infectious department and treated with ceftriaxone plus metronidazole (anti-anaerobes) and mannitol (reduce intracranial pressure). Her symptoms improved and currently received rehabilitation treatment. CONCLUSION: We herein report a rare case involving meningitis caused by infection of oral anaerobes. The mNGS can accurately detect the pathogens of infectious diseases.

Mutational screening of Greek patients with axonal Charcot-Marie-Tooth disease using targeted next-generation sequencing: Clinical and molecular spectrum delineation.

BACKGROUND AND AIMS: Axonal forms of Charcot-Marie-Tooth disease (CMT) are classified as CMT2, distal hereditary motor neuropathy (dHMN) or hereditary sensory neuropathy (HSN) and can be caused by mutations in over 100 genes. We presently aimed to investigate for the first time the genetic landscape of axonal CMT in the Greek population. METHODS: Sixty index patients with CMT2, dHMN or HSN were screened by a combination of Sanger sequencing (GJB1) and next-generation sequencing custom-made gene panel covering 24 commonly mutated genes in axonal CMT. RESULTS: Overall, 20 variants classified as pathogenic or likely pathogenic were identified in heterozygous state in 20 index cases, representing 33.3% of the cohort. Of these, 14 were known pathogenic/likely pathogenic and six were designated as such according to ACMG classification, after in silico evaluation, testing for familial segregation and further literature review. The most frequently involved genes were GJB1 (11.7%), MPZ (5%) and MFN2 (5%), followed by DNM2 (3.3%) and LRSAM1 (3.3%). Single cases were identified with mutations in BSCL2, HSPB1 and GDAP1. INTERPRETATION: A wide phenotypic variability in terms of severity and age of onset was noted. Given the limited number of genes tested, the diagnostic yield of the present panel compares favourably with studies in other European populations. Our study delineates the genetic and phenotypic variability of inherited axonal neuropathies in the Greek population and contributes to the pathogenicity characterization of further variants linked to axonal neuropathies.

Subgingival microbial diversity and respiratory decline: A cross-sectional study.

AIM: To investigate whether there is an association between subgingival microbial diversity and reduced respiratory function. MATERIALS AND METHODS: A group of dentate 58-72-year-old men in Northern Ireland had a comprehensive periodontal examination including subgingival plaque sampling. DNA was extracted from plaque samples and the V1-V3 regions of the 16S rRNA gene were analysed by high-throughput sequencing and a microbial diversity index (MDI) was derived. Spirometry measurements were made using a wedge bellows spirometer. The primary outcome variable of interest was the percentage of predicted forced expiratory volume in 1 s (% predicted FEV1 ). Analysis included multiple linear regression with adjustment for various confounders. RESULTS: Five-hundred and seven men were included in the analysis. The mean age was 63.6 years (SD = 3.1). Of these, 304 (60.0%) men had no or mild periodontitis, 105 (20.7%) had moderate periodontitis and 98 (19.3%) had severe periodontitis. Multiple linear regression analysis showed that a one unit increase in MDI was associated with a 0.71% loss (95% confidence interval: 0.06%-1.35%; p = .03) in % predicted FEV1 after adjustment for all confounders. CONCLUSIONS: In this group of dentate men from Northern Ireland, subgingival microbial diversity was associated with reduced respiratory function.

Gut Microbiota in Children with Hand Foot and Mouth Disease on 16S rRNA Gene Sequencing.

Hand foot and mouth disease (HFMD) is a contagious and seasonal viral disease in children. The gut microbiota of HFMD children is not clear now. The study aimed to explore the gut microbiota of HFMD children. The 16S rRNA gene of the gut microbiota of ten HFMD patients and ten healthy children were sequenced on the NovaSeq and PacBio platforms respectively. There were significant differences in gut microbiota between the patients and healthy children. The diversity and abundance of gut microbiota in HFMD patients were lower than that in healthy children. The species Roseburia inulinivorans and Romboutsia timonensis were more abundant in healthy children than those in HFMD patients, which suggests that the two species may be used as probiotics for adjusting the gut microbiota of HFMD patients. Meanwhile, the results of 16S rRNA gene sequences from the two platforms were different. The NovaSeq platform identified more microbiota and has the characteristics of high throughput, short time and low price. However, the NovaSeq platform has low resolution at the species level. The PacBio platform has high resolution based on its long reads length, which is more suitable for species-level analysis. But, the shortcomings of the high price and low throughput of PacBio still need to be overcome. With the development of sequencing technology, the reduction in sequencing price and the increase in throughput will promote the third-generation sequencing technology used in the study of gut microbes.

Microbiome in paired root apices and periapical lesions and its association with clinical signs in persistent apical periodontitis using next-generation sequencing.

AIM: To assess and compare the microbiome of paired root apices and periapical lesions from cases with failed endodontic treatment and to associate the microbiome and bacterial metabolic pathways in both sites with asymptomatic apical periodontitis (AAP) and symptomatic apical periodontitis (SAP), using next-generation sequencing (NGS). METHODOLOGY: Matched root apices and periapical lesions of patients with failed root canal treatments were surgically extracted. Specimens were cryopulverized, bacterial DNA was extracted and the V3-V4 hypervariable regions of the 16 S rRNA gene were amplified and sequenced using the Illumina Miseq platform. Diversity and community composition were studied in the paired samples, as well as in AAP and SAP cases. Diversity indices were compared in each case by means of the Wilcoxon matched-pairs signed rank and Mann-Whitney U tests. Differences in the community composition were explored with multivariate statistical analysis and Linear discriminant analysis Effect Size (LEfSe). Bacterial functional study was performed through the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. RESULTS: Twenty-one paired apices and lesions were successfully sequenced and analysed, identifying a total of 21 phyla and 600 genera. A higher alpha-diversity was observed in the periapical lesions, although no global differences in the community composition between the two sites were found (p = .87), the most prevalent genera being Fusobacterium, Porphyromonas and Streptococcus. Prevotella, Clostridiales_vadinBB60_group, Bosea, Phreatobacter, Afipia and Xanthobacteriaceae_unclassified were enriched in SAP samples, while Pseudopropionibacterium, Campylobacter and Peptoniphilus were significantly more abundant in AAP cases (p < .05). Metabolic pathways involved in the amino acid metabolism or degradation and flagellum assembly were more abundant in SAP samples, whereas glucose metabolism-related pathways were associated with AAP. CONCLUSIONS: The bacterial community composition was similar in the apices and periapical lesions. The microbiome was different in AAP and SAP samples, gram-negative bacteria showing higher relative abundances in SAP cases. An association was observed between amino acid degradation and flagellum assembly pathways, and the development of tenderness to percussion or palpation.

Amplicon-based next-generation sequencing for comparative analysis of root canal microbiome of teeth with primary and persistent/secondary endodontic infections.

OBJECTIVES: To compare the root canal microbiome profiles of primary and persistent/secondary infections using high-throughput sequencing with the help of a reliable bioinformatics algorithm. MATERIALS AND METHODS: Root canal samples of 10 teeth in the primary endodontic infection (PEI) group and 10 teeth in the persistent/secondary endodontic infection (SEI) group were included resulting in a total of 20 samples. After DNA extraction from the samples, sequencing was performed on the Illumina MiSeq platform. Pair-end Illumina reads were imported to QIIME 2; amplicon sequence variants (ASVs) generated by DADA2 were mapped to GreenGenes database. Weighted UniFrac distances were calculated and principal coordinates analysis (PCoA) was used to compare beta diversity patterns. The multiple response permutation procedure (MRPP), the analysis of similarities (ANOSIM), and permutational multivariate analysis of variance (adonis) were conducted for testing group differences. Linear discriminant analysis effect size (LEfSe) analysis was utilized to identify differentially abundant taxa between the groups. The linear discriminant analysis (LDA) score threshold was set to 4.0. RESULTS: Within the Gram-negative facultative anaerobic Gammaproteobacteria class outgroup, two orders (Pasteurellales, Vibrionales) and two families (Pasteurellaceae, Vibrionaceae) were significantly more abundant in the PEI group, whereas Gram-positive bacteria, Actinomycetales order, and Gram-positive anaerobic taxa, one genus (Olsenella) and one species (Olsenella uli), were identified as significantly more abundant in the SEI group. CONCLUSIONS: A few taxa were differentially abundant within either the PEI or SEI group. CLINICAL RELEVANCE: Reliable bioinformatic tools are needed to define microbial profiles of endodontic infections. Based on a limited number of samples, no distinct variation was determined between the bacterial diversity of initial and recurrent endodontic infections.

Identification of novel susceptibility genes for non-syndromic cleft lip with or without cleft palate using NGS-based multigene panel testing.

For non-syndromic cleft lip with or without cleft palate (ns-CL/P), the proportion of heritability explained by the known risk loci is estimated to be about 30% and is captured mainly by common variants identified in genome-wide association studies. To contribute to the explanation of the "missing heritability" problem for orofacial clefts, a candidate gene approach was taken to investigate the potential role of rare and private variants in the ns-CL/P risk. Using the next-generation sequencing technology, the coding sequence of a set of 423 candidate genes was analysed in 135 patients from the Polish population. After stringent multistage filtering, 37 rare coding and splicing variants of 28 genes were identified. 35% of these genetic alternations that may play a role of genetic modifiers influencing an individual's risk were detected in genes not previously associated with the ns-CL/P susceptibility, including COL11A1, COL17A1, DLX1, EFTUD2, FGF4, FGF8, FLNB, JAG1, NOTCH2, SHH, WNT5A and WNT9A. Significant enrichment of rare alleles in ns-CL/P patients compared with controls was also demonstrated for ARHGAP29, CHD7, COL17A1, FGF12, GAD1 and SATB2. In addition, analysis of panoramic radiographs of patients with identified predisposing variants may support the hypothesis of a common genetic link between orofacial clefts and dental abnormalities. In conclusion, our study has confirmed that rare coding variants might contribute to the genetic architecture of ns-CL/P. Since only single predisposing variants were identified in novel cleft susceptibility genes, future research will be required to confirm and fully understand their role in the aetiology of ns-CL/P.