Background

The widespread availability of next generation genome sequencing technologies has enabled a wide range of variant detection applications, especially in cancer and inborn genetic disorders. For model systems and microorganisms, the same technology may be used to discover the causative mutations for any phenotype, including those generated in response to chemical perturbation. In the case of pathogenic organisms, these approaches have allowed the determination of drug targets by means of resistance selection followed by genome sequencing.

Results

MinorityReport is open source software written in python that facilitates the comparison of any two sets of genome alignments for the purpose of rapidly identifying the spectrum of nonsynonymous changes, insertions or deletions, and copy number variations in a presumed mutant relative to its parent. Specifically, MinorityReport relates mapped sequence reads in SAM format output from any alignment tool for both the mutant and parent genome, relative to a reference genome, and produces the set of variants that distinguishes the mutant from the parent, all presented in an intuitive, straightforward report format. MinorityReport features tunable parameters for evaluating evidence and a scoring system that prioritizes reported variants based on relative proportions of read counts supporting the variant in the mutant versus parent data sets. The utility of MinorityReport is demonstrated using previously published publicly available data sets to find the determinants of resistance for novel anti-malarial drugs.

Conclusions

MinorityReport is readily available (github: JeremyHorst/MinorityReport) to identify the genetic mechanisms of drug resistance in Plasmodium, genotype-phenotype relationships in human diads, or genomic variations between any two related organisms.

Abstract Background Immunologic responses of the tooth to caries begin with odontoblasts recognizing carious bacteria. Inflammatory propagation eventually leads to tooth pulp necrosis and danger to health. The present study aims to determine cytokine gene expression profiles generated within human teeth in response to dental caries in vivo and to build a mechanistic model of these responses and the downstream signaling network. Results We demonstrate profound differential up-regulation of inflammatory genes in the odontoblast layer (ODL) in human teeth with caries in vivo, while the pulp remains largely unchanged. Interleukins, chemokines, and all tested receptors thereof were differentially up-regulated in ODL of carious teeth, well over one hundred-fold for 35 of 84 genes. By interrogating reconstructed protein interaction networks corresponding to the differentially up-regulated genes, we develop the hypothesis that pro-inflammatory cytokines highly expressed in ODL of carious teeth, IL-1β, IL-1α, and TNF-α, carry the converged inflammatory signal. We show that IL1β amplifies antimicrobial peptide production in odontoblasts in vitro 100-fold more than lipopolysaccharide, in a manner matching subsequent in vivo measurements. Conclusions Our data suggest that ODL amplifies bacterial signals dramatically by self-feedback cytokine-chemokine signal-receptor cycling, and signal convergence through IL1R1 and possibly others, to increase defensive capacity including antimicrobial peptide production to protect the tooth and contain the battle against carious bacteria within the dentin.

Purpose: American Academy of Pediatric Dentistry guidelines recommend treatment of primary teeth with 38 percent silver diamine fluoride (SDF) as a noninvasive option to arrest active dental caries lesions. A significant outcome of SDF treatment are lesions that clinically harden and become more resistant to further decay. Many practicing dentists believe that this increased hardening is due to the reaction of silver and fluoride with carious dentin. The purpose of this study was to focus on the structural and chemical effects of silver diamine fluoride treatment on the native tooth. Methods: In SDF-treated cavitated dentin lesions in teeth subsequently extracted for orthodontic reasons, the authors observed continuous, filamentous silver densities formed in situ from 50 to 2,100 μm in length and 0.25 to 7.0 μm in diameter using high-resolution synchrotron X-ray microcomputer tomography and field emission scanning electron microscopy. These microwires fill voids in the lesion caused by disease and permeate through surrounding dentinal tubules. Results: Spectroscopy confirmed that the chemical composition of the observed microwires is predominantly silver. Conclusions: These observations suggest mechanistic explanations for the structural reinforcement of carious dentin in addition to remineralization. It is hypothesized that silver diamine fluoride may achieve its antimicrobial functions by biochemical interactions and through its inherent ability to integrate into the native tooth structure.

Purpose: The purpose of this study was to measure the shear bond strength (SBS) of glass ionomer cement (GIC) to artificial carious dentin with and without silver diamine fluoride (SDF) treatment. Methods: Permanent molars were sectioned and demineralized to create artificial carious lesions. In five groups, the demineralization of dentin, application of SDF, use of conditioner, and elapsed time between the placement of SDF and restoration were tested for differences in SBS using an UltraTester machine. Statistical analysis was done using the Kruskal-Wallis test and Tukey-Kramer multiple comparison tests. Results: The highest bond strength was found when GIC was placed on conditioned and demineralized dentin treated with SDF one week earlier. Treatment with SDF and use of conditioner did not statistically affect the SBS of GIC to demineralized dentin. Statistically significant increases in bond strength were found when one week elapsed between SDF application and GIC placement. The lowest bond strength was found with immediate GIC application onto SDF-treated demineralized dentin. Conclusions: These in vitro findings suggest that silver diamine fluoride treatment does not significantly affect the bond strength of glass ionomer cement to dentin lesions, and improved retention is obtained by allowing SDF solution to set for one week prior to GIC placement.

Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite's genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.

Purpose: American Academy of Pediatric Dentistry guidelines recommend treatment of primary teeth with 38 percent silver diamine fluoride (SDF) as a noninvasive option to arrest active dental caries lesions. A significant outcome of SDF treatment are lesions that clinically harden and become more resistant to further decay. Many practicing dentists believe that this increased hardening is due to the reaction of silver and fluoride with carious dentin. The purpose of this study was to focus on the structural and chemical effects of silver diamine fluoride treatment on the native tooth. Methods: In SDF-treated cavitated dentin lesions in teeth subsequently extracted for orthodontic reasons, the authors observed continuous, filamentous silver densities formed in situ from 50 to 2,100 μm in length and 0.25 to 7.0 μm in diameter using high-resolution synchrotron X-ray microcomputer tomography and field emission scanning electron microscopy. These "microwires" fill voids in the lesion caused by disease and permeate through surrounding dentinal tubules. Results: Spectroscopy confirmed that the chemical composition of the observed microwires is predominantly silver. Conclusions: These observations suggest mechanistic explanations for the structural reinforcement of carious dentin in addition to remineralization. It is hypothesized that silver diamine fluoride may achieve its antimicrobial functions by biochemical interactions and through its inherent ability to integrate into the native tooth structure.

Objective

To identify associations between microbes and host genes in cats with feline chronic gingivostomatitis (FCGS), a debilitating inflammatory oral mucosal disease with no known cause, compared with healthy cats and cats with periodontitis (control cats).

Animals

19 control cats and 23 cats with FCGS.

Procedures

At least 1 caudal oral mucosal swab specimen was obtained from each cat. Each specimen underwent unbiased metatranscriptomic next-generation RNA sequencing (mNGS). Filtered mNGS reads were aligned to all known genetic sequences from all organisms and to the cat transcriptome. The relative abundances of microbial and host gene read alignments were compared between FCGS-affected cats and control cats and between FCGS-affected cats that did and did not clinically respond to primary treatment. Assembled feline calicivirus (FCV) genomes were compared with reverse transcription PCR (RT-PCR) primers commonly used to identify FCV.

Results

The only microbe strongly associated with FCGS was FCV, which was detected in 21 of 23 FCGS-affected cats but no control cats. Problematic base pair mismatches were identified between the assembled FCV genomes and RT-PCR primers. Puma feline foamy virus was detected in 9 of 13 FCGS-affected cats that were refractory to treatment and 5 healthy cats but was not detected in FCGS-affected cats that responded to tooth extractions. The most differentially expressed genes in FCGS-affected cats were those associated with antiviral activity.

Conclusions and clinical relevance

Results suggested that FCGS pathogenesis has a viral component. Many FCV strains may yield false-negative results on RT-PCR-based assays. Coinfection of FCGS-affected cats with FCV and puma feline foamy virus may adversely affect response to treatment.

Enamel matrix self-assembly has long been suggested as the driving force behind aligned nanofibrous hydroxyapatite formation. We tested if amelogenin, the main enamel matrix protein, can self-assemble into ribbon-like structures in physiologic solutions. Ribbons 17 nm wide were observed to grow several micrometers in length, requiring calcium, phosphate, and pH 4.0-6.0. The pH range suggests that the formation of ion bridges through protonated histidine residues is essential to self-assembly, supported by a statistical analysis of 212 phosphate-binding proteins predicting 12 phosphate-binding histidines. Thermophoretic analysis verified the importance of calcium and phosphate in self-assembly. X-ray scattering characterized amelogenin dimers with dimensions fitting the cross-section of the amelogenin ribbon, leading to the hypothesis that antiparallel dimers are the building blocks of the ribbons. Over 5-7 days, ribbons self-organized into bundles composed of aligned ribbons mimicking the structure of enamel crystallites in enamel rods. These observations confirm reports of filamentous organic components in developing enamel and provide a new model for matrix-templated enamel mineralization.

Objectives:

The Stopping Cavities Trial investigated effectiveness and safety of 38% silver diamine fluoride in arresting caries lesions.

Materials and Methods:

Double-blind randomized placebo-controlled superiority trial with 2 parallel groups. Oregon preschools. 66 preschool children with ≥1 lesion. 38% silver diamine fluoride or placebo (blue-tinted water), applied topically to the lesion. The primary endpoint was caries arrest (lesion inactivity, Nyvad criteria) 14- 21 days post intervention. Dental plaque was collected from all children, and microbial composition was assessed by RNA sequencing from 2 lesions and 1 unaffected surface before treatment and at follow-up for 3 children from each group.

Results and Conclusion:

Mean fraction of arrested caries lesions in the silver diamine fluoride group was higher (0.72; 95% CI; 0.55, 0.84) than in the placebo group (0.05; 95% CI; 0.00, 0.16). Confirmatory analysis using generalized estimating equation log-linear regression, accounting for the number of treated surfaces and length of follow-up, indicated the fraction of arrested caries was significantly higher in the treatment group (relative risk, 17.3; 95% CI: 4.3 to 69.4). No harms were observed. RNA sequencing analysis identified no consistent changes in relative abundance of caries-associated microbes, nor emergence of antibiotic or metal resistance gene expression. Topical 38% silver diamine fluoride was effective and safe in arresting cavities in preschool children. The treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.

Trial Registration:

ClinicalTrials.gov NCT02536040 .

Clinical Significance

In this clinical trial, 72% of caries lesions were arrested by silver diamine fluoride, with no harms. Contrary to the presumed antibacterial mechanism, lesion bacterial composition changed negligibly. This simple topical treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.

Objectives

The Stopping Cavities Trial investigated effectiveness and safety of 38% silver diamine fluoride in arresting caries lesions.

Materials and methods

The study was a double-blind randomized placebo-controlled superiority trial with 2 parallel groups. The sites were Oregon preschools. Sixty-six preschool children with ≥1 lesion were enrolled. Silver diamine fluoride (38%) or placebo (blue-tinted water), applied topically to the lesion. The primary endpoint was caries arrest (lesion inactivity, Nyvad criteria) 14-21days post intervention. Dental plaque was collected from all children, and microbial composition was assessed by RNA sequencing from 2 lesions and 1 unaffected surface before treatment and at follow-up for 3 children from each group.

Results and conclusion

Average proportion of arrested caries lesions in the silver diamine fluoride group was higher (0.72; 95% CI; 0.55, 0.84) than in the placebo group (0.05; 95% CI; 0.00, 0.16). Confirmatory analysis using generalized estimating equation log-linear regression, based on the number of arrested lesions and accounting for the number of treated surfaces and length of follow-up, indicates the risk of arrested caries was significantly higher in the treatment group (relative risk, 17.3; 95% CI: 4.3 to 69.4). No harms were observed. RNA sequencing analysis identified no consistent changes in relative abundance of caries-associated microbes, nor emergence of antibiotic or metal resistance gene expression. Topical 38% silver diamine fluoride is effective and safe in arresting cavities in preschool children.

Clinical significance

The treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.