Exome sequencing identifies a recurrent de novo ZSWIM6 mutation associated with acromelic frontonasal dysostosis.

Acromelic frontonasal dysostosis (AFND) is a rare disorder characterized by distinct craniofacial, brain, and limb malformations, including frontonasal dysplasia, interhemispheric lipoma, agenesis of the corpus callosum, tibial hemimelia, preaxial polydactyly of the feet, and intellectual disability. Exome sequencing of one trio and two unrelated probands revealed the same heterozygous variant (c.3487C>T [p. Arg1163Trp]) in a highly conserved protein domain of ZSWIM6; this variant has not been seen in the 1000 Genomes data, dbSNP, or the Exome Sequencing Project. Sanger validation of the three trios confirmed that the variant was de novo and was also present in a fourth isolated proband. In situ hybridization of early zebrafish embryos at 24 hr postfertilization (hpf) demonstrated telencephalic expression of zswim6 and onset of midbrain, hindbrain, and retinal expression at 48 hpf. Immunohistochemistry of later-stage mouse embryos demonstrated tissue-specific expression in the derivatives of all three germ layers. qRT-PCR expression analysis of osteoblast and fibroblast cell lines available from two probands was suggestive of Hedgehog pathway activation, indicating that the ZSWIM6 mutation associated with AFND may lead to the craniofacial, brain and limb malformations through the disruption of Hedgehog signaling.

MinorityReport, software for generalized analysis of causal genetic variants.

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.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

UCSF Protocol for Caries Arrest Using Silver Diamine Fluoride: Rationale, Indications and Consent.

The Food and Drug Administration recently cleared silver diamine fluoride for reducing tooth sensitivity. Clinical trials document arrest and prevention of dental caries by silver diamine fluoride. This off-label use is now permissible and appropriate under U.S. law. A CDT code was approved for caries arresting medicaments for 2016 to facilitate documentation and billing. We present a systematic review, clinical indications, clinical protocol and consent procedure to guide application for caries arrest treatment.

UCSF Protocol for Caries Arrest Using Silver Diamine Fluoride: Rationale, Indications and Consent.

The Food and Drug Administration recently cleared silver diamine fluoride for reducing tooth sensitivity. Clinical trials document arrest and prevention of dental caries by silver diamine fluoride. This off-label use is now permissible and appropriate under U.S. law. A CDT code was approved for caries arresting medicaments for 2016 to facilitate documentation and billing. We present a systematic review, clinical indications, clinical protocol and consent procedure to guide application for caries arrest treatment.

A human homeotic transformation resulting from mutations in PLCB4 and GNAI3 causes auriculocondylar syndrome.

Auriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic "question-mark" ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans.

Self-assembly of filamentous amelogenin requires calcium and phosphate: from dimers via nanoribbons to fibrils.

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.

Caries induced cytokine network in the odontoblast layer of human teeth.

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-1ß, IL-1α, and TNF-α, carry the converged inflammatory signal. We show that IL1ß 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.

IGF1R variants associated with isolated single suture craniosynostosis.

The genetic contribution to the pathogenesis of isolated single suture craniosynostosis is poorly understood. The role of mutations in genes known to be associated with syndromic synostosis appears to be limited. We present our findings of a candidate gene resequencing approach to identify rare variants associated with the most common forms of isolated craniosynostosis. Resequencing of the coding regions, splice junction sites, and 5' and 3' untranslated regions of 27 candidate genes in 186 cases of isolated non-syndromic single suture synostosis revealed three novel and two rare sequence variants (R406H, R595H, N857S, P190S, M446V) in insulin-like growth factor I receptor (IGF1R) that are enriched relative to control samples. Mapping the resultant amino acid changes to the modeled homodimer protein structure suggests a structural basis for segregation between these and other disease-associated mutations found in IGF1R. These data suggest that IGF1R mutations may contribute to the risk and in some cases cause single suture craniosynostosis.

Use of unbiased metagenomic and transcriptomic analyses to investigate the association between feline calicivirus and feline chronic gingivostomatitis in domestic cats.

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.

Clinical Instructions for Using Silver Diamine Fluoride (SDF) in Dental Caries Management.

Manufacturer instructions for 38% silver diamine fluoride (SDF) are limited to current FDA clearance for tooth desensitization. There is a need for instructions to provide best-practice recommendations for off-label use of SDF for caries prevention and arrest. METHODS: The authors considered existing clinical approaches to the use of 38% SDF at pH 10 for the prevention and arrest of active dental caries, in light of the best current evidence. Application of SDF, with or without subsequent direct restoration, is included. The content was reviewed by stakeholders including but not limited to those listed on the consensus statement (Appendix A, below). RESULTS: 38% SDF for the prevention and arrest of active caries lesions, as well as compatibility with common direct restorative materials, such as glass-ionomer cement and resin composite, has a foundation in the scientific literature. A practical decision-flow diagram and accompanying best practices for treatment of caries lesions, based on clinical access and intention to restore, were developed based on available evidence and expert clinical observation when no evidence was available. CONCLUSIONS: Based on the best available evidence, a logical approach can be adopted regarding the practical use of 38% SDF for caries prevention and arrest. PRACTICAL IMPLICATIONS: SDF used as per these instructions for prevention on high-risk tooth surfaces and arrest of active caries lesions has a place in the practitioner's dental caries management armamentarium. When SDF is applied to active lesions, it can be used with or without subsequent restoration, depending on clinical context, expert judgment, and patient input.

A protein sequence meta-functional signature for calcium binding residue prediction.

The diversity of characterized protein functions found amongst experimentally interrogated proteins suggests that a vast array of unknown functions remains undiscovered. These protein functions are imparted by specific geometric distributions of amino acid residue chemical moieties, each contributing a functional interaction. We hypothesize that individual residue function contributions are predictable through sequence analytic knowledge based algorithms, and that they can be recombined to understand composite protein function by predicting spatial relation in tertiary structure. We assess the former by training a meta-functional signature algorithm to specifically predict calcium ion binding residues from protein sequence. We estimate the latter by testing for match between predictive contribution of positions in predicted secondary structures and patterns of side chain proximity forced by secondary structure moieties. Specific training for calcium binding results in 83% area under the receiver operator characteristic curve added value over random (AUCoR) and p<10(-300) significance as measured by Kendall's τ in ten fold cross validation for parallel sets of 811 residues in 336 proteins and 696 residues in 299 proteins. Training for generalized function results in 63% AUCoR and p≅10(-221) for the same tests. Including inference of side chain proximity improves predictive ability by 2% AUCoR consistently. The results demonstrate that protein meta-functional signatures can be trained to predict specific protein functions by considering amino acid identity and structural features accessible from sequence, laying the groundwork for composite sequence based function site prediction.

Managing dental caries against the backdrop of COVID-19: approaches to reduce aerosol generation.

The COVID-19 pandemic resulted in severe limitation and closure of dental practices in many countries. Outside of the acute (peak) phases of the disease, dentistry has begun to be practised again. However, there is emerging evidence that SARS-CoV-2 can be transmitted via airborne routes, carrying implications for dental procedures that produce aerosol. At the time of writing, additional precautions are required when a procedure considered to generate aerosol is undertaken.This paper aims to present evidence-based treatments that remove or reduce the generation of aerosols during the management of carious lesions. It maps aerosol generating procedures (AGPs), where possible, to alternative non-AGPs or low AGPs. This risk reduction approach overcomes the less favourable outcomes associated with temporary solutions or extraction-only approaches. Even if this risk reduction approach for aerosol generation becomes unnecessary in the future, these procedures are not only suitable but desirable for use as part of general dental care post-COVID-19.

Shear Bond Strength of Glass Ionomer Cement to Silver Diamine Fluoride-Treated Artificial Dentinal Caries.

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.

Enhanced Tooth Structure Via Silver Microwires Following Treatment with 38 Percent Silver Diamine Fluoride.

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.

A novel method for predicting and using distance constraints of high accuracy for refining protein structure prediction.

The principal bottleneck in protein structure prediction is the refinement of models from lower accuracies to the resolution observed by experiment. We developed a novel constraints-based refinement method that identifies a high number of accurate input constraints from initial models and rebuilds them using restrained torsion angle dynamics (rTAD). We previously created a Bayesian statistics-based residue-specific all-atom probability discriminatory function (RAPDF) to discriminate native-like models by measuring the probability of accuracy for atom type distances within a given model. Here, we exploit RAPDF to score (i.e., filter) constraints from initial predictions that may or may not be close to a native-like state, obtain consensus of top scoring constraints amongst five initial models, and compile sets with no redundant residue pair constraints. We find that this method consistently produces a large and highly accurate set of distance constraints from which to build refinement models. We further optimize the balance between accuracy and coverage of constraints by producing multiple structure sets using different constraint distance cutoffs, and note that the cutoff governs spatially near versus distant effects in model generation. This complete procedure of deriving distance constraints for rTAD simulations improves the quality of initial predictions significantly in all cases evaluated by us. Our procedure represents a significant step in solving the protein structure prediction and refinement problem, by enabling the use of consensus constraints, RAPDF, and rTAD for protein structure modeling and refinement.

Novel paradigms for drug discovery: computational multitarget screening.

An established paradigm in current drug development is (i) to identify a single protein target whose inhibition is likely to result in the successful treatment of a disease of interest; (ii) to assay experimentally large libraries of small-molecule compounds in vitro and in vivo to identify promising inhibitors in model systems; and (iii) to determine whether the findings are extensible to humans. This complex process, which is largely based on trial and error, is risk-, time- and cost-intensive. Computational (virtual) screening of drug-like compounds simultaneously against the atomic structures of multiple protein targets, taking into account protein-inhibitor dynamics, might help to identify lead inhibitors more efficiently, particularly for complex drug-resistant diseases. Here we discuss the potential benefits of this approach, using HIV-1 and Plasmodium falciparum infections as examples. We propose a virtual drug discovery 'pipeline' that will not only identify lead inhibitors efficiently, but also help minimize side-effects and toxicity, thereby increasing the likelihood of successful therapies.

Protein meta-functional signatures from combining sequence, structure, evolution, and amino acid property information.

Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

Prevention of Dental Caries by Silver Diamine Fluoride.

The use of silver diamine fluoride (SDF) for management of dental caries has gained considerable attention due to recent regulatory clearance in the United States. The primary focus of policies, presentations, and publications has been the arrest of caries lesions (cavities) because of the material's unique ability to non-invasively achieve this elusive and clinically important goal. However, SDF also has proven efficacy in prevention, ie, decreasing the incidence of new caries lesions. Analysis of nine clinical trials in children shows that SDF prevented 61% of new lesions compared to controls. To prevent one new caries lesion, clinicians need to treat four primary teeth (one patient) or 12.1 permanent molars (three patients) with SDF. The preventive effect appears to be immediate and maintains at the same fraction over time. Direct comparisons of SDF applied once per year with alternative treatments show that SDF is more effective than other topical fluorides placed two to four times per year and more cost-effective than dental sealants. Enamel lesions may be even more responsive than cavitated dentin lesions. Annual application of SDF to high-risk surfaces (eg, mesial surfaces of permanent first molars where the distal surface of the second primary molar is carious) in patients with any risk of new caries lesions appears to be the most cost-effective approach available to prevent dental caries. SDF is an underutilized evidence-based preventive agent for dental caries.

Fluorides and Other Preventive Strategies for Tooth Decay.

We focus on scalable public health interventions that prevent and delay the development of caries and enhance resistance to dental caries lesions. These interventions should occur throughout the life cycle, and need to be age appropriate. Mitigating disease transmission and enhancing resistance are achieved through use of various fluorides, sugar substitutes, mechanical barriers such as pit-and-fissure sealants, and antimicrobials. A key aspect is counseling and other behavioral interventions that are designed to promote use of disease transmission-inhibiting and tooth resistance-enhancing agents. Advocacy for public water fluoridation and sugar taxes is an appropriate dental public health activity.