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Objectives The goal of endodontic therapy is to prevent apical periodontitis. This is achieved by biomechanical preparation, microbial control using endodontic irrigants, and complete obturation of the canal space. In order to prevent possible post‐obturation complications and for an added antimicrobial effect, substantivity is a desired characteristic of endodontic irrigants. Currently the most commonly used endodontic irrigant that produces an antibacterial substantivity effect is chlorohexidine (CHX). Silver diamine fluoride (SDF) is a topically applied agent for managing dental caries and has shown to stop caries lesion progression. The objective of this study was to compare the antimicrobial substantivity effect of 3.8% SDF against other commonly used endodontic irrigants such as 2% CHX and 6.25% Sodium hypochlorite (NaOCl). Material and methods Using a diffusion disc assay we determined the antimicrobial activities of 38%, 3.8%, 0.38%, and 0.038% of SDF against the bacterium Enterococcus faecalis OG1RF. Subsequently, we compared the levels of colonization of E. faecalis by scanning electron microscopy (SEM) at 1.5‐ and 3‐week time intervals on dentin pretreated with 3.8% SDF, 6.25% NaOCl, 2% CHX or sterile phosphate buffered saline (PBS). Results The diffusion disc assay demonstrated that 38% and 3.8% of SDF inhibited the growth of E. faecalis. Moreover, the substantivity of 3.8% SDF (p

Mutations in WNT10A have frequently been reported as etiologic for tooth agenesis (TA). However, the effects of WNT10A variation on gene/protein function and contribution to TA phenotypes remain poorly understood. Here, we performed bioinformatic and functional characterization analysis of WNT10A variants. In silico prediction of variant function was performed with VIPUR for all WNT10A missense variants reported in the Exome Aggregation Consortium database. Functional characterization experiments were then performed for selected WNT10A variants previously associated with TA. Expression vectors for wild-type and mutant WNT10A were made and transfected into stem cells from human exfoliated deciduous teeth (SHED) for evaluation of gene/protein function, WNT signaling activity, and effects on expression of relevant genes. While 75% of WNT10A variants were predicted neutral, most of the TA-associated variants received deleterious scores by potentially destabilizing or preventing the disulfide bond formation required for proper protein function. WNT signaling was significantly decreased with 8 of 13 variants tested, whereas wild-type–like activity was retained with 4 of 13 variants. WNT10A-mutant cells (T357I, R360C, and R379C mutants) showed reduced or impaired binding affinity to FZD5, suggesting a potential mechanism for the decreased WNT signaling. Mutant cells also had decreased WNT10A protein expression in comparison to wild-type cells. mRNA expression of PAX9, MSX1, AXIN2, and RUNX2 (known tooth development genes) was perturbed in mutant cells and quite significantly for PAX9 and RUNX2. Transcriptome analysis of wild-type and T357I-mutant cells identified 36 differentially expressed genes (26 downregulated, 10 upregulated) involved in skeletal system development and morphogenesis and pattern specification. WNT10A variants deemed pathogenic for TA likely affect protein folding and/or stabilization, leading to decreased WNT signaling and concomitant dysregulated expression of relevant genes. These findings may allow for improved interpretation of TA phenotypes upon clinical diagnosis while providing important insights toward the development of future tooth replacement therapies.

Spotted fever group Rickettsia (SFGR) is one among the aetiologies that cause fever of unknown origin in Angola. Despite their occurrence, there is little information about its magnitude in this country either because it is misdiagnosed or due to the lack of diagnostic resources. For this purpose, eighty-seven selected malaria- A nd yellow fever-negative serum specimens collected between February 2016 and March 2017 as part of the National Laboratory of Febrile Syndromes, from patients with fever (=37.5°C) for at least 4 days and of unknown origin, were screened for Rickettsia antibodies through an immunofluorescence assay (IFA). Serological results were interpreted according to the 2017 guidelines for the detection of Rickettsia spp. Three seroreactive patients had detectable IgM antibodies to Rickettsia with an endpoint titre of 32 and IgG antibodies with endpoint titres of 128 and 256. These findings supported a diagnosis of Rickettsia exposure amongst these patients and highlight that rickettsioses may be among the cause of unknown febrile syndromes in Angola. Therefore, physicians must be aware of this reality and must include this vector-borne disease as part of aetiologies that should be considered and systematically tested in order to delineate appropriate strategies of diagnostic and control of Rickettsia in Angola. P. Barradas (SFRH/BD/116449/2016) acknowledges the Portuguese Foundation for Science and Technology (FCT) for financial support. IPATIMUP integrates the i3S Research Unit, which was partially supported by FCT. -is work was funded by FEDER Funds -rough the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT, under the project number PEst-C/SAU/LA0003/2013. -is paper was published under the framework of the European Social Fund, Human Resources Development Operational Programme (2007–2013) (POSDRU/159/1.5/S/136893). -e authors would like to thank Dr. Joana de Morais, INIS, for supporting this work and all the surveillance technicians involved in sample collection.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect for which only ~20% of the underlying genetic variation has been identified. Variants in noncoding regions have been increasingly suggested to contribute to the missing heritability. In this study, we investigated whether variation in craniofacial enhancers contributes to NSCLP. Candidate enhancers were identified using VISTA Enhancer Browser and previous publications. Prioritization was based on patterning defects in knockout mice, deletion/duplication of craniofacial genes in animal models and results of whole exome/whole genome sequencing studies. This resulted in 20 craniofacial enhancers to be investigated. Custom amplicon-based sequencing probes were designed and used for sequencing 380 NSCLP probands (from multiplex and simplex families of nonHispanic white (NHW) and Hispanic ethnicities) using Illumina MiSeq. The frequencies of identified variants were compared to ethnically-matched European (CEU) and Los Angeles Mexican (MXL) control genomes and used for association analyses. Variants in mm427/MSX1 and hs1582/SPRY1 showed genome-wide significant association with NSCLP (p≤ 6.4 × 10(−11)). In silico analysis showed that these enhancer variants may disrupt important transcription factor binding sites. Haplotypes involving these enhancers and also mm435/ABCA4 were significantly associated with NSCLP, especially in NHW (p≤ 6.3 × 10(−7)). Importantly, group-wise burden analysis showed several enhancer combinations significantly over-represented in NSCLP individuals revealing novel NSCLP pathways and supporting a polygenic inheritance model. Our findings support the role of craniofacial enhancer sequence variation in the etiology of NSCLP.

Nonsyndromic cleft lip and palate (NSCLP) is one of the most common craniofacial anomalies in humans, affecting more than 135,000 newborns worldwide. NSCLP has a multifactorial etiology with more than 50 genes postulated to play an etiologic role. The genetic pathway comprised of Pbx-Wnt-p63-Irf6 genes was shown to control facial morphogenesis in mice and proposed as a regulatory pathway for NSCLP. Based on these findings, we investigated whether variation in PBX1, PBX2, and TP63, and their proposed interactions were associated with NSCLP. Fourteen single nucleotide variants (SNVs) in/nearby PBX1, PBX2, and TP63 were genotyped in 780 NSCLP families of nonHispanic white (NHW) and Hispanic ethnicities. Family-based association tests were performed for individual SNVs stratified by ethnicity and family history of NSCLP. Gene-gene interactions were also tested. A significant association was found for PBX2 rs3131300 and NSCLP in combined Hispanic families (p = .003) while nominal association was found for TP63 rs9332461 in multiplex Hispanic families (p = .005). Significant haplotype associations were observed for PBX2 in NHW (p = .0002) and Hispanic families (p = .003), and for TP63 in multiplex Hispanic families (.003). An independent case-control group was used to validate findings, and significant associations were found with PBX1 rs6426870 (p = .007) and TP63 rs9332461 (p = .03). Gene-gene interactions were detected between PBX1/PBX2/TP63 with IRF6 in NHW families, and between PBX1 with WNT9B in both NHW and Hispanic families (p

(This article belongs to the Special Issue Parasitic Diseases from Wild Animals with Emphasis in Zoonotic Infections) Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors’ knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock–wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field. This work was supported by the project UIDB/CVT/00772/2020 funded by the Fundação para a Ciência e Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

Simple Summary Helicobacter pylori and H. suis are associated with gastric pathologies in humans. To obtain better insights into the potential role of wild boars as reservoirs of these pathogens, gastric samples of 14 animals were tested for the presence of H. pylori and H. suis DNA. Two wild boars were found PCR-positive for H. pylori and one for H. suis. This indicates that these microorganisms may colonize the stomach of wild boars. Abstract Helicobacter pylori (H. pylori) is a Gram-negative bacterium that infects half of the human population worldwide, causing gastric disorders, such as chronic gastritis, gastric or duodenal ulcers, and gastric malignancies. Helicobacter suis (H. suis) is mainly associated with pigs, but can also colonize the stomach of humans, resulting in gastric pathologies. In pigs, H. suis can induce gastritis and seems to play a role in gastric ulcer disease, seriously affecting animal production and welfare. Since close interactions between domestic animals, wildlife, and humans can increase bacterial transmission risk between species, samples of gastric tissue of 14 free range wild boars (Sus scrofa) were evaluated for the presence of H. pylori and H. suis using PCR. Samples from the antral gastric mucosa from two animals were PCR-positive for H. pylori and another one for H. suis. These findings indicate that these microorganisms were able to colonize the stomach of wild boars and raise awareness for their putative intervention in Helicobacter spp. transmission cycle.

OBJECTIVE To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA). SETTINGS AND SAMPLE POPULATION Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants. MATERIALS AND METHODS Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant. RESULTS Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P

Introduction Understanding the physicochemical and biological properties of endodontic sealers is important for endodontic treatment planning. This study evaluated the properties of EndoSequence BC Sealer HiFlow (BCH; Brasseler USA, Savannah, GA), EndoSequence BC Sealer (BC, Brasseler USA), and AH Plus (AHP; Dentsply DeTrey, Konstanz, Germany). The effect of temperature on the setting time and flow of these sealers was also evaluated. Methods The setting time, flow, radiopacity, pH, solubility, and calcium release were investigated following ISO guidelines. The morphology and chemical composition of the sealers were evaluated by scanning electron microscopy and energy-dispersive spectroscopy. The antibacterial activity of sealers was tested against 2 strains of Enterococcus faecalis. Sealer cytotoxicity and the effects on messenger RNA expression of proinflammatory and mineralization genes were also investigated. Data analysis was performed using analysis of variance, Tukey, Kruskal-Wallis, and Dunn multiple comparison tests. P ≤ .05 was considered statistically significant. Results The setting time and flow rate of all sealers were affected by heat (P ≤ .05). The setting times and solubility of BCH and BC were significantly higher than AHP (P ≤ .0001). The radiopacity of AHP was higher than BCH and BC (P ≤ .0001). All sealers were alkaline and had antibacterial effects. Cell viability was higher for BCH and BC than AHP (P ≤ .0001). No significant differences in messenger RNA expression of proinflammatory and mineralization genes were observed. Conclusions Overall, BCH and BC had similar physicochemical and biological properties. The observed high solubility of BCH and BC as well as the high cytotoxicity of AHP might negatively impact the clinical performance of these materials. The application of heat affected the setting time and flow of all sealers.

Crenosoma striatum is a host-specifi c metastrongiloid nematode causing respiratory tract disease in hedgehogs (Erinaceus europaeus). Since few studies have reported C. striatum in hedgehogs and little genetic data is available concerning this lungworm, this study aimed to determine the occurrence of C. striatum in a population sample of hedgehogs from Portugal, additionally providing morphological, histological and molecular data. From 2017 to 2018 a survey of infection was carried out in 11 necropsied hedgehogs. Worms were extracted from fresh lung tissues and microscopically evaluated. Molecular characterization of partial mitochondrial (12S rRNA) and nuclear (18S rRNA) genes was performed. The presence of lungworms in pulmonary tissues of five hedgehogs (45.5%) was detected. Morphological and histopathological analyses evidenced adult forms of nematodes consistent with C. striatum. Molecular characterization of 18S rRNA genes confirmed the classifi cation as C. striatum. Also, novel genetic data characterizing the mitochondrial (12S rRNA) gene of C. striatum is presented. This is the first report of C. striatum infection in hedgehogs of Portugal. The findings here reported provide new insights regarding the geographic distribution and the molecular identification of this lungworm species. P. Barradas (SFRH/BD/116449/2016) and Ana R. Flores (SFRH/BD/116373/2016) acknowledge the Portuguese Foundation for Science and Technology (FCT) for financial support. IPATIMUP integrates the i3S Research Unit, which is partially supported by FEDER through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT, under the project number PEst-C/SAU/LA0003/2013.

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus‐STAT3 bimolecular fluorescence complementation assay that allows the visualization and study of STAT3 dimerization and protein‐protein interactions in living cells. Inactivating mutations on residues susceptible to post‐translational modifications (PTMs) (K49R, K140R, K685R, Y705F and S727A) changed significantly the intracellular distribution of unstimulated STAT3 dimers when the dimers were formed by STAT3 molecules that carried different mutations (ie they were “asymmetric”). Some of these asymmetric dimers changed the proliferation rate of HeLa cells. Our results indicate that asymmetric PTMs on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. We put forward these observations as a working hypothesis, since confirming the existence of asymmetric STAT3 homodimers in nature is extremely difficult, and our own experimental setup has technical limitations that we discuss. However, if our hypothesis is confirmed, its conceptual implications go far beyond STAT3, and could advance our understanding and control of signaling pathways.

Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980-2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0-100% (BZ and ML), 0-17% (LEV) and 0-73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.Importance croissante de la résistance aux anthelminthiques chez les ruminants européens : création et méta-analyse d’une base de données ouverte.Les helminthes sont omniprésents dans les systèmes de production de ruminants au pâturage et sont responsables de coûts et de pertes de production importants. La résistance aux anthelminthiques (RA) des parasites est maintenant répandue dans toute l’Europe, bien qu’il existe encore des lacunes dans nos connaissances dans certaines régions et certains pays. La RA est une menace majeure pour la pérennité de la production moderne de ruminants, en diminuant la productivité, en compromettant la santé et le bien-être des animaux, et en augmentant les émissions de gaz à effet de serre au travers d’une augmentation du parasitisme et des intrants agricoles. Une meilleure compréhension de l’étendue de la RA en Europe est nécessaire pour développer et préconiser des approches de lutte antiparasitaire plus durables. Une base de données intégrant des informations publiées et non publiées en Europe concernant la RA des nématodes gastro-intestinaux (NGI) et de la douve du foie (Fasciola hepatica) a été compilée par les membres de l’action européenne COST « COMBAR » (« Combattre la résistance aux anthelminthiques chez les ruminants ») et combinée avec les données d’une précédente étude systématique concernant la RA des NGI. Au total, 197 publications sur la RA des NGI étaient disponibles pour analyse, représentant 535 études dans 22 pays et couvrant la période 1980–2020. Des signalements de RA étaient présents sur tout le continent européen et certains rapports indiquaient une forte prévalence nationale. Les estimations heuristiques pondérées par la taille de l’échantillon de la prévalence de la RA en Europe sur toute la période d’étude, stratifiées par classe d’anthelminthiques, variaient de 0 à 48 %. La prévalence régionale (nationale) estimée était très hétérogène, variant entre 0 % et 100 % selon le secteur de l’élevage et la classe d’anthelminthique, et augmentait généralement avec les efforts de recherche dans le pays. Dans les quelques pays disposant de données longitudinales adéquates, il y avait une tendance à l’augmentation de la RA au fil du temps pour toutes les classes d’anthelminthiques des NGI : les résultats agrégés chez les ovins et caprins depuis 2010 révèlent une prévalence moyenne de résistance aux benzimidazoles (BZ) de 86 %, aux lactones macrocycliques sauf moxidectine (ML) de 52 %, au lévamisole (LEV) de 48 % et à la moxidectine (MOX) de 21 %. Tous les genres principaux de NGI ont survécu au traitement dans diverses études. Chez les bovins, la prévalence de la RA variait selon les classes d’anthelminthiques de 0 à 100 % (BZ et ML), 0 à 17 % (LEV) et 0 à 73 % (MOX), et Cooperia et Ostertagia ont survécu aux traitements. Une RA suspectée chez F. hepatica a été signalée dans 21 études portant sur 6 pays. Pour les NGI, et encore plus pour F. hepatica, il y avait un biais d’échantillonnage en faveur des exploitations individuelles suspectées de RA, et l’effort de recherche était biaisé vers l’Europe occidentale et en particulier le Royaume-Uni. La capture continue des résultats futurs dans la base de données, en direct, les efforts pour éviter les biais dans le recrutement des exploitations, des tests plus précis pour la RA et une meilleure appréciation de l’importance de la RA parmi l’industrie agricole et les décideurs politiques, soutiendront des analyses plus sophistiquées des facteurs contribuant à la RA, et des stratégies efficaces pour ralentir sa propagation.

OBJECTIVE To investigate the association of single nucleotide variants in the candidate genes WNT10A, WNT10B and GREM2 with isolated tooth agenesis. SETTING AND SAMPLE POPULATION A total of 435 Caucasian individuals (88 cases with isolated tooth agenesis and 347 unrelated controls) were ascertained at the University of Texas Health Science Center at Houston School of Dentistry. Clinical and radiographic examination by orthodontists confirmed the diagnosis of tooth agenesis. Genetic evaluation excluded syndromic forms of tooth agenesis. MATERIALS AND METHODS Saliva samples were collected as source of genomic DNA. Fourteen variants in/nearby WNT10A, WNT10B and GREM2 were genotyped to test for association with tooth agenesis. Genotyping was performed using TaqMan chemistry in a real-time polymerase chain reaction assay. Allelic and haplotype frequencies were compared among cases and controls using chi-square tests as implemented in PLINK v.1.06. Bonferroni correction was used and P ≤ 0.004 indicates statistical differences between groups. RESULTS Significant associations were found between individual SNPs and SNP combinations in WNT10A, WNT10B and GREM2 SNPs with isolated tooth agenesis (P

Tooth agenesis (TA), the failure of development of one or more permanent teeth, is a common craniofacial abnormality observed in different world populations. The genetic etiology of TA is heterogeneous; more than a dozen genes have been associated with isolated or nonsyndromic TA, and more than 80 genes with syndromic forms. In this study, we applied whole exome sequencing (WES) to identify candidate genes contributing to TA in four Turkish families. Likely pathogenic variants with a low allele frequency in the general population were identified in four disease-associated genes, including two distinct variants in TSPEAR, associated with syndromic and isolated TA in one family each; a variant in LAMB3 associated with syndromic TA in one family; and a variant in BCOR plus a disease-associated WNT10A variant in one family with syndromic TA. With the notable exception of WNT10A (Tooth agenesis, selective, 4, MIM #150400), the genotype-phenotype relationships described in the present cohort represent an expansion of the clinical spectrum associated with these genes: TSPEAR (Deafness, autosomal recessive 98, MIM #614861), LAMB3 (Amelogenesis imperfecta, type IA, MIM #104530; Epidermolysis bullosa, junctional, MIMs #226700 and #226650), and BCOR (Microphthalmia, syndromic 2, MIM #300166). We provide evidence supporting the candidacy of these genes with TA, and propose TSPEAR as a novel nonsyndromic TA gene. Our data also suggest potential multilocus genomic variation, or mutational burden, in a single family, involving the BCOR and WNT10A loci, underscoring the complexity of the genotype-phenotype relationship in the common complex trait of TA.

AIM To investigate the effects of combinations of several irrigants on the roughness and wettability of dentine, adhesion of Enterococcus faecalis and Candida albicans and adsorption of chlorhexidine (CHX) to the dentine. METHODOLOGY Bovine dentine samples were prepared and their surface roughness standardized. The samples were distributed in groups (n = 10) and subjected to one of the following irrigation protocols: G1 - saline solution; G2 - sodium hypochlorite (NaOCl); G3 - NaOCl + ethylenediaminetetraacetic acid (EDTA); G4 - NaOCl + peracetic acid (PAA); G5 - NaOCl + 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP); G6 - NaOCl + EDTA + CHX; G7 - NaOCl + PAA + CHX; G8 - NaOCl + HEDP + CHX; and G9 - mixture of NaOCl + HEDP. After treatments, roughness and wettability were measured. In order to evaluate the adhesion of microorganisms to dentine, new dentine samples were prepared and after 2 h of contact with the microorganisms, were analysed using a confocal laser scanning microscope and the number of microorganisms adhering to the surfaces were determined. Absorption spectra were collected by attenuated total reflectance of Fourier-transform infrared spectroscopy before and after immersion of other dentine samples in each solution of G6, G7 and G8 and in a solution of 2% CHX at various time intervals. The areas of the band associated with CHX with the peak at 1492 cm-1 were calculated between 1479 and 1500 cm-1 of the spectral range. The data obtained in all experiments were subjected to one-way ANOVA (α 0.05), whilst EDTA and PAA did (P

Orofacial development is a multifaceted process involving tightly regulated genetic signaling networks, that when perturbed, lead to orofacial abnormalities including cleft lip and/or cleft palate. We and others have shown an association between the cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) gene and nonsyndromic cleft lip with or without cleft palate (NSCLP). Further, we demonstrated that knockdown of Crispld2 in zebrafish alters neural crest cell migration patterns resulting in abnormal jaw and palate development. In this study, we performed RNA profiling in zebrafish embryos and identified 249 differentially expressed genes following knockdown of Crispld2. In silico pathway analysis identified a network of seven genes previously implicated in orofacial development for which differential expression was validated in three of the seven genes (CASP8, FOS, and MMP2). Single nucleotide variant (SNV) genotyping of these three genes revealed significant associations between NSCLP and FOS/rs1046117 (GRCh38 chr14:g.75746690 T > C, p = 0.0005) in our nonHispanic white (NHW) families and MMP2/rs243836 (GRCh38 chr16:g.55534236 G > A; p = 0.002) in our Hispanic families. Nominal association was found between NSCLP and CASP8/rs3769825 (GRCh38 chr2:g.202111380 C > A; p

Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets.Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values.05 were considered statistically significant.Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG.Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis.

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Background and purpose Adiponectin, the most abundant peptide secreted by adipocytes, is involved in the regulation of energy metabolism and vascular physiology. Thus, we investigated the effects of exogenous administration of adiponectin on metabolism, vascular reactivity and perivascular adipose tissue (PVAT) of rat mesenteric arteries in high-fat fed Wistar rats. Experimental approach The effects of adiponectin were investigated on nitric oxide (NO)-dependent and independent vasorelaxation in isolated rat mesenteric arteries from 12-month-old male Wistar rats fed with high-fat diet during 4 months (HFD) and compared with age-matched control Wistar rats (W12m). Adiponectin (2.7 mg) was administered by continuous infusion with a minipump (96 µg/day), implanted subcutaneously, for 28 days before sacrifice. Key results Chronic adiponectin treatment significantly reduced body weight, total cholesterol, free fatty acids, fasting glucose and area under the curve of intraperitoneal glucose tolerance test when compared with HFD rats. It also normalized NO-dependent vasorelaxation incrementing endothelial nitric oxide synthase (eNOS) phosphorylation in mesenteric arteries of HFD rats. In aged (W12m) and HFD rats PVAT significantly increased the expression of chemokines and pro-inflammatory adipokines, the latter of which are important contributors to endothelial dysfunction. Importantly, adiponectin was able to significantly reduce them. Conclusions and implications These results show that adiponectin was able to normalize endothelial function by a mechanism that involves an increment in eNOS phoshorylation and a decrement in PVAT inflammation. Detailed characterization of the adiponectin signaling pathway in the vasculature and perivascular fat is likely to provide novel tools in the management of atherosclerosis and metabolic disease.

Helminth and protozoan infections are responsible for important diseases in grazing sheep, which can be especially threatening in an autochthonous breed at risk of extinction like the Churra Galega Mirandesa Portuguese sheep breed. The aim of the present study was to determine the diversity, prevalence and burden of gastrointestinal parasites in these sheep and to assess the effects of deworming practices, cohabiting animals on the farm and feed management. Coprological qualitative and quantitative analysis (flotation, natural sedimentation and McMaster method) were used to identify and quantify gastrointestinal parasites and a questionnaire was designed and applied. A total of 512 faecal samples were collected from 49 flocks, and 49 replies to the questionnaire were received. Parasites were identified in 100% of the flocks, and in 97% of the samples. The genera or species that have been morphologically identified were: strongyle-type, Nematodirus spp., Skrjabinema spp., Moniezia expansa, Moniezia benedeni, Trichuris spp., Capillaria spp., Eimeria spp., Dicrocoelium spp. and Fasciola hepatica. This is the first report in Portugal of Skrjabinema spp. The burden of parasites’ oocysts and eggs per gram in faecal samples ranged, respectively, from 50 to 17,550 for Eimeria spp., and from 50 to 6250 for strongyle-type eggs. Factors affecting parasitic infections were evaluated using a multivariate logistic regression. Grazing time and a lack of anthelmintic treatment were positively associated with Nematodirus spp. infection. This study showed that there is a high prevalence and diversity of gastrointestinal parasites in the Churra Galega Mirandesa sheep breed.

Signal Transducer and Activator of Transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus-STAT3 bimolecular fluorescence complementation (BiFC) assay that allows the visualization and study of STAT3 dimerization and protein-protein interactions in living cells. Inactivating mutations on residues susceptible to post-translational modifications (K49R, K140R, K685R, Y705F and S727A) did not alter the basal dimerization of unstimulated STAT3, but changed significantly the intracellular distribution of STAT3 dimers. Surprisingly, the distribution of specific asymmetric STAT3 dimers (i.e. the STAT3 molecules carry different mutations) was different from symmetric dimers. Our results indicate that asymmetric post-translational modifications on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. This set of Venus-STAT3 BiFC constructs provides yet unexplored means to advance our understanding of STAT3 behavior and function in cancer and beyond.

Tooth agenesis is the most common developmental abnormality of the human dentition characterized by the congenital absence of one or more permanent teeth. Oligodontia is the term used to describe severe tooth agenesis, where six or more permanent teeth are missing. The WNT gene pathway regulates multiple developmental processes during craniofacial and tooth development, and variations in WNT pathway genes have been reported in individuals with tooth agenesis. In this study, we investigated the association of 37 SNPs in/nearby 12 WNT pathway genes (WNT3, WNT3A, WNT5A, WNT8A, WNT9B, WNT10A, WNT11, AXIN1, AXIN2, APC, LRP5, LRP6) with oligodontia in 22 multiplex families. Genotypes were generated using Taqman chemistry in a real-time polymerase chain reaction assay. Family-based association tests were performed using FBAT. Pairwise-haplotype analysis was also performed. Bonferroni correction was used to adjust for multiple testing and P-values ≤ 0.001 were considered statistically significant. We found nominal association for AXIN2 rs7591, located in the 3’ UTR, with oligodontia (P=0.04). In silico analysis of SNP function predicted a binding site for miR-205 with potential impact on AXIN2 expression. Although modest, these results continue to support a role for AXIN2 in the etiology of familial tooth agenesis.

Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine.

Increased matrix metalloproteinases (MMPs) activity is a hallmark of periapical granulomas. However, the factors underlying the MMPs expression modulation in healthy and diseased periapical tissues remains to be determined. Objective In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. Material and Methods MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p

Introduction Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methods Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. Results All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. Conclusions The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions.

Introduction The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. Methods Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti–glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). Results Treg inhibition by anti–glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. Conclusions Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species. The authors would like to thank the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” for partial financial support to this study.

TBX21-1993T/C (rs4794067) polymorphism increases the transcriptional activity of the Tbx21, essential for interferon gamma (IFNg) transcription, but its functional impact on development Th1- response in vivo remains unclear, as well its potential influence over inflammatory osteolytic conditions, such as periapical lesions. Therefore, this study comprises a case-control and functional investigation of Tbx21 genetic variations impact on Th1 response in vivo and in vitro, and its impact on periapical lesions risk and outcome, performed with a population of healthy controls (H; N = 283) and patients presenting periapical lesions (L; N = 188) or deep caries (DC; N = 152). TBX21-1993T/C genotyping demonstrated that the polymorphic allele C, as well TC/TC+CC genotypes, was significantly less frequent in the L patients compared to H and DC groups. Additionally, gene expression analysis demonstrates that T-cell-specific T-box transcription factor (Tbet) and IFNg transcripts levels were downregulated whereas IL-17 levels were upregulated in the TBX21-1993 C carriers (TC/TC+CC) in comparison with the TT group. Also, while TT and TC+CC genotypes are equally prevalent in the lesions presenting low IFN/IL17 ratio, a significant decrease in polymorphic TC+CC genotypes was observed in lesions presenting intermediate and high IFN/IL17 ratio. In vitro experiments confirmed the predisposition to Th1 polarization associated with TBX21-1993, since PBMC CD4 T cells from T allele carriers produce higher IFNg levels upon CD3/CD28 stimulation than the C group, in both standard/neutral and Th1-polarizing culture conditions. In conclusion, the TBX21-1993 T allele and TC/CC genotypes predispose to Th1-type immune response development in vitro, influence immune response polarization in vivo, and consequently account for the risk for apical periodontitis development.

Despite much progress in understanding the genetics of syndromic tooth agenesis (TA), the causes of the most common, isolated TA remain elusive. Recent studies have identified novel genes and variants contributing to the etiology of TA, and revealed new pathways in which tooth development genes belong. Further, the use of new research approaches including next-generation sequencing has provided increased evidence supporting an oligogenic inheritance model for TA, and may explain the phenotypic variability of the condition. In this review, we present current knowledge about the genetic mechanisms underlying syndromic and isolated TA in humans, and highlight the value of incorporating next-generation sequencing approaches to identify causative and/or modifier genes that contribute to the etiology of TA.

Tooth development is regulated by multiple genetic pathways, which ultimately drive the complex interactions between the oral epithelium and mesenchyme. Disruptions at any time point during this process may lead to failure of tooth development, also known as tooth agenesis (TA). TA is a common craniofacial abnormality in humans and represents the failure to develop one or more permanent teeth. Many genes and potentially subtle variants in these genes contribute to the TA phenotype. We report the clinical and genetic impact of a rare homozygous ANTXR1 variant (c.1312C>T), identified by whole exome sequencing (WES), in a consanguineous Turkish family with tooth agenesis. Mutations in ANTXR1 have been associated with GAPO (growth retardation, alopecia, pseudoanodontia, and optic atrophy) syndrome and infantile hemangioma, however no clinical characteristics associated with these conditions were observed in our study family. We detected the expression of Antxr1 in oral and dental tissues of developing mouse embryos, further supporting a role for this gene in tooth development. Our findings implicate ANTXR1 as a candidate gene for isolated TA, suggest the involvement of specific hypomorphic alleles, and expand the previously known ANTXR1-associated phenotypes.

The chronic inflammatory immune response triggered by the infection of the tooth root canal system results in the local upregulation of RANKL, resulting in periapical bone loss. While RANKL has a well-characterized role in the control of bone homeostasis/pathology, it can play important roles in the regulation of the immune system, although its possible immunoregulatory role in infectious inflammatory osteolytic conditions remains largely unknown. Here, we used a mouse model of infectious inflammatory periapical lesions subjected to continuous or transitory anti-RANKL inhibition, followed by the analysis of lesion outcome and multiple host response parameters. Anti-RANKL administration resulted in arrest of bone loss but interfered in the natural immunoregulation of the lesions observed in the untreated group. RANKL inhibition resulted in an unremitting proinflammatory response, persistent high proinflammatory and effector CD4 response, decreased regulatory T-cell (Treg) migration, and lower levels of Treg-related cytokines IL-10 and TGFb. Anti-RANKL blockade impaired the immunoregulatory process only in early disease stages, while the late administration of anti-RANKL did not interfere with the stablished immunoregulation. The impaired immunoregulation due to RANKL inhibition is characterized by increased delayed-type hypersensitivity in vivo and T-cell proliferation in vitro to the infecting bacteria, which mimic the effects of Treg inhibition, reinforcing a possible influence of RANKL on Treg-mediated suppressive response. The adoptive transfer of CD4+FOXp3+ Tregs to mice receiving anti-RANKL therapy restored the immunoregulatory capacity, attenuating the inflammatory response in the lesions, reestablishing normal T-cell response in vivo and in vitro, and preventing lesion relapse upon anti-RANKL therapy cessation. Therefore, while RANKL inhibition efficiently limited the periapical bone loss, it promoted an unremitting host inflammatory response by interfering with Treg activity, suggesting that this classic osteoclastogenic mediator plays a role in immunoregulation.

Previously reported co-occurrence of colorectal cancer (CRC) and tooth agenesis (TA) and the overlap in disease-associated gene variants suggest involvement of similar molecular pathways. Here, we took an unbiased approach and tested genome-wide significant CRC-associated variants for association with isolated TA. Thirty single nucleotide variants (SNVs) in CRC-predisposing genes/loci were genotyped in a discovery dataset composed of 440 individuals with and without isolated TA. Genome-wide significant associations were found between TA and ATF1 rs11169552 (P = 4.36 × 10−10) and DUSP10 rs6687758 (P = 1.25 × 10−9), and positive association found with CASC8 rs10505477 (P = 8.2 × 10−5). Additional CRC marker haplotypes were also significantly associated with TA. Genotyping an independent dataset consisting of 52 cases with TA and 427 controls confirmed the association with CASC8. Atf1 and Dusp10 expression was detected in the mouse developing teeth from early bud stages to the formation of the complete tooth, suggesting a potential role for these genes and their encoded proteins in tooth development. While their individual contributions in tooth development remain to be elucidated, these genes may be considered candidates to be tested in additional populations.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

Objective Nonsyndromic cleft lip with or without cleft palate (NSCL±P) is a common craniofacial anomaly of complex etiology in people. WNT pathway genes have important roles during craniofacial development, and an association of WNT genes with NSCL±P has been demonstrated in different populations. The aim of this study was to evaluate the association between polymorphisms in WNT3 and WNT9B genes and CL/P in Brazilian families. Patients Seventy nuclear families composed of an affected child and the child's unaffected parents were examined clinically. Saliva samples were collected for molecular analyses. Design Three single nucleotide polymorphisms (SNPs) in the WNT3 gene and two in WNT9B were investigated in real-time polymerase chain reaction using TaqMan chemistry. The Family-Based Association Test and the transmission disequilibrium test were used to verify the association between each marker allele and NSCL±P. The level of significance was established at P ≤ .01 after Bonferroni correction. Results A positive association was detected between NSCL±P and SNP rs1530364 in the WNT9B gene. Haplotype analysis showed an association of WNT3 and WNT9B haplotypes. No association was detected between NSCL±P and individual SNPs in WNT3. Conclusion Our study further supports the involvement of WNT9B as a cleft susceptibility gene in Brazilian families experiencing NSCL±P. Although additional studies are still necessary to unveil the exact mechanism by which WNT genes would contribute to NSCL±P, allelic polymorphisms in these genes and their interactions may partly explain the variance of individual susceptibility to NSCL±P.

Previously reported co-occurrence of colorectal cancer (CRC) and tooth agenesis (TA) and the overlap in disease-associated gene variants suggest involvement of similar molecular pathways. In this study, we took an unbiased approach and tested genome-wide significant CRC-associated variants for association with isolated TA. Thirty single nucleotide variants (SNVs) in CRC-predisposing genes/loci were genotyped in a discovery dataset composed of 440 individuals with and without isolated TA. Genome-wide significant associations were found between TA and DUSP10 rs6687758 (P=1.25 × 10−9) and ATF1 rs11169552 (P=4.36 × 10−10), with strong association found with CASC8 rs10505477 (P=8.2 × 10−5). Additional CRC marker haplotypes were also significantly associated with TA (PCASC8.Atf1 and Dusp10 expression was detected in the mouse developing teeth from early bud stages to the formation of the complete tooth, suggesting a potential role for these genes and their encoded proteins in tooth development. Our findings suggest Atf1 and Dusp10 as new tooth development genes, while having a role in colorectal cancer. While their individual contributions in tooth development remain to be elucidated, these genes may be considered additional candidates to be tested in future human genetic studies.

Background Oligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition. Methods We applied whole-exome sequencing (WES) to identify the cause of oligodontia in a 9-year-old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype. Results We identified a novel compound heterozygous missense mutation in WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression. Conclusions Our results reveal a novel compound heterozygous variant in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.

Food intake and energy expenditure are closely regulated by several mechanisms which involve peripheral organs and nervous system, in order to maintain energy homeostasis.

Dogs play many roles and their presence within people’s houses has increased. In rural settings dog faeces are not removed from the streets, representing an environmental pollution factor. Our aim was to evaluate the occurrence of environmental contamination with zoonotic intestinal parasites of three groups of dogs in Ponte de Lima, Portugal, with a particular emphasis on Echinococcus granulosus. We collected 592 dog faecal samples from the environment, farm and hunting dogs. Qualitative flotation coprological analysis was performed and the frequency in the positive samples ranged between 57.44% and 81.19% in different groups. We isolated up to four different parasites in one sample and detected seven intestinal parasitic species, genera or families overall. Ancylostomatidae was the most prevalent parasite, followed by Trichuris spp., Toxocara spp., Isospora spp., Dipylidium caninum, Taeniidae and Toxascaris leonina. Taeniidae eggs were analyzed with the PCR technique and revealed not to be from Echinococcus. The parasite prevalence and the diversity of zoonotic parasites found were high, which calls for a greater awareness of the problem among the population, especially hunters. Promoting research at the local level is important to plan control strategies. Health education should be developed with regard to farmers and hunters, and a closer collaboration between researchers, practitioners and public health authorities is needed.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect of complex etiology. Several genes have been implicated in the etiology of NSCL/P, although only a few have been replicated across datasets. ARHGAP29 was suggested as a candidate gene for NSCL/P as it is located in close proximity to ABCA4 (1p22), a gene previously identified in a GWAS of NSCL/P. Rare, potentially damaging, coding variants in ARHGAP29 were found in NSCL/P cases, and its expression was detected during murine craniofacial development. In this study, we investigated whether variations in ARHGAP29 were associated with NSCL/P in our family based dataset. Five SNPs flanking and within ARHGAP29 were genotyped in our NSCL/P datasets consisting of simplex and multiplex families of non Hispanic white (NHW, primarily European) and Hispanic ethnicities. Results showed strong association of three ARHGAP29 SNPs with NSCL/P in the NHW families. Two intronic SNPs (rs1541098 and rs3789688) showed strong association with NSCL/P in all NHW families (P=0.0005 and P=0.0002, respectively), and simplex NHW families (P=0.003 for both SNPs). A SNP in the 3’ UTR (rs1576593) also showed strong association with NSCL/P in all NHW families (P=0.002), and the multiplex subset (P=0.002). ARHGAP29 SNP haplotypes were also associated with NSCL/P. Evidence of gene-gene interaction was found between ARHGAP29 and additional cleft susceptibility genes. This study further supports ARHGAP29 as a candidate gene for human NSCL/P in families of Caucasian descent.

Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions.We analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry.The expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P.00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P.005). Higher concentrations of LPS led to increased HSP expression in macrophages (P.0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages.The observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide additional insights into the process of periapical lesion development.

Evidence from biological and human studies strongly supports a role for MMP and TIMP genes as candidate genes for non-syndromic cleft lip with or without cleft palate (NSCL/P). We previously showed the association of promoter polymorphisms in MMP3 (rs3025058 and rs522616) and TIMP2 (rs8179096) with NSCL/P. In this study, we examined the functional significance of these polymorphisms. A specific DNA-protein complex for MMP3 rs522616 A was detected, and this allele by itself showed greater promoter activity than the G allele. However, the effect of rs522616 was ultimately regulated by the rs3025058 allele on the background. For TIMP2 rs8179096, the T allele showed a 2.5-fold increase in promoter activity when compared with allele C, whereas both C and T alleles were found to bind to nuclear factor kappa B. Our results provide new evidence that promoter polymorphisms in MMP3 and TIMP2 are functional and may affect gene transcription with possible effects on craniofacial development leading to NSCL/P.

In recent years, the knowledge generated by decoding the human genome has allowed groundbreaking genetic research to better understand genomic architecture and heritability in healthy and disease states. The vast amount of data generated over time and yet to be generated provides the basis for translational research towards the development of preventive and therapeutic strategies for many conditions. In this special issue, we highlight the discoveries of disease-associated and protective DNA variations in common human diseases and developmental disorders.

Single nucleotide polymorphisms are genome variations that can be used as population-specific markers to infer genetic background and population origin. The Brazilian population is highly admixed due to immigration from several other populations. In particu- lar, the state of Sao Paulo is recognized for the presence of Japanese individuals who seem likely to have contributed to a substantial proportion of ancestry in the modern Brazilian population. In the present study, we analyzed allele and genotype frequencies and associations of the -418G.C (rs8179090) single nucleotide polymorphism in the TIMP2 gene promoter in Brazilian and Japanese subjects, as well as in Japanese descendants from southeastern Brazil. The allele and genotype frequency analyses among groups demonstrated statistical significance ( P,0.001) and the rare C allele was not found in 200 unrelated Brazilian subjects, while its distribution in Japanese individuals and Japanese descendants was 18.7% and 10.3%, respectively. Individuals with the C allele were about 70 times more likely to be Japanese and 41 times more likely to be Japanese descendants compared with Brazilian individuals (odds ratio 69.09, 95% confidence interval 9.35-510 for Japanese; and odds ratio 41.38, 95% confi - dence interval 4.80-356.15 for Japanese descendants). Taken together, these findings suggest that subjects carrying the C allele, located in the -418G.C single nucleotide polymorphism of the TIMP2 gene, have a high probability of being Japanese or Japanese descendants. In addition to other genetic polymorphisms, the -418G.C TIMP2 polymorphism could be a population marker to assist in predicting Japanese ancestry, both in Japanese individuals and in admixed populations.

Heat shock proteins (HSPs) protect cells under adverse conditions such as infection, inflammation, and disease. The differential expression of HSPs in human periapical granulomas suggests a potential role for these proteins in periapical lesion development, which may contribute to different clinical outcomes. Therefore, we hypothesized that polymorphisms in HSP genes leading to perturbed gene expression and protein function may contribute to an individual's susceptibility to periapical lesion development.Subjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Texas School of Dentistry at Houston and at the University of Pittsburgh. Genomic DNA samples of 400 patients were sorted into 2 groups: 183 cases with deep carious lesions and periapical lesions (cases) and 217 cases with deep carious lesions but without periapical lesions (controls). Eight single nucleotide polymorphisms (SNPs) in HSPA4, HSPA6, HSPA1L, HSPA4L, and HSPA9 genes were selected for genotyping. Genotypes were generated by end point analysis by using Taqman chemistry in a real-time polymerase chain reaction assay. Allele and genotype frequencies were compared among cases and controls by using χ(2) and Fisher exact tests as implemented in PLINK v.1.07. In silico analysis of SNP function was performed by using Polymorphism Phenotyping V2 and MirSNP software.Overall, SNPs in HSPA1L and HSPA6 showed significant allelic association with cases of deep caries and periapical lesions (P .05). We also observed altered transmission of HSPA1L SNP haplotypes (P = .03). In silico analysis of HSPA1L rs2075800 function showed that this SNP results in a glutamine-to-lysine substitution at position 602 of the protein and might affect the stability and function of the final protein.Variations in HSPA1L and HSPA6 may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing apical periodontitis.

Craniofacial anomalies, and in particular oral-facial clefts, are major human birth defects with a worldwide frequency of 1 in 700 live births and substantial clinical impact. The possible etiologies are many, including single-gene disorders, chromosome aberrations, exposure to teratogens, and sporadic conditions of unknown cause (1). Oral-facial clefts can be further classified as nonsyndromic (isolated) or syndromic based on the presence of other structural anomalies. Approximately 30% of all clefts are associated with one of more than 400 described syndromes (2) while the remaining 70% are isolated defects. It is generally accepted that cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) are genetically distinct phenotypes. CL/P is more common, affecting 1–2/1000 births and presenting considerable differences in prevalence, with Native Americans and Asians showing the highest rate and Africans the lowest. On the other hand, CPO is less common, with a prevalence of approximately 1/1500–2000 births in Caucasians, less variable among different ethnic backgrounds (3). These observations suggest that the relative contribution of individual susceptibility genes may vary across different populations, thus reinforcing the need of replication of association studies in different populations. Further, numerous lines of evidence now suggest that the phenotypic spectrum of nonsyndromic CL/P is more complex than previously realized and therefore genetic studies should include a more accurate description of the cleft phenotype, such as cleft type and laterality, as well as presence of subclinical phenotypes, such as defects in the orbicularis oris muscle and dental anomalies (4,5). Several loci and genes - including, but not limited to, MSX1, IRF6, CRISPLD2, FOXE1, AXIN2 , and members of the WNT, FGF, and MMP gene families - have been associated with oral clefts (6–17). Additionally, recent advances in research methodologies have accelerated the discovery of loci conferring susceptibility to isolated CL/P through the use of genome-wide association studies (GWAS). The first three GWAS found strong evidence for association of an intergenic marker (rs987525) in the 8q24 chromosomal region with CL/P (18–20), and this association has been independently validated in additional populations, including a population from Brazil (21). Recently, a third GWAS identified associations with markers in/nearby ABCA4 and MAFB genes, located on chromosomes 1p22.1 and 20q11.1-q13.1, respectively, with CL/P in multiple populations (22). In two subsequent studies, the originally associated SNP in ABCA4 (rs540026) was associated with increased risk of CL/P in US and South American populations (23, 24) whereas a SNP in MAFB (rs13041247) was associated with increased risk of CL/P in Chinese (25). Intriguingly, a study with a Nigerian population did not find evidence of association for either ABCA4 or MAFB genes with CL/P (26). Due to allelic heterogeneity among populations, in order to validate the findings of genetic association studies, it is necessary to independently attempt to replicate these findings in multiple populations. Hence, to further investigate a possible role for ABCA4 and MAFB in the susceptibility to CL/P in a population from Brazil, we tested the previously associated SNPs in ABC4 (rs560426 and rs481931), and MAFB (rs13041247 and rs11696257) genes (Table 1) for association with CL/P in our case-control dataset. Table 1 Details of the SNPs investigated in this study.

BACKGROUND Oral clefts are common congenital anomalies and result from defects during embryogenesis. The complex etiology is evident by the number of genes and signaling pathways involved in craniofacial development. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for tissue remodeling during craniofacial development. METHODS In this study, we investigated the association of polymorphisms in 14 biologically relevant MMP and TIMP genes in 494 individuals with oral clefts and 413 control individuals from Brazil. Genotypes were generated using Taqman chemistry. Analyses were performed using PLINK software. RESULTS Polymorphisms in MMP3 (rs522616) and TIMP2 (rs8179096) showed significant association with all cleft types (all clefts, cleft lip/palate, and cleft palate; p ≤ 0.002). An additional family-based dataset (881 case-parent trios) from the United States was used for confirmation of the association findings (p < 0.05). Analysis of gene-gene interaction suggests that MMP3 and TIMP2 may interactively contribute to a cleft phenotype. CONCLUSIONS This study provides new evidence that variation in MMP3 may contribute to nonsyndromic oral clefts and further supports the involvement of TIMP2 as a cleft susceptibility gene. Although additional studies are still necessary to unveil the exact mechanism by which MMP3 and TIMP2 would contribute to a cleft phenotype, allelic polymorphisms in these genes and their interactions may partly explain the variance of individual susceptibility to oral clefts. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

Matrix metalloproteinases (MMPs) play a key role in the tissue destruction characteristic of chronic periodontitis. The purpose of this study was to investigate the association of MMP and TIMP polymorphisms with chronic periodontitis in two populations.A total of 34 polymorphisms spanning 12 MMP and 2 TIMP genes were genotyped in 401 individuals from Brazil (99 cases with chronic periodontitis and 302 controls), and 274 individuals from the US (70 cases and 204 controls). Individuals were considered cases if presenting at least three teeth exhibiting sites of clinical attachment loss ≥ 5 mm in two different quadrants. Controls were characterized by absence of clinical attachment loss and no sites with probing depth3 mm. MMP3 and TIMP1 mRNA expression was evaluated in healthy and diseased periodontal tissues.TIMP1 showed association with chronic periodontitis in the Brazilian population (for rs5906435, p = 0.0004), whereas MMP3 showed association in the US population (for rs679620, p = 0.0003; and rs650108, p = 0.002) and in the Brazilian population (for rs639752, p = 0.005). MMP3 and TIMP1 mRNA expression was significantly higher in diseased tissues when compared to control tissues.Our results further support a role for variations in MMP3 in chronic periodontitis and report a novel association with TIMP1. These genes may be considered additional candidate genes for chronic periodontitis.

Background: The proinflammatory chemokine interleukin (IL)-8 is important in the regulation of the inflammatory response. Analyses of the single nucleotide polymorphism (SNP) reference sequence (rs) 4073 showed that the A allele upregulated IL-8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis. Methods: Genotyping was performed by a standard polymerase chain reaction–restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control subjects and patients with chronic periodontitis; analyses were adjusted by multivariate logistic regression modeling. A real-time polymerase chain reaction performance was used to detect levels of the IL-8 mRNA. Results: The analysis pointed to a statistically significant association of chronic periodontitis with the heterozygous TA genotype (P = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control group versus 48% in the periodontitis group). The higher levels of the IL-8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (P = 0.03). Conclusion: The SNP rs4073 was associated with chronic periodontitis in non-smoker Brazilian subjects because the frequency of the A allele was higher in the disease group than in the control group, and the TA genotype was associated with increased levels of IL-8 mRNA transcripts. J Periodontol 2011;82:893-899.