Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

Conditional expression of NanoLuc luciferase through a multimodular system offers rapid detection of antimalarial drug activity.

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.

Effect of periodontal therapy on insulin resistance in adults with dysglycemia and periodontitis: a systematic review and meta-analysis.

OBJECTIVES: This systematic review and meta-analysis aimed to address whether non-surgical periodontal therapy (NSPT) can affect insulin resistance, estimated by the homeostasis model assessment (HOMA), in adults with prediabetes or type 2 diabetes mellitus and periodontitis. MATERIALS AND METHODS: Six electronic databases and the gray literature were systematically searched for interventional studies reporting NSPT effect on insulin resistance. Seven studies met the eligibility criteria to be synthesized in the qualitative analysis, six reporting change in HOMA-IR, three reporting change in HOMA-%S, and two in HOMA-ß. Among them, four were pooled in a meta-analysis of standardized mean difference (SMD) of HOMA-IR; comparing pre- and post-intervention values, three were pooled considering HOMA-%S as outcome, and two studies were summarized considering SMD of HOMA-%S between intervention and control groups. HOMA-ß results were qualitatively synthetized. RESULTS: With low level of certainty, NSPT significantly reduced HOMA-IR, when compared with pre-intervention data (SMD, -0.35, 95% CI -0.63 to 0.07, p=0.02). There were no significant changes in HOMA-%S or in HOMA-ß scores. The level of certainty was very low and moderate, respectively. CONCLUSIONS: Assertions about a causal link between NSPT and insulin resistance are weak and conflicting, although our more robust results point out to the absence of effect. . CLINICAL RELEVANCE: Because further high-quality studies assessing the relationship between periodontitis and insulin resistance are need, the findings of the current systematic review are limited to give recommendations for clinicians. However, while identifying a lack of research in humans with T2D concerning periodontitis and insulin resistance, this study reinforces the need of multicenter well-designed randomized clinical trials.

New perspectives in a microstructure study of the bovine and bubaline claw.

The techniques of microtomography (Micro-CT), confocal laser scanner microscopy (CLSM), atomic force microscopy (AFM), nanoindentation - Vickers hardness (Nano-VH) and X-ray fluorescence (XRF) are undeniably important to the modern study of bovine podiatry. These techniques are also employed in engineering, physics and in the assessment of biomaterials used in reconstructive or experimental surgeries in bovine and bubaline claws. Although studies involving these analyses are still inconspicuous in veterinary medicine, these technologies represent a new paradigm in this area, enabling the development of new lines of research. The objective of this review is to gather information about the microstructural aspects of bovine and bubaline claws, concerning the intratubular and extratubular keratin, which is responsible for the physical and mechanical structure of the claw capsule. This study elucidates different methods used to evaluate the hooves of healthy and sick animals through a micrometric analysis and nano-scale analyses. We would like to emphasise that the described techniques can be applied to study other species.

Flavonoids from Pterogyne nitens as Zika virus NS2B-NS3 protease inhibitors.

Although the widespread epidemic of Zika virus (ZIKV) and its neurological complications are well-known there are still no approved drugs available to treat this arboviral disease or vaccine to prevent the infection. Flavonoids from Pterogyne nitens have already demonstrated anti-flavivirus activity, although their target is unknown. In this study, we virtually screened an in-house database of 150 natural and semi-synthetic compounds against ZIKV NS2B-NS3 protease (NS2B-NS3p) using docking-based virtual screening, as part of the OpenZika project. As a result, we prioritized three flavonoids from P. nitens, quercetin, rutin and pedalitin, for experimental evaluation. We also used machine learning models, built with Assay Central® software, for predicting the activity and toxicity of these flavonoids. Biophysical and enzymatic assays generally agreed with the in silico predictions, confirming that the flavonoids inhibited ZIKV protease. The most promising hit, pedalitin, inhibited ZIKV NS2B-NS3p with an IC50 of 5 µM. In cell-based assays, pedalitin displayed significant activity at 250 and 500 µM, with slight toxicity in Vero cells. The results presented here demonstrate the potential of pedalitin as a candidate for hit-to-lead (H2L) optimization studies towards the discovery of antiviral drug candidates to treat ZIKV infections.

Clinical efficacy of subgingivally delivered propolis as an adjuvant to nonsurgical periodontal treatment of periodontitis: A systematic review and meta-analysis.

This systematic review and meta-analysis evaluated randomized and nonrandomized studies that assessed the effect of local subgingival propolis as an adjunct to nonsurgical periodontal therapy (NSPT) in the treatment of periodontitis. A detailed search was carried out in Cochrane Library, Embase, LILACS, LIVIVO, PubMed, Scopus, and Web of Science, with no time or language restrictions. A grey literature search was also conducted. The methodology of included studies was evaluated by the Cochrane RoB2 tool. The certainty of each clinical outcome was assessed by the GRADE system. Meta-analyses of mean difference were conducted using the random-effects model, through RevMan 5.4 software. Six studies met the eligibility criteria to be synthesized in the qualitative analysis, and three studies were included in the meta-analysis. The subgingival application of propolis as an adjunct to NSPT improved probing pocket depth (PPD), clinical attachment level (CAL), and bleeding on probing (BOP) in most of the assessed studies. The overall mean difference in PPD reduction was 1.49 mm, 30-45 days after treatment, and 0.8 mm, 90 days after treatment, with very low level of certainty. The overall bias was scored as high risk for all included studies. The adjunctive use of locally delivered propolis associated to NSPT to treat periodontitis may improve periodontal clinical parameters, compared with NSPT alone/placebo. However, the evidence was not strong enough to safely base any clinical recommendation.

Salivary metabolites to detect patients with cancer: a systematic review.

Novel adjunctive screening aids are needed to reduce the morbidity and mortality related to cancer, and every effort should be made for early diagnosis. This systematic review aimed to evaluate salivary metabolites and their diagnostic value in patients with cancer.The systematic review was performed in two phases and included studies that focused on the diagnostic value of salivary metabolites in humans with solid malignant neoplasms. Five electronic databases were searched, and the risk of bias in individual studies was evaluated using the revised Quality Assessment of Diagnostic Accuracy Studies criteria (QUADAS-2). All procedures were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Of the 1151 studies retrieved, 25 were included; 13 studies used targeted and 12 untargeted metabolomics approaches. Most studies included patients with breast and oral cancer. Except for one, all studies had case-control designs, and none fulfilled all quality assessments. Overall, 140 salivary metabolites were described. The most frequently reported metabolites were alanine, valine, and leucine. Among the 11 studies that reported diagnostic test accuracy (DTA) values, proline, threonine, and histidine in combination and monoacylglycerol alone demonstrated the highest DTA for breast cancer. Combined choline, betaine, pipecolinic acid, and L-carnitine showed better discriminatory performance for early oral cancer.This systematic review highlights the current evidence on salivary metabolites that may be used as a future strategy to diagnose cancer. Further studies including larger sample sizes with confirmation of the results by untargeted analysis are warranted.

Curcumin downregulates the PI3K-AKT-mTOR pathway and inhibits growth and progression in head and neck cancer cells.

Curcumin, a polyphenol isolated from the rhizome of Curcuma longa, has been studied because of its antioxidant, antimicrobial, and antiinflammatory properties. This study aimed to evaluate the effects of curcumin on head and neck cancer (HNC) cell lines and how it modulates the PI3K-AKT-mTOR signaling pathway. Dose-response curves for curcumin were established for hypopharynx carcinoma (FaDu), tongue carcinoma (SCC-9), and keratinocytes (HaCaT) cell lines and IC50 values were calculated. Cell cycle and cell death were investigated through flow cytometry. Cytoskeleton organization was assessed through phalloidin+FITC staining. qPCR array and western blot were performed to analyze gene and protein expression. Curcumin reduced cell viability in a dose-dependent and selective manner, induced cell death on SCC-9 cells (necrosis/late apoptosis: 44% curcumin vs. 16.4% vehicle), and arrested cell cycle at phase G2 /M on SCC-9 and FaDu (G2 : SCC-9-19.1% curcumin vs. 13.4% vehicle; FaDu-37.8% curcumin vs. 12.9% vehicle). Disorganized cytoskeleton and altered cell morphology were observed. Furthermore, curcumin downregulated the PI3K-AKT-mTOR signaling pathway by modifying the expression of key genes and proteins. These findings highlight the promising therapeutic potential of curcumin to inhibit HNC growth and progression and to modulate the PI3K-AKT-mTOR pathway.

Development of natural polymeric microcapsules for antimicrobial drug delivery: triclosan loaded chitosan and alginate-based microcapsules.

The goal of this work was the development of natural polymeric microcapsules for antimicrobial drug delivery - triclosan loaded alginate and chitosan-based microcapsules for potential coating applications in substrates such as textiles or plastics. Microcapsules containing 2.5% (w/w) or 3% (w/w) triclosan in both core and matrix were synthesized and evaluated by Fourier-transform infrared spectroscopy, scanning electron microscopy, confocal microscopy, differential scanning calorimetry, thermogravimetry, and antimicrobial activity. The microcapsules produced featured spherical and mostly irregularly-shaped surfaces composed by an alginate core in a chitosan outer matrix, as revealed by confocal microscopy, and antimicrobial activity against S. aureus and E. coli with inhibition halos up to 60 mm and 25 mm respectively, granted by a triclosan loading of 61.66%. The thermal analysis suggested that the polymers protected the active substance from temperature-induced degradation. In conclusion, these microcapsules may be applied toward antimicrobial functionalization of plastics, textiles and other materials.

Effects of turmeric and curcumin on oral mucositis: A systematic review.

The aim of this study was to evaluate the effects of turmeric and curcumin in the management of oral mucositis in cancer patients undergoing chemo and/or radiotherapy. The systematic review was reported according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses. The search was performed in the following database: Cochrane Library, LILACS, LIVIVO, PubMed, Scopus, and Web of Science. A gray literature search was undertaken using Google Scholar, Open Grey, and ProQuest. The methodology of included studies was evaluated by the Meta-Analysis of Statistics Assessment and Review Instrument. After a two-step selection process, four randomized and one nonrandomized clinical trials were included in the analysis. Two studies were categorized as low and three as moderate risk of bias. Turmeric/curcumin was applied topically as a gel or as a mouthwash. Patients treated with turmeric/curcumin experienced reduced grade of mucositis, pain, erythema intensity, and ulcerative area. Current evidence suggests that topical application of turmeric or curcumin is effective in controlling signs and symptoms of oral mucositis. Thus, further investigation is required to confirm the promising effect of turmeric and curcumin in oral inflammatory lesions.

Surface ozone climatology of South Eastern Brazil and the impact of biomass burning events.

In the austral spring, biomass fires affect a vast area of South America each year. We combined in situ ozone (O3) data, measured in the states of São Paulo and Paraná, Brazil, in the period 2014-2017, with aerosol optical depth, co-pollutants (NOx, PM2.5 and PM10) and air backtrajectories to identify sources, transport and geographical patterns in the air pollution data. We applied cluster analysis to hourly O3 data and split the investigation area of approximately 290,000 km2 into five groups with similar features in terms of diurnal, weekly, monthly and seasonal O3 concentrations. All groups presented a peak in September and October, associated with the fire activities and enhanced photochemistry. The highest mean O3 concentrations were measured inland whilst, besides having lower concentrations, the coastal group was also associated with the smallest diurnal and seasonal variations. The latter was attributed to lower photochemical activity due to frequently occurring overcast weather situation. The mean annual regional contribution of O3 over the area was 61 µg/m3, with large seasonal and intersite variabilities (from 35 to 84 µg/m3). The long-range transport of smoke contributed with between 23 and 41% of the total O3 during the pollution events. A pollution outbreak in September 2015 caused many-fold increases in O3, PM2.5 and PM10 across the investigation area, which exceeded the World Health Organisation recommendations. We show that the regional transport of particulates and gas due to biomass burning overlays the local emissions in already highly polluted cities. Such an effect can outweigh local measures to curb anthropogenic air pollution in cities.

Heterocycle Thiazole Compounds Exhibit Antifungal Activity through Increase in the Production of Reactive Oxygen Species in the Cryptococcus neoformans-Cryptococcus gattii Species Complex.

Human cryptococcosis can occur as a primary or opportunistic infection and develops as an acute, subacute, or chronic systemic infection involving different organs of the host. Given the limited therapeutic options and the occasional resistance to fluconazole, there is a need to develop novel drugs for the treatment of cryptococcosis. In this report, we describe promising thiazole compounds 1, 2, 3, and 4 and explore their possible modes of action against Cryptococcus To this end, we show evidence of interference in the Cryptococcus antioxidant system. The tested compounds exhibited MICs ranging from 0.25 to 2 µg/ml against Cryptococcus neoformans strains H99 and KN99α. Interestingly, the knockout strains for Cu oxidase and sarcosine oxidase were resistant to thiazoles. MIC values of thiazole compounds 1, 2, and 4 against these mutants were higher than for the parental strain. After the treatment of C. neoformans ATCC 24067 (or C. deneoformans) and C. gattii strain L27/01 (or C. deuterogattii) with thiazoles, we verified an increase in intracellular reactive oxygen species (ROS). Also, we verified the synergistic interactions among thiazoles and menadione, which generates superoxides, with fractional inhibitory concentrations (FICs) equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazole compounds 1, 2, 3, and 4, respectively. In addition, thiazoles exhibited antagonistic interactions with parasulphonatephenyl porphyrinato ferrate III (FeTPPS). Thus, in this work, we showed that the action of these thiazoles is related to an interference with the antioxidant system. These findings suggest that oxidative stress may be primarily related to the accumulation of superoxide radicals.

Antifungal and antioxidant activity of fatty acid methyl esters from vegetable oils.

Fatty acid methyl esters (FAMEs) were obtained from vegetable oils of soybean, corn and sunflower. The current study was focused on evaluating the antifungal activity of FAMEs mainly against Paracoccidioides spp., as well as testing the interaction of these compounds with commercial antifungal drugs and also their antioxidant potential. FAMEs presented small IC50 values (1.86-9.42 µg/mL). All three FAMEs tested showed antifungal activity against isolates of Paracoccidioides spp. with MIC values ranging from 15.6-500 µg/mL. Sunflower FAMEs exhibited antifungal activity that extended also to other genera, with an MIC of 15.6 µg/mL against Candida glabrata and C. krusei and 31.2 µg/mL against C. parapsilosis. FAMEs exhibited a synergetic effect with itraconazole. The antifungal activity of the FAMEs against isolates of Paracoccidioides spp. is likely due to the presence of methyl linoleate, the major compound present in all three FAMEs. The results obtained indicate the potential of FAMEs as sources for antifungal and antioxidant activity.

Host-derived salivary biomarkers in diagnosing periodontal disease: systematic review and meta-analysis.

AIM: To systematically evaluate the accuracy of host-derived salivary biomarkers in the diagnosis of periodontal disease based on the given sensitivity and specificity information. MATERIALS AND METHODS: Studies were eligible for inclusion if they had compared the diagnostic application of salivary biomarkers with clinical examination of periodontal disease. A detailed search was performed in five databases without restrictions on subject age, chronology, or language. Additionally, a partial grey-literature search was conducted. The revised Quality Assessment of Diagnostic Accuracy Studies tool and Meta-analysis were used to evaluate the selected studies. RESULTS: From the 905 screened studies, four were included in the qualitative and quantitative analysis. One biomarker, macrophage inflammatory protein-1α (MIP-1α), had excellent diagnostic accuracy and two, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), showed acceptable diagnostic values. However, the only biomarker considered excellent was evaluated in a single study, which may reduce the robustness of the results. CONCLUSION: There is currently limited evidence to confirm the diagnostic capability of salivary biomarkers in the clinical assessment of periodontal disease. Notwithstanding, the summary findings showed the growing importance of salivary biomarker, and can guide larger, well-controlled, diagnostic accuracy studies. Likewise, although not conclusive, MIP-1α, IL-1ß, and IL-6 may be promising biomarkers for future studies.

Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations.

BACKGROUND: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. METHODS: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. RESULTS: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. CONCLUSIONS: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations.

Acute kidney injury after snakebite accident treated in a Brazilian tertiary care centre.

AIM: Acute kidney injury (AKI) is one of the main causes of morbidity and mortality in cases of envenomation by venomous snakes. The present study was carried out to investigate the clinical and laboratory manifestations in accidents with venomous snakes and the risk factors associated with AKI in these accidents. METHODS: A retrospective study was carried out with patients victims of snakebite admitted to a reference centre. AKI was defined according to the RIFLE and AKIN criteria. RESULTS: A total of 276 patients were included, of which 230 (83.7%) were males. AKI was observed in 42 cases (15.2%). The mean genus involved in the accidents was Bothrops (82.2%). Mean age of patients with AKI was higher than in patients without AKI (43 ± 20 vs. 34 ± 21 years, P = 0.015). The time elapsed between the accident and medical care was higher in the AKI group (25 ± 28 vs. 14 ± 16h, P = 0.034), as well as the time elapsed between the accident and the administration of antivenom (30.7 ± 27 vs. 15 ± 16 h, P = 0.01). Haemodialysis was required in 30% of cases and complete renal function recovery was observed in 54.8% of cases at hospital discharge. There were four deaths, none of which had AKI. Factors associated with AKI were haemorrhagic abnormalities (P = 0.036, OR = 6.718, 95% CI: 1.067-25.661) and longer length of hospital stay (P = 0.004, OR = 1.69, 95% CI 1.165-2.088). CONCLUSION: Acute kidney injury is an important complication of snakebite accidents, showing low mortality, but high morbidity, which can lead to partial renal function recovery.

Impairment Assessment in Adult ADHD and Related Disorders: Current Opinions From Clinic and Research.

OBJECTIVES: Assessing functional impairment is one of the essential components in the clinical evaluation of ADHD in adulthood, serving both diagnostic and outcome evaluation purposes. However, clinicians and researchers may face challenges in selecting suitable instruments due to variations in accessibility and quality of instruments. METHODS: We conducted an online survey involving an international group of 92 respondents engaged in clinical practice and/or research on ADHD. The survey aimed to evaluate current practices in assessing impairment in adult ADHD and related disorders, while also identifying areas requiring adaptation or potential new developments. RESULTS: Our findings revealed that clinicians and researchers utilize a diverse range of instruments for assessing impairment in adults with ADHD, including some that may lack adequate properties for this purpose. Notably, dissatisfaction with current practice standards was expressed, underscoring the need for novel assessment approaches and improved psychometric properties. CONCLUSION: It is evident that research endeavors are warranted to either refine existing measures or devise new ones for assessing functional impairment in adult ADHD. Emphasis should be placed on disseminating instruments that enhance accessibility in both research and clinical settings, and facilitate streamlined administration and interpretation.

Multiple habitats drive the functional diversity of fish assemblages in a tropical estuary.

Understanding estuarine diversity patterns is crucial to highlight the ecological value of coastal ecosystems for fish assemblages. To increase our knowledge, we investigated the functional diversity of fish assemblages in five estuarine habitats (sandy beaches, mudflats, seagrass meadows, mangrove fringes, and estuarine riparian vegetation) in a tropical estuary of Brazil. Functional diversity metrics were assessed considering seven fish functional traits and calculated using functional indices, PCoA (functional spaces), and community-weighted mean (CWM). Then, a unified RLQ and fourth-corner analysis were used to evaluate environment-trait relationships. A total of 27,036 individuals of 119 species were recorded in all habitats. Functional diversity showed similar trends to estuarine habitats, which were more driven by the spatial configuration rather than by their structure, emphasizing the importance of environmental heterogeneity. There was a greater occupation of functional space to habitats located in the lower estuary compared to the upper estuary. Furthermore, body shapes and trophic guilds were the most common traits related to changes in functional diversity between habitats. The RLQ analysis revealed differences in trait composition between habitats influenced by salinity and transparency, although the fourth corner method did not show a significant relationship between fish functional traits and environmental variables. Our results suggest that the mosaic of habitats support the high functional diversity of fishes in tropical estuaries.

Transdentinal effects of S-PRG fillers on odontoblast-like cells.

OBJECTIVES: To investigate the transdentinal effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) fillers on odontoblast-like cells. METHODS: An eluate of S-PRG fillers was obtained by dissolving the particles in distilled water (1:1 m/v). Dentin discs with similar permeability were mounted into artificial pulp chambers and MDPC-23 cells were seeded on their pulpal surface. The occlusal surface was treated with (n = 10): ultrapure water (negative control - NC), hydrogen peroxide (positive control - PC), S-PRG eluate exposure for 1 min (S-PRG 1 min), or S-PRG filler eluate exposure for 30 min (S-PRG 30 min). After 24 h, cell viability (alamarBlue) and morphology (SEM) were evaluated. The extract obtained from transdentinal diffusion was applied to MDPC-23 pre-cultured in plates for another 24 h to evaluate viability (alamarBlue, 1, 3, and 7 days), gene expression of Col1a1, Alpl, Dspp, and Dmp1 (RT-qPCR, 1 and 7 days), and mineralization (Alizarin Red, 7 days). Data were analyzed with ANOVA (α = 5 %). RESULTS: While S-PRG 1 min did not differ from NC, S-PRG 30 min reduced 17.9 % viability of cells from discs. S-PRG treatments resulted in low cell detaching from dentin, and the remaining cells exhibited typical morphology or minor cytoplasmic contraction. S-PRG 30 min slightly increased cell viability (6 %) 1 day after contact with the extract. S-PRG treatments upregulated the expression of the investigated genes, especially after 1 day. S-PRG 30 min stimulated mineralization activity by 39.7 %. CONCLUSIONS: S-PRG filler eluate does not cause transdentinal cytotoxicity on odontoblast-like cells, and long-term exposure can stimulate their dentinogenic-related mineralization activity. SIGNIFICANCE: The transdentinal elution of ions from S-PRG fillers is not expected to be harmful to the dental pulp and may exert bioactive effects by inducing dentin matrix deposition through the metabolism of underlying odontoblasts.

Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.