Biocompatibility of bulk-fill resins in vitro.

OBJECTIVES: This study aims to evaluate the cytotoxicity and genotoxicity of three different extracts obtained from Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill and Coltene Fill-Up! resins. MATERIALS AND METHODS: The cytotoxicity was determined on 3T3 fibroblast cells using the MTT and crystal violet assays. The genotoxicity was determined using a cytokinesis-block micronucleus assay. RESULTS: The cytotoxicity of the resin extracts on 3T3 mouse fibroblasts was found to be dose-dependent with both the MTT and crystal violet assays. Extracts concentrated above 1% were cytotoxic according to the MTT assay. The Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill, and Coltene Fill-Up! resins reached the LD50 at concentrations of 60%, 50%, and 20%, respectively, and showed genotoxicity rates that were 2-5 times, 3-8 times, and 4-15 times higher than the negative control, respectively. CONCLUSIONS: Coltene Fill-Up! resin extracts were the most cytotoxic and genotoxic, followed by Tetric Evoceram® Bulk Fill and Filtek™ One Bulk Fill. CLINICAL RELEVANCE: The analyzed bulk-fill resins showed differences in in vitro biocompatibility, and the Filtek™ One Bulk Fill was found to be the safest for clinical use.

The Incidence of Dental Caries in Children with Down Syndrome: A Systematic Review and Meta-Analysis.

Scientific evidence regarding the incidence of dental caries in Down syndrome (DS) patients is limited and sometimes presents divergent opinions among authors, making it difficult to reach definitive conclusions. We aimed to evaluate the caries incidence in the DS pediatric population and compare it against healthy controls. The search was performed using 4 universal databases: Cochrane, B-on, Biomed, and PubMed. The selected articles were synthesized and subsequently evaluated according to an adaptation of the Quality Assessment Checklist for Prevalence Studies risk of bias tool, and analysis charts were performed by the Risk of Bias visualization tool (ROBVIS). Statistics and graphs were performed by Open Meta Analyst and JASP software. The confounding effect on caries incidence of the following factors was evaluated through meta-regression: age, Male/Female (M/F) ratio, DMFT, dmft, and study geographic location. Overall, the incidence of caries in the DS population was 49.9%, whereas in the control population was 63.4%. The M/F ratio, DMFT, and dmft significantly affected the incidence of DS individuals (p-value < 0.05). The evidence regarding the lower pooled incidence of caries in individuals with DS regarding controls is limited by the few scientific reports available and cross-section designs. Therefore, further studies are needed to confirm these results.

High Instantaneous Inhibitory Potential of Bictegravir and the New Spiro-ß-Lactam BSS-730A for HIV-2 Isolates from RAL-Naïve and RAL-Failing Patients.

Integrase inhibitors (INIs) are an important class of drugs for treating HIV-2 infection, given the limited number of drugs active against this virus. While the clinical efficacy of raltegravir and dolutegravir is well established, the clinical efficacy of bictegravir for treating HIV-2 infected patients has not been determined. Little information is available regarding the activity of bictegravir against HIV-2 isolates from patients failing raltegravir-based therapy. In this study, we examined the phenotypic and matched genotypic susceptibility of HIV-2 primary isolates from raltegravir-naïve and raltegravir-failing patients to raltegravir, dolutegravir, and bictegravir, and to the new spiro-ß-lactam BSS-730A. The instantaneous inhibitory potential (IIP) was calculated to help predict the clinical activity of bictegravir and BSS-730A. Isolates from raltegravir-naïve patients were highly sensitive to all INIs and BSS-730A. Combined integrase mutations E92A and Q148K conferred high-level resistance to raltegravir, and E92Q and T97A conferred resistance to raltegravir and dolutegravir. The antiviral activity of bictegravir and BSS-730A was not affected by these mutations. BSS-730A displayed strong antiviral synergism with raltegravir. Mean IIP values at Cmax were similar for all INIs and were not significantly affected by resistance mutations. IIP values were significantly higher for BSS-730A than for INIs. The high IIP values of bictegravir and BSS-730A for raltegravir-naïve and raltegravir-resistant HIV-2 isolates highlight their potential value for treating HIV-2 infection. Overall, the results are consistent with the high clinical efficacy of raltegravir and dolutegravir for HIV-2 infection and suggest a promising clinical profile for bictegravir and BSS-730A.

APOBEC3B Potently Restricts HIV-2 but Not HIV-1 in a Vif-Dependent Manner.

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

Polymerizable Matrix Metalloproteinases' Inhibitors with Potential Application for Dental Restorations.

Collagen cleavage by matrix metalloproteinase (MMP) is considered a major cause of dental resins long term failure. Most MMP inhibitors display significant toxicity and are unsuitable for dental resins' applications. Here we report a study of a new class of inhibitors that display the unique property of being co-polymerizable with other vinyl compounds present in commercial dental resins, limiting their release and potential toxicity. Computational affinity towards the active site of different MMP-1; -2; -8; -9 and -13 of several compounds showed interesting properties and were synthesized. These free compounds were tested concerning their toxicity upon contact with two different cell types, with no substantial decrease in cell viability at high concentrations. Even so, compound's safety can be further improved upon copolymerization with commercial dental resins, limiting their release.

The effects of addition of functional monomers and molecular imprinting on dual drug release from intraocular lens material.

Although cataract surgery is considered a safe procedure, post-surgery complications such as endophthalmitis and ocular inflammation, may occur. To prevent this, antibiotics and anti-inflammatories are prescribed in the form of eye drops during the post-operatory period, but they lead to a low drug bioavailability in target tissues. The objective of this work is to develop an intraocular lens (IOL) material to deliver simultaneously one antibiotic, moxifloxacin (MXF), and one anti-inflammatory, diclofenac (DFN), in therapeutic concentrations to prevent both complications. The IOL material was modified through the incorporation of functional monomers, as well as molecular imprinting with both drugs using the same functional monomers, namely acrylic acid (AA), methacrylic acid (MAA), 4-vinylpiridine (4-VP) and a combination of MAA + 4-VP. The best results were obtained with MAA. Molecular imprinting did not influence the drug release, except with AA. Application of a mathematical model predicted that the released MXF and DFN concentrations would stay above the pre-determined MIC of S. aureus and S. epidermidis and the minimum values of IC50 of COX-1 and COX-2, for 9 and 14 days, respectively. Antibacterial tests showed that the released antibiotic remained active. The physical properties of the drug-loaded MAA-hydrogel remained adequate. The developed system proved to be non-irritant and non-cytotoxic.

Biocompatibility of self-adhesive resin cement with fibroblast cells.

STATEMENT OF PROBLEM: Dental cements that release monomers that negatively impact adjacent oral soft tissues may adversely affect clinical outcomes. However, in vitro studies evaluating the cytotoxic and genotoxic potential of substances released from dental cements are lacking. PURPOSE: The purpose of this in vitro study was to define and compare the cytotoxicity and genotoxicity of the eluates of a self-adhesive resin cement (RelyX Unicem 2 Automix) autopolymerized and light polymerized with 2 other types of luting cements: a glass ionomer cement (Ketac Cem Easymix) and a resin-modified glass ionomer cement (Ketac Cem Plus). MATERIAL AND METHODS: The eluates were prepared, and 3T3 mouse fibroblast cells were exposed for 24 hours to serial eluate dilutions of the 3 types of cement. Cytotoxicity was determined by using a cell viability assessment through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays. Genotoxic effects were determined by using the cytokinesis-block micronucleus assay. RESULTS: Cell viability was higher in the presence of the glass ionomer cement eluate than of the resin-modified glass ionomer cement and resin cement eluates. A pronounced decrease in viability was found when the cells were exposed to undiluted samples of resin-modified glass ionomer cement (around 50%) or resin cement (around 80% to 90%). No significant difference in cell viability was found between autopolymerized and light-polymerized resin cements. All cements induced a dose-dependent response of mononucleated cell formation. However, only the resin cements showed double strand breaks significant differences in the deoxyribonucleic acid (DNA) molecules against the basal DNA lesions that occurred spontaneously. CONCLUSIONS: The glass ionomer cement was not found to be cytotoxic or genotoxic, whereas the eluates derived from the resin-modified glass ionomer cement and resin cement, independently of the polymerization method, were cytotoxic in fibroblast cells. Maximum cytotoxicity was observed in the presence of resin cement, which also showed genotoxicity, independently of being light polymerized.

In vitro and in silico evaluations of resin-based dental restorative material toxicity.

OBJECTIVES: This study aims to evaluate the cytocompatibility of three provisional restoration materials and predict neurotoxic potential of their monomers. These materials are Tab 2000® (methyl methacrylate based), ProTemp 4™ (bis-acrylic based) and Structur 3® (urethane dimethacrylate based). MATERIALS AND METHODS: Resin samples were incubated in a cell culture medium and the cytotoxic effects of these extracts were studied in 3T3 fibroblast cells through MTT and crystal violet assays as well as ROS assessment. The presence of relevant leached monomers was determined by HPLC. Additionally, the blood-brain barrier (BBB) permeability to these resin-based monomers was predicted using ACD/Labs algorithms model. RESULTS: Cell survival rates were compared with the resin extracts, and Structur 3® was statistically significant different from the others (p < 0.001) at all-time incubation periods. All materials induced a dose-dependent loss of cell viability; however, only Structur 3 extracts were cytotoxic against 3T3 fibroblasts. The highest cytotoxic effect (77%, p < 0.001) was observed at 24 h incubation period, which may be associated with the presence of urethane dimethacrylate (UDMA) leached monomers. Furthermore, the computational model showed that most monomers under study are expectedly capable of crossing the BBB. CONCLUSIONS: Our results showed that Structur 3® is not cytocompatible with our cell model and UDMA is a potential neurotoxic compound. CLINICAL RELEVANCE: These results indicate that only ProTemp 4™ and Tab 2000® are safe for provisional restorations.

High Hydrostatic Pressure as Sterilization Method for Drug-Loaded Intraocular Lenses.

Sterilization is a key step in the manufacturing of drug-loaded intraocular lenses (IOLs). Two of the most used methods to sterilize commercial IOLs are steam heat and gamma radiation. However, when the IOLs are loaded with drugs, the adequacy of those methods must be questioned because sterilization may affect the activity of the drugs and/or the drug release. Recently, high hydrostatic pressure (HHP), which is increasingly used in the food industry, has been applied in the sterilization of gels for medical applications. The objective of this work was to assess the performance of HHP in the sterilization of a commercial acrylic material used for the production of IOLs, both without and with loaded drugs. Bare samples and samples loaded with an antibiotic and two anti-inflammatories were tested, and the results were compared to those obtained with conventional sterilization methods. HHP not only sterilized highly contaminated samples but also enhanced drug loading and did not affect significantly the hydrogel properties. Gamma radiation degraded the drugs in solution; thus, it is adequate only for dry sample sterilization. Steam heat did not affect the release profiles but cannot be applied to temperature-sensitive drugs. We concluded that HHP may advantageously substitute steam heat and gamma radiation in the sterilization of drug-loaded IOLs.

Assessment of the Cavidi ExaVir Load Assay for Monitoring Plasma Viral Load in HIV-2-Infected Patients.

HIV plasma viral load is an established marker of disease progression and of response to antiretroviral therapy, but currently there is no commercial assay validated for the quantification of viral load in HIV-2-infected individuals. We sought to make the first clinical evaluation of Cavidi ExaVir Load (version 3) in HIV-2-infected patients. Samples were collected from a total of 102 individuals living in Cape Verde, and the HIV-2 viral load was quantified by both ExaVir Load and a reference in-house real-time quantitative PCR (qPCR) used in Portugal in 91 samples. The associations between viral load and clinical prognostic variables (CD4+ T cell counts and antiretroviral therapy status) were similar for measurements obtained using ExaVir Load and qPCR. There was no difference between the two methods in the capacity to discriminate between nonquantifiable and quantifiable HIV-2 in the plasma. In samples with an HIV-2 viral load quantifiable by both methods (n = 27), the measurements were highly correlated (Pearson r = 0.908), but the ExaVir Load values were systematically higher relative to those determined by qPCR (median difference, 0.942 log10 copies/ml). A regression model was derived that enables the conversion of ExaVir Load results to those that would have been obtained by the reference qPCR. In conclusion, ExaVir Load version 3 is a reliable commercial assay to measure viral load in HIV-2-infected patients and therefore a valuable alternative to the in-house assays in current use.

Effect of diabetes mellitus type 2 on salivary glucose--a systematic review and meta-analysis of observational studies.

BACKGROUND: Early screening of type 2 diabetes mellitus (DM) is essential for improved prognosis and effective delay of clinical complications. However, testing for high glycemia often requires invasive and painful blood testing, limiting its large-scale applicability. We have combined new, unpublished data with published data comparing salivary glucose levels in type 2 DM patients and controls and/or looked at the correlation between salivary glucose and glycemia/HbA1c to systematically review the effectiveness of salivary glucose to estimate glycemia and HbA1c. We further discuss salivary glucose as a biomarker for large-scale screening of diabetes or developing type 2 DM. METHODS AND FINDINGS: We conducted a meta-analysis of peer-reviewed published articles that reported data regarding mean salivary glucose levels and/or correlation between salivary glucose levels and glycemia or HbA1c for type 2 DM and non-diabetic individuals and combined them with our own unpublished results. Our global meta-analysis of standardized mean differences on salivary glucose levels shows an overall large positive effect of type 2 DM over salivary glucose (Hedge's g = 1.37). The global correlation coefficient (r) between salivary glucose and glycemia was large (r = 0.49), with subgroups ranging from medium (r = 0.30 in non-diabetics) to very large (r = 0.67 in diabetics). Meta-analysis of the global correlation between salivary glucose and HbA1c showed an overall association of medium strength (r = 0.37). CONCLUSIONS: Our systematic review reports an overall meaningful salivary glucose concentration increase in type 2 DM and a significant overall relationship between salivary glucose concentration and associated glycemia/HbA1c values, with the strength of the correlation increasing for higher glycemia/HbA1c values. These results support the potential of salivary glucose levels as a biomarker for type 2 DM, providing a less painful/invasive method for screening type 2 DM, as well as for monitoring blood glucose levels in large cohorts of DM patients.

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division.

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G.

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.

Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity.

We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy.