NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Resin tags formation by modified Renewal MI formulations in a carious dentine model.

Objectives: To determine which components in a new restorative material (Renewal MI) improve its ability to form resin tags within demineralized dentine. Methods: Varied components included polylysine (PLS), monocalcium phosphate (MCP), powder to liquid ratio (PLR), 4-methacryloyloxyethyl trimellitate anhydride (4META), and polypropylene glycol dimethacrylate (PPGDMA). Urethane dimethacrylate (UDMA), containing PPGDMA (24 wt%) and 4META (3 wt%), was mixed with glass filler with MCP (8 wt%) and PLS (5 wt%). PLR was 3:1 or 5:1. Reducing MCP and/or PLS to 4 and 2 wt% respectively or fully removing MCP, PLS, 4META or PPGDMA gave 16 formulations in total. Renewal MI, Z250 (with or without Scotchbond Universal adhesive) and Activa were used as commercial comparators. Collagen discs were obtained by totally demineralizing 2 mm thick, human, premolar, coronal dentine discs by immersion in formic acid (4M) for 48 h. The restorative materials were then applied on top (n = 3), before dissolving the collagen in sodium hypochlorite (15%). SEM/EDX was employed to determine resin tags length, composition, and surface coverage. Results: Tags were >400, 20 and 200 µm and covered 62, 55 and 39% of the adhesion interface for Renewal MI, Scotchbond and Activa, respectively. With experimental formulations, they were 200 and >400 µm long with high vs. low PLR and composed primarily of polymerized monomers. Percentages of the adhesion interface covered varied between 35 and 84%. Reducing PLS or MCP caused a decline in coverage that was linear with their concentrations. Reducing MCP had lesser effect when PLS or PLR were low. Removal of 4META caused a greater reduction in coverage than PPGDMA removal. Conclusion: PLS, MCP, 4META, PPGDMA and low PLR together enhance Renewal MI tags formation in, and thereby sealing of, demineralized dentine.

The effect of varying monocalcium phosphate and polylysine levels on dental composite properties.

OBJECTIVES: The aim was to quantify effects of polylysine (PLS, 2 or 5 wt%) and monocalcium phosphate (MCP, 4 or 8 wt%) on properties of dental composites. METHODS: Light-activated, lower surface polymerisation kinetics versus sample depth (1-4 mm) of 4 formulations were quantified using ATR-FTIR. Water sorption and solubility (at 1 week) were assessed following ISO/4049. PLS release (over 1 month) and biaxial flexural strength (over 6 months) of fully-cured, water-immersed, 1 mm thick discs were determined. Surface mineral precipitation, following immersion in simulated body fluid (SBF), was assessed by SEM. Z250 was used as a conventional composite comparator. RESULTS: With 40s light exposure, increasing depth (from 1 to 4 mm) led to enhanced delay before polymerisation (from 3 to 17s) and decreased final conversion (72-66%) irrespective of PLS and MCP level. Increasing PLS and MCP raised solubility (4-13 µg/mm3). Water sorption (between 32 and 55 µg/mm3) and final PLS release (8-13% of disc content) were raised primarily by increasing PLS. Higher PLS also reduced strength. Strength reached minimum values (69-94 MPa) at 3 months. Surface mineral deposition was enhanced by increased MCP. For Z250, polymerisation delays (3-6s) and final conversions (55-54%) at 1-4 mm depth, solubility (0 µg/mm3), water sorption (16 µg/mm3) and strength (180 MPa) were all significantly different. CONCLUSION: Delay time increased whilst final conversion decreased with thicker samples. Higher PLS enhances its percentage release, but lower level is required to keep water sorption, solubility and mechanical properties within ISO 4049 recommendations. Doubling MCP raises solubility and enhances minerals reprecipitation with minimal mechanical property compromise.

The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L.

NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.

Renewal MI Dental Composite Etch and Seal Properties.

This study's aim was to assess whether the Renewal MI composite can self-etch enamel, seal sound cavities, and stabilize demineralized dentine. Etching was assessed using scanning electron microscopy (SEM). Cavity sealing was quantified using the ISO-11405 dye microleakage test. Demineralized dentine stabilization was evaluated by visualizing resin tag formation, enzyme activity and mineral precipitation at the adhesion interface. Renewal MI provided a mild etching of sound enamel in comparison with 37% phosphoric acid. It provided a comparable seal of sound cavities to Z250/Scotchbond Universal adhesive and a superior seal to Activa, Fuji IX and Fuji II LC. With demineralized dentine, Renewal MI formed 300-400 µm resin tags covering 63% of the adhesion interface compared with 55 and 39% for Z250/Scotchbond and Activa. Fuji IX and Fuji II LC formed no resin tags. A higher tag percentage correlated with lower surface enzyme activity. Unlike Activa and Fuji II LC, Renewal MI promoted mineral precipitation from simulated body fluid, occluding adjacent dentinal tubules within 6 months. These novel etching and sealing properties may facilitate Renewal MI's application in minimally invasive dentistry.

Early Polylysine Release from Dental Composites and Its Effects on Planktonic Streptococcus mutans Growth.

The study aim was to assess the effect of incorporating polylysine (PLS) filler at different mass fractions (0.5, 1 and 2 wt%) on PLS release and Streptococcus mutans planktonic growth. Composite containing PLS mass and volume change and PLS release upon water immersion were assessed gravimetrically and via high-performance liquid chromatography (HPLC), respectively. Disc effects on bacterial counts in broth initially containing 8 × 105 versus 8 × 106 CFU/mL Streptococcus mutans UA159 were determined after 24 h. Survival of sedimented bacteria after 72 h was determined following LIVE/DEAD staining of composite surfaces using confocal microscopy. Water sorption-induced mass change at two months increased from 0.7 to 1.7% with increasing PLS concentration. Average volume increases were 2.3% at two months whilst polylysine release levelled at 4% at 3 weeks irrespective of composite PLS level. Early percentage PLS release, however, was faster with higher composite content. With 0.5, 1 and 2% polylysine initially in the composite filler phase, 24-h PLS release into 1 mL of water yielded 8, 25 and 93 ppm respectively. With initial bacterial counts of 8 × 105 CFU/mL, this PLS release reduced 24-h bacterial counts from 109 down to 108, 107 and 102 CFU/mL respectively. With a high initial inoculum, 24-h bacterial counts were 109 with 0, 0.5 or 1% PLS and 107 with 2% PLS. As the PLS composite content was raised, the ratio of dead to live sedimented bacteria increased. The antibacterial action of the experimental composites could reduce residual bacteria remaining following minimally invasive tooth restorations.

Effect of Novel Antibacterial Composites on Bacterial Biofilms.

Continuing cariogenic bacterial growth demineralizing dentine beneath a composite filling is the most common cause of tooth restoration failure. Novel composites with antibacterial polylysine (PLS) (0, 4, 6, or 8 wt%) in its filler phase were therefore produced. Remineralising monocalcium phosphate was also included at double the PLS weight. Antibacterial studies involved set composite disc placement in 1% sucrose-supplemented broth containing Streptococcus mutans (UA159). Relative surface bacterial biofilm mass (n = 4) after 24 h was determined by crystal violet-binding. Live/dead bacteria and biofilm thickness (n = 3) were assessed using confocal laser scanning microscopy (CLSM). To understand results and model possible in vivo benefits, cumulative PLS release from discs into water (n = 3) was determined by a ninhydrin assay. Results showed biofilm mass and thickness decreased linearly by 28% and 33%, respectively, upon increasing PLS from 0% to 8%. With 4, 6, and 8 wt% PLS, respectively, biofilm dead bacterial percentages and PLS release at 24 h were 20%, 60%, and 80% and 85, 163, and 241 µg/disc. Furthermore, initial PLS release was proportional to the square root of time and levelled after 1, 2, and 3 months at 13%, 28%, and 42%. This suggested diffusion controlled release from water-exposed composite surface layers of 65, 140, and 210 µm thickness, respectively. In conclusion, increasing PLS release initially in any gaps under the restoration to kill residual bacteria or longer-term following composite/tooth interface damage might help prevent recurrent caries.

Polymerization kinetics stability, volumetric changes, apatite precipitation, strontium release and fatigue of novel bone composites for vertebroplasty.

PURPOSE: The aim was to determine effects of diluent monomer and monocalcium phosphate monohydrate (MCPM) on polymerization kinetics and volumetric stability, apatite precipitation, strontium release and fatigue of novel dual-paste composites for vertebroplasty. MATERIALS AND METHODS: Polypropylene (PPGDMA) or triethylene (TEGDMA) glycol dimethacrylates (25 wt%) diluents were combined with urethane dimethacrylate (70 wt%) and hydroxyethyl methacrylate (5 wt%). 70 wt% filler containing glass particles, glass fibers (20 wt%) and polylysine (5 wt%) was added. Benzoyl peroxide and MCPM (10 or 20 wt%) or N-tolyglycine glycidyl methacrylate and tristrontium phosphate (15 wt%) were included to give initiator or activator pastes. Commercial PMMA (Simplex) and bone composite (Cortoss) were used for comparison. ATR-FTIR was used to determine thermal activated polymerization kinetics of initiator pastes at 50-80°C. Paste stability, following storage at 4-37°C, was assessed visually or through mixed paste polymerization kinetics at 25°C. Polymerization shrinkage and heat generation were calculated from final monomer conversions. Subsequent expansion and surface apatite precipitation in simulated body fluid (SBF) were assessed gravimetrically and via SEM. Strontium release into water was assessed using ICP-MS. Biaxial flexural strength (BFS) and fatigue properties were determined at 37°C after 4 weeks in SBF. RESULTS: Polymerization profiles all exhibited an inhibition time before polymerization as predicted by free radical polymerization mechanisms. Initiator paste inhibition times and maximum reaction rates were described well by Arrhenius plots. Plot extrapolation, however, underestimated lower temperature paste stability. Replacement of TEGDMA by PPGDMA, enhanced paste stability, final monomer conversion, water-sorption induced expansion and strontium release but reduced polymerization shrinkage and heat generation. Increasing MCPM level enhanced volume expansion, surface apatite precipitation and strontium release. Although the experimental composite flexural strengths were lower compared to those of commercially available Simplex, the extrapolated low load fatigue lives of all materials were comparable. CONCLUSIONS: Increased inhibition times at high temperature give longer predicted shelf-life whilst stability of mixed paste inhibition times is important for consistent clinical application. Increased volumetric stability, strontium release and apatite formation should encourage bone integration. Replacing TEGDMA by PPGDMA and increasing MCPM could therefore increase suitability of the above novel bone composites for vertebroplasty. Long fatigue lives of the composites may also ensure long-term durability of the materials.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1ß via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1ß is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1ß secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1ß is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1ß. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1ß regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1ß from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1ß release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1ß release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2ß, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1ß release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Monomer conversion, dimensional stability, strength, modulus, surface apatite precipitation and wear of novel, reactive calcium phosphate and polylysine-containing dental composites.

PURPOSE: The aim was to assess monomer conversion, dimensional stability, flexural strength / modulus, surface apatite precipitation and wear of mono / tri calcium phosphate (CaP) and polylysine (PLS)-containing dental composites. These were formulated using a new, high molecular weight, fluid monomer phase that requires no polymerisation activator. MATERIALS AND METHODS: Urethane and Polypropylene Glycol Dimethacrylates were combined with low levels of an adhesion promoting monomer and a light activated initiator. This liquid was mixed with a hybrid glass containing either 10 wt% CaP and 1 wt% PLS (F1) or 20 wt% CaP and 2 wt% PLS (F2). Powder to liquid mass ratio was 5:1. Commercial controls included Gradia Direct Posterior (GD) and Filtek Z250 (FZ). Monomer conversion and polymerisation shrinkage were calculated using Fourier Transform Infrared (FTIR). Subsequent volume increases in water over 7 weeks were determined using gravimetric studies. Biaxial flexural strength (BFS) / modulus (BFM) reduction and surface apatite precipitation upon 1 and 4 weeks immersion in water versus simulated body fluid (SBF) were assessed using a mechanical testing frame and scanning electron microscope (SEM). Mass / volume loss and surface roughness (Ra) following 7 weeks water immersion and subsequent accelerated tooth-brush abrasion were examined using gravimetric studies and profilometer. RESULTS: F1 and F2 exhibited much higher monomer conversion (72%) than FZ (54%) and low calculated polymerization shrinkage (2.2 vol%). Final hygroscopic expansions decreased in the order; F2 (3.5 vol%) > F1 (1.8 vol%) ~ Z250 (1.6 vol%) > Gradia (1.0 vol%). BFS and BFM were unaffected by storage medium type. Average BFS / BFM upon 4 weeks immersion reduced from 144 MPa / 8 GPa to 107 MPa / 5 GPa for F1 and 105 MPa / 6 GPa to 82 MPa / 4 GPa for F2. Much of this change was observed in the first week of immersion when water sorption rate was high. Surface apatite layers were incomplete at 1 week, but around 2 and 15 micron thick for F1 and F2 respectively following 4 weeks in SBF. Mass and volume loss following wear were equal. Average results for F1 (0.5%), F2 (0.7%), and FZ (0.5%) were comparable but lower than that of GD (1%). Ra, however, decreased in the order; F1 (15 µm) > F2 (11 µm) > GD (9 µm) > FZ (5 µm). CONCLUSIONS: High monomer conversion in combination with large monomer size and lack of amine activator should improve cytocompatibility of the new composites. High monomer molecular weight and powder content enables low polymerisation shrinkage despite high conversion. Increasing active filler provides enhanced swelling to balance shrinkage, which, in combination with greater surface apatite precipitation, may help seal gaps and reduce bacterial microleakage. High monomer conversion also ensures competitive mechanical / wear characteristics despite enhanced water sorption. Furthermore, increased active filler could help reduce surface roughness upon wear.

Dental Composites with Calcium / Strontium Phosphates and Polylysine.

PURPOSE: This study developed light cured dental composites with added monocalcium phosphate monohydrate (MCPM), tristrontium phosphate (TSrP) and antimicrobial polylysine (PLS). The aim was to produce composites that have enhanced water sorption induced expansion, can promote apatite precipitation and release polylysine. MATERIALS AND METHODS: Experimental composite formulations consisted of light activated dimethacrylate monomers combined with 80 wt% powder. The powder phase contained a dental glass with and without PLS (2.5 wt%) and/or reactive phosphate fillers (15 wt% TSrP and 10 wt% MCPM). The commercial composite, Z250, was used as a control. Monomer conversion and calculated polymerization shrinkage were assessed using FTIR. Subsequent mass or volume changes in water versus simulated body fluid (SBF) were quantified using gravimetric studies. These were used, along with Raman and SEM, to assess apatite precipitation on the composite surface. PLS release was determined using UV spectroscopy. Furthermore, biaxial flexural strengths after 24 hours of SBF immersion were obtained. RESULTS: Monomer conversion of the composites decreased upon the addition of phosphate fillers (from 76 to 64%) but was always higher than that of Z250 (54%). Phosphate addition increased water sorption induced expansion from 2 to 4% helping to balance the calculated polymerization shrinkage of ~ 3.4%. Phosphate addition promoted apatite precipitation from SBF. Polylysine increased the apatite layer thickness from ~ 10 to 20 µm after 4 weeks. The novel composites showed a burst release of PLS (3.7%) followed by diffusion-controlled release irrespective of phosphate addition. PLS and phosphates decreased strength from 154 MPa on average by 17% and 18%, respectively. All formulations, however, had greater strength than the ISO 4049 requirement of > 80 MPa. CONCLUSION: The addition of MCPM with TSrP promoted hygroscopic expansion, and apatite formation. These properties are expected to help compensate polymerization shrinkage and help remineralize demineralized dentin. Polylysine can be released from the composites at early time. This may kill residual bacteria.

Poly(propylene glycol) and urethane dimethacrylates improve conversion of dental composites and reveal complexity of cytocompatibility testing.

OBJECTIVES: To determine the effects of various monomers on conversion and cytocompatibility of dental composites and to improve these properties without detrimentally affecting mechanical properties, depth of cure and shrinkage. METHODS: Composites containing urethane dimethacrylate (UDMA) or bisphenol A glycidyl methacrylate (Bis-GMA) with poly(propylene glycol) dimethacrylate (PPGDMA) or triethylene glycol dimethacrylate (TEGDMA) were characterized using the following techniques: conversion (FTIR at 1 and 4mm depths), depth of cure (BS EN ISO 4049:2009 and FTIR), shrinkage (BS EN ISO 17304:2013 and FTIR), strength and modulus (biaxial flexural test) and water sorption. Cytocompatibility of composites and their liquid phase components was assessed using three assays (resazurin, WST-8 and MTS). RESULTS: UDMA significantly improved conversion, BFS and depth of cure compared to Bis-GMA, without increasing shrinkage. UDMA was cytotoxic at lower concentrations than Bis-GMA, but extracts of Bis-GMA-containing composites were less cytocompatible than of those containing UDMA. PPGDMA improved conversion and depth of cure compared to TEGDMA, without detrimentally affecting shrinkage. TEGDMA was shown by all assays to be highly toxic. Resazurin, but not WST-8 and MTS, suggested that PPGDMA exhibited improved cytocompatibility compared to TEGDMA. SIGNIFICANCE: The use of UDMA and PPGDMA results in composites with excellent conversion, depth of cure and mechanical properties, without increasing shrinkage. Composites containing UDMA appear to be slightly more cytocompatible than those containing Bis-GMA. These monomers may therefore improve the material properties of dental restorations, particularly bulk fill materials. The effect of diluent monomer on cytocompatibility requires further investigation.