Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention.

Dental caries is the most common human disease caused by oral biofilms despite the widespread use of fluoride as the primary anticaries agent. Recently, an FDA-approved iron oxide nanoparticle (ferumoxytol, Fer) has shown to kill and degrade caries-causing biofilms through catalytic activation of hydrogen peroxide. However, Fer cannot interfere with enamel acid demineralization. Here, we show notable synergy when Fer is combined with stannous fluoride (SnF2), markedly inhibiting both biofilm accumulation and enamel damage more effectively than either alone. Unexpectedly, we discover that the stability of SnF2 is enhanced when mixed with Fer in aqueous solutions while increasing catalytic activity of Fer without any additives. Notably, Fer in combination with SnF2 is exceptionally effective in controlling dental caries in vivo, even at four times lower concentrations, without adverse effects on host tissues or oral microbiome. Our results reveal a potent therapeutic synergism using approved agents while providing facile SnF2 stabilization, to prevent a widespread oral disease with reduced fluoride exposure.

Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention.

Dental caries (tooth decay) is the most prevalent human disease caused by oral biofilms, affecting nearly half of the global population despite increased use of fluoride, the mainstay anticaries (tooth-enamel protective) agent. Recently, an FDA-approved iron oxide nanozyme formulation (ferumoxytol, Fer) has been shown to disrupt caries-causing biofilms with high specificity via catalytic activation of hydrogen peroxide, but it is incapable of interfering with enamel acid demineralization. Here, we find notable synergy when Fer is combined with stannous fluoride (SnF 2 ), markedly inhibiting both biofilm accumulation and enamel damage more effectively than either alone. Unexpectedly, our data show that SnF 2 enhances the catalytic activity of Fer, significantly increasing reactive oxygen species (ROS) generation and antibiofilm activity. We discover that the stability of SnF 2 (unstable in water) is markedly enhanced when mixed with Fer in aqueous solutions without any additives. Further analyses reveal that Sn 2+ is bound by carboxylate groups in the carboxymethyl-dextran coating of Fer, thus stabilizing SnF 2 and boosting the catalytic activity. Notably, Fer in combination with SnF 2 is exceptionally effective in controlling dental caries in vivo , preventing enamel demineralization and cavitation altogether without adverse effects on the host tissues or causing changes in the oral microbiome diversity. The efficacy of SnF 2 is also enhanced when combined with Fer, showing comparable therapeutic effects at four times lower fluoride concentration. Enamel ultrastructure examination shows that fluoride, iron, and tin are detected in the outer layers of the enamel forming a polyion-rich film, indicating co-delivery onto the tooth surface. Overall, our results reveal a unique therapeutic synergism using approved agents that target complementary biological and physicochemical traits, while providing facile SnF 2 stabilization, to prevent a widespread oral disease more effectively with reduced fluoride exposure.

Underestimation of myocardial blood flow by dynamic perfusion CT: Explanations by two-compartment model analysis and limited temporal sampling of dynamic CT.

PURPOSE: Previous studies using dynamic perfusion CT and volume perfusion CT (VPCT) software consistently underestimated the stress myocardial blood flow (MBF) in normal myocardium to be 1.1-1.4 ml/min/g, whilst the O 15-water PET studies demonstrated the normal stress MBF of 3-5 ml/min/g. We hypothesized that the MBF determined by VPCT (MBF-VPCT) is actually presenting the blood-to-myocardium transfer constant, K1. In this study, we determined K1 using Patlak plot (K1-Patlak) and compared the results with MBF-VPCT. MATERIAL AND METHODS: 17 patients (66 ± 9 years, 7 males) with suspected coronary artery disease (CAD) underwent stress dynamic perfusion CT, followed by rest coronary CT angiography (CTA). Arterial input and myocardial output curves were analyzed with Patlak plot to quantify myocardial K1. Significant CAD was defined as >50% stenosis on CTA. A simulation study was also performed to investigate the influence of limited temporal sampling in dynamic CT acquisition on K1 using the undersampling data generated from MRI. RESULTS: There were 3 patients with normal CTA, 7 patients with non-significant CAD, and 7 patients with significant CAD. K1-patlak was 0.98 ± 0.35 (range 0.22-1.67) ml/min/g, whereas MBF-VPCT was 0.83 ± 0.23 (range 0.34-1.40) ml/min/g. There was a linear relationship between them: (MBF-VPCT) = 0.58 x (K1-patlak) + 0.27 (r(2) = 0.65, p < 0.001). The simulation study done on MRI data demonstrated that Patlak plot substantially underestimated true K1 by 41% when true K1 was 2.0 ml/min/g with the temporal sampling of 2RR for arterial input and 4RR for myocardial output functions. CONCLUSIONS: The results of our study are generating hypothesis that MBF-VPCT is likely to be calculating K1-patlak equivalent, not MBF. In addition, these values may be substantially underestimated because of limited temporal sampling rate.

Serum phosphate is an independent predictor of the total aortic calcification volume in non-hemodialysis patients undergoing cardiovascular surgery.

BACKGROUND: A high serum phosphate level is a well-known risk factor for vascular calcification (VC) in patients on hemodialysis (HD). However, the association between the serum phosphate level and VC in non-HD patients is unclear. Our aim was to assess the impact of serum phosphate level on aortic calcification (AC) volume in non-HD patients undergoing cardiovascular surgery. METHODS: A total of 117 patients who underwent thoracoabdominal computed tomography as a preoperative general evaluation before cardiovascular surgery were enrolled. The total AC volume was quantified using the volume-rendering method by extracting the area ≥130HU within the entire aorta. The total AC volume index (AC-VI) was estimated as the total AC volume divided by the body surface area. RESULTS: In the 117 patients (64.7±13.1 years, 39% women), the median total AC-VI was 1.23mL/m(2). The mean estimated glomerular filtration rate (eGFR), adjusted serum calcium levels, and serum phosphate levels were 63.8±19.9mL/min/1.73m(2), 9.1±0.4mg/dL, and 3.6±0.6mg/dL, respectively. When the patients were classified into four quartiles based on their total AC-VI value, the serum phosphate level showed a positive correlation with a probability of being in the highest AC-VI quartile (R(2)=0.0146, p=0.0383) whereas the adjusted serum calcium level did not show a significant correlation (R(2)=0.0040, p=0.2615). A similar relationship between the serum phosphate level, adjusted serum calcium level, and AC-VI was confirmed when the total AC-VI was divided into the thoracic AC-VI and abdominal AC-VI. Multivariate analysis indicated that the serum phosphate level was an independent positive predictor of higher total AC-VI quartiles (ß=0.8013, p=0.0160). CONCLUSIONS: An increase in serum phosphate level was associated with an increased AC burden in non-HD patients undergoing cardiovascular surgery.

Dose reduction in dynamic CT stress myocardial perfusion imaging: comparison of 80-kV/370-mAs and 100-kV/300-mAs protocols.

OBJECTIVES: To determine the effect of reduced 80-kV tube voltage with increased 370-mAs tube current on radiation dose, image quality and estimated myocardial blood flow (MBF) of dynamic CT stress myocardial perfusion imaging (CTP) in patients with a normal body mass index (BMI) compared with a 100-kV and 300-mAs protocol. METHODS: Thirty patients with a normal BMI (<25 kg/m(2)) with known or suspected coronary artery disease underwent adenosine-stress dual-source dynamic CTP. Patients were randomised to 80-kV/370-mAs (n = 15) or 100-kV/300-mAs (n = 15) imaging. Maximal enhancement and noise of the left ventricular (LV) cavity, contrast-to-noise ratio (CNR) and MBF of the two groups were compared. RESULTS: Imaging with 80-kV/370-mAs instead of 100-kV/300-mAs was associated with 40% lower radiation dose (mean dose-length product, 359 ± 66 vs 628 ± 112 mGy[Symbol: see text]cm; P < 0.001 ) with no significant difference in CNR (34.5 ± 13.4 vs 33.5 ± 10.4; P = 0.81) or MBF in non-ischaemic myocardium (0.95 ± 0.20 vs 0.99 ± 0.25 ml/min/g; P = 0.66). Studies obtained using 80-kV/370-mAs were associated with 30.9% higher maximal enhancement (804 ± 204 vs 614 ± 115 HU; P < 0.005), and 31.2% greater noise (22.7 ± 3.5 vs 17.4 ± 2.6; P < 0.001). CONCLUSIONS: Dynamic CTP using 80-kV/370-mA instead of 100-kV/300-mAs allowed 40% dose reduction without compromising image quality or MBF. Tube voltage of 80-kV should be considered for individuals with a normal BMI. KEY POINTS: • CT stress perfusion imaging (CTP) is increasingly used to assess myocardial function. • Dynamic CTP is feasible at 80-kV in patients with normal BMI. • An 80-kV/370-mAs protocol allows 40% dose reduction compared with 100-kV/300-mAs. • Contrast-to-noise ratio and myocardial blood flow of the two protocols were comparable.

E2f1-deficient NOD/SCID mice have dry mouth due to a change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland.

Non-obese diabetic (NOD) mice have been used as a model for dry mouth. NOD mice lacking the gene encoding E2f1, a transcription factor, develop hyposalivation more rapidly progressively than control NOD mice. However, the model mice are associated with an underlying disease such as diabetes. We have now established E2f1-deficient NOD/severe combined immunodeficiency disease (NOD/SCID.E2f1(-/-)) mice to avoid the development of diabetes (Matsui-Inohara et al., Exp Biol Med (Maywood) 234(12):1525-1536, 2009). In this study, we investigated the pathophysiological features of dry mouth using NOD/SCID.E2f1(-/-) mice. In NOD/SCID.E2f1(-/-) mice, the volume of secreted saliva stimulated with pilocarpine is about one third that of control NOD/SCID mice. In behavioral analysis, NOD/SCID.E2f1(-/-) mice drank plenty of water when they ate dry food, and the frequency and time of water intake were almost double compared with control NOD/SCID mice. Histological analysis of submandibular glands with hematoxylin-eosin stain revealed that NOD/SCID.E2f1(-/-) mice have more ducts than NOD/SCID mice. In western blot analysis, the expression of aquaporin 5 (AQP5), a marker of acinar cells, in parotid and in submandibular glands of NOD/SCID.E2f1(-/-) mice was lower than in NOD/SCID mice. Immunohistochemical analysis of parotid and submandibular acini revealed that the localization of AQP5 in NOD/SCID.E2f1(-/-) mice differs from that in NOD/SCID mice; AQP5 was leaky and diffusively localized from the apical membrane to the cytosol in NOD/SCID.E2f1(-/-) mice. The ubiquitination of AQP5 was detected in submandibular glands of NOD/SCID.E2f1(-/-) mice. These findings suggest that the change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland cause the pathogenesis of hyposalivation in NOD/SCID.E2f1(-/-) mice.

Roles of salivary components in Streptococcus mutans colonization in a new animal model using NOD/SCID.e2f1-/- mice.

Streptococcus mutans plays an important role in biofilm formation on the tooth surface and is the primary causative agent of dental caries. The binding of S. mutans to the salivary pellicle is of considerable etiologic significance and is important in biofilm development. Recently, we produced NOD/SCID.e2f1(-/-) mice that show hyposalivation, lower salivary antibody, and an extended life span compared to the parent strain: NOD.e2f1(-/-). In this study we used NOD/SCID.e2f1(-/-) 4 or 6 mice to determine the roles of several salivary components in S. mutans colonization in vivo. S. mutans colonization in NOD/SCID.e2f1(-/-) mice was significantly increased when mice were pre-treated with human saliva or commercial salivary components. Interestingly, pre-treatment with secretory IgA (sIgA) at physiological concentrations promoted significant colonization of S. mutans compared with sIgA at higher concentrations, or with human saliva or other components. Our data suggest the principal effects of specific sIgA on S. mutans occur during S. mutans colonization, where the appropriate concentration of specific sIgA may serve as an anti-microbial agent, agglutinin, or an adherence receptor to surface antigens. Further, specific sIgA supported biofilm formation when the mice were supplied 1% sucrose water and a non-sucrose diet. The data suggests that there are multiple effects exerted by sIgA in S. mutans colonization, with synergistic effects evident under the condition of sIgA and limited nutrients on colonization in NOD/SCID.e2f1(-/-) mice. This is a new animal model that can be used to assess prevention methods for dental biofilm-dependent diseases such as dental caries.

Mice deficient in N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase are unable to synthesize chondroitin/dermatan sulfate containing N-acetylgalactosamine 4,6-bissulfate residues and exhibit decreased protease activity in bone marrow-derived mast cells.

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.

E2F-1-deficient NOD/SCID mice developed showing decreased saliva production.

The non-obese diabetic mouse (NOD) is the most characterized model used to study insulin-dependent type 1 diabetes mellitus (IDDM) and Sjoögren's syndrome (SS). In a previous report, we found NOD.E2f1(-/-) mice show a greater progressive development to IDDM and SS compared to NOD mice. Our previous data indicated a progressive decrease in regulatory T cells (CD4(+)CD25(+)) and a decrease in the systemic secretion systems for insulin, and saliva was associated with the progression of IDDM and SS. Therefore, to define the mechanism of early-onset IDDM SS in E2F-1 deficient NOD mice required further investigation by producing E2F-1 deficient NOD/SCID mice in which the T and B cells do not develop. The purpose here was to analyze the essential function of the E2F-1 molecule in the development of IDDM and SS; and the dysfunction of the pancreas islet and salivary gland in the NOD background using NOD/SCID mice. We produced NOD/SCID.E2f1(-/-) mice using homologous recombination; determined diabetes development; measured saliva and insulin production; and performed a histological analysis. The deficient mice showed a decreasing volume of saliva; no infiltration of lymphocytes into salivary glands; no development of diabetes; and no protein localization of FGFR-2b in the ducts of the salivary gland that regulates submandibular gland proliferation and morphogenesis. Therefore, we considered a deficiency in E2F-1 induces a decrease in regulatory T cells and an increase in auto-reactive T cells; however, the E2F-1 deficiency is not associated with T and B cells-independent dysfunction of pancreatic beta cell in insulin secretion. Further, the E2F-1 deficiency is associated with T and B cells-independent dysfunction of the salivary gland exhibits a decrease in saliva production volume. We suggest E2F-1 may be also associated with the differentiation of exocrine cells in the duct where FGFR-2b is expressed in the salivary gland. The E2F-1 deficient NOD/SCID mouse model is useful for showing the development of the salivary gland; and is also useful for various experiments in humanized mice.

E2F1-deficient NOD/SCID mice are an experimental model for dry mouth.

Saliva contains a wide variety of secretory proteins, including alpha-amylase, lysozyme, peroxidase, immunoglobulins, and mucins. Hyposecretion of saliva and consequent dry mouth will lead to severe dental caries, periodontal disease, and mucosal infections, resulting in degrade of quality of life. Polyposia development is one of sign usually seen in dry mouth patients. However, little is reported in dry mouth-model animal regarding the entire process of polyposia development. We investigated the behavior of polyposia in E2F1-deficient non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice, as a dry mouth-model. E2F1-deficient NOD/SCID mice secreted small amount of saliva under the stimulation with a cholinergic agonist, pilocarpine, compared with control mice. The frequency of water intake by E2F-1-deficient NOD/SCID mice was more than that by control mice. These results suggest that E2F-1-deficient NOD/SCID mice show a behavior similar to polyposia and are very useful experimental model of dry mouth patients.