Penetration and bactericidal efficacy of two oral care products in an oral biofilm model.

PURPOSE: To investigate the immediate penetration and bactericidal effect of two oral care products marketed in China on an intact natural plaque biofilm model at different time points. METHODS: Eight subjects (aged 20 to 30 years; Turesky Plaque Index Score 2 to 3) were enrolled in the study according to the inclusion criteria. Plaque accumulators were worn by the subjects for 6 and 48 hours for harvesting the dental biofilm. Then the biofilms from different groups were stained with the LIVE/DEAD BacLight fluorescence system to investigate the changes in thickness and fluorescence intensity of living bacteria in biofilm 5 and 15 minutes post-treatment with a mouthrinse containing 0.074% cetylpyridinium chloride (1-minute treatment) or a toothpaste supernatant containing 1.16% stannous chloride (2-minute treatment). In addition, a specific Sn2+ probe was utilized to evaluate the penetration of Sn2+ in the biofilm. Fluorescent images were collected using confocal laser scanning microscopy. Analysis of covariance was used for statistical analyses. All comparisons were two-sided using a 5% level of significance. RESULTS: The thickness of generated plaque biofilm increased gradually from 7.352±4.22 µm at 6 hours to 16.73±7.38 µm at 48 hours (P< 0.05), whereas the thickness and fluorescence intensity of living bacteria stayed unchanged over time. After the treatment of toothpaste supernatant, the ratios of living bacteria thickness and fluorescence intensity of 6- and 48-hour plaque biofilm were significantly decreased (P< 0.05). Treatment of mouthrinse reduced the ratio of living bacteria thickness, but showed no significant impact on overall fluorescence intensity of living bacteria. For 48-hour biofilm, toothpaste supernatant significantly reduced fluorescence intensity of living bacteria from outer layer through inner layer, whereas the mouthrinse showed bactericidal effect only in the outer layer and middle layer. A wide distribution of Sn2+ was shown in the biofilm with the treatment of the tested toothpaste. CLINICAL SIGNIFICANCE: This biofilm model proved to be useful and appropriate for pre-clinical testing of anti-plaque agents. A brief exposure of the biofilm to the tested toothpaste produced significant losses in bacteria viability across outer-middle-inner layers. The tested mouthrinse exerted its bactericidal effect mostly in outer and middle layers of biofilm. The penetration of Sn2+ in the biofilm performed an important function in the bactericidal effect of the toothpaste.

Fluorescence turn-on detection of Sn2+ in live eukaryotic and prokaryotic cells.

Sn(2+) is usually added to toothpaste to prevent dental plaque and oral disease. However, studies of its physiological role and bacteriostatic mechanism are restricted by the lack of versatile Sn(2+) detection methods applicable to live cells, including Streptococcus mutans. Here we report two Sn(2+) fluorescent probes containing a rhodamine B derivative as a fluorophore, linked via the amide moiety to N,N-bis(2-hydroxyethyl)ethylenediamine (R1) and tert-butyl carbazate group (R2), respectively. These probes can selectively chelate Sn(2+) and show marked fluorescence enhancement due to the ring open reaction of rhodamine induced by Sn(2+) chelation. The probes have high sensitivity and selectivity for Sn(2+) in the presence of various relevant metal ions. Particularly, both R1 and R2 can target lysosomes, and R2 can probe Sn concentrations in lysosomes with rather acidic microenvironment. Furthermore, these two probes have low toxicity and can be used as imaging probes for monitoring Sn(2+) not only in live KB cells (eukaryotic) but also in Streptococcus mutans cells (prokaryotic), which is a useful tool to study the physiological function of Sn(2+) in biological systems.

Interactions and effects of a stannous-containing sodium fluoride dentifrice on oral pathogens and the oral microbiome.

Numerous studies have investigated the effects of stannous ions on specific microbes and their efficacy in reducing dental plaque. Nonetheless, our understanding of their impact on the oral microbiome is still a subject of ongoing exploration. Therefore, this study sought to evaluate the effects of a stannous-containing sodium fluoride dentifrice in comparison to a zinc-containing sodium fluoride dentifrice and a control group on intact, healthy oral biofilms. Utilizing the novel 2bRAD-M approach for species-resolved metagenomics, and FISH/CLSM with probes targeting periodontal and caries associated species alongside Sn2+ and Zn2+ ions, we collected and analyzed in situ biofilms from 15 generally healthy individuals with measurable dental plaque and treated the biofilms with dentifrices to elucidate variations in microbial distribution. Although significant shifts in the microbiome upon treatment were not observed, the use of a stannous-containing sodium fluoride dentifrice primarily led to an increase in health-associated commensal species and decrease in pathogenic species. Notably, FISH/CLSM analysis highlighted a marked reduction in representative species associated with periodontitis and caries following treatment with the use of a stannous-containing sodium fluoride dentifrice, as opposed to a zinc-containing sodium fluoride dentifrice and the control group. Additionally, Sn2+ specific intracellular imaging reflected the colocalization of Sn2+ ions with P. gingivalis but not with other species. In contrast, Zn2+ ions exhibited non-specific binding, thus suggesting that Sn2+ could exhibit selective binding toward pathogenic species. Altogether, our results demonstrate that stannous ions could help to maintain a healthy oral microbiome by preferentially targeting certain pathogenic bacteria to reverse dysbiosis and underscores the importance of the continual usage of such products as a preventive measure for oral diseases and the maintenance of health.

Structures of Anabaena calcium-binding protein CcbP: insights into Ca2+ signaling during heterocyst differentiation.

Ca2+-binding proteins play pivotal roles in both eukaryotic and prokaryotic cells. CcbP from cyanobacterium Anabaena sp. strain PCC 7120 is a major Ca2+-binding protein involved in heterocyst differentiation, a process that forms specialized nitrogen-fixing cells. The three-dimensional structures of both Ca2+-free and Ca2+-bound forms of CcbP are essential for elucidating the Ca2+-signaling mechanism. However, CcbP shares low sequence identity with proteins of known structures, and its Ca2+-binding sites remain unknown. Here, we report the solution structures of CcbP in both Ca2+-free and Ca2+-bound forms determined by nuclear magnetic resonance spectroscopy. CcbP adopts an overall new fold and contains two Ca2+-binding sites with distinct Ca2+-binding abilities. Mutation of Asp38 at the stronger Ca2+-binding site of CcbP abolished its ability to regulate heterocyst formation in vivo. Surprisingly, the ß-barrel subdomain of CcbP, which does not participate in Ca2+-binding, topologically resembles the Src homology 3 (SH3) domain and might act as a protein-protein interaction module. Our results provide the structural basis of the unique Ca2+ signaling mechanism during heterocyst differentiation.

Association of blood pressure in the first-week of hospitalization and long-term mortality in patients with acute left ventricular myocardial infarction.

BACKGROUND: Previous studies have shown that optimal blood pressure (BP) control is necessary to outcomes in patients with acute myocardial infarction (AMI). Acute left ventricular MI is a prevalent type of AMI with poor prognosis. We aimed to analyze the associations between BP control in the first 7 days of hospitalization and long-term mortality specific to patients with isolated left ventricular MI. METHODS: A total of 3108 acute left ventricular MI patients were included in this analysis. The average BP on the first seven days of hospitalization was categorized into 10-mmHg increments. The primary and secondary outcomes were all-cause death and cardiac death, respectively. Cox models were used to assess the association of outcomes with BP during hospitalization. RESULTS: The median length-of-stay was 7 (IQR 6-10) days. The relationship between systolic BP (SBP) or diastolic BP (DBP) followed a U-shaped curve association with outcomes. All-cause mortality was higher in patients with lower SBP (≤90 mmHg) (adjusted hazard ratios (HRs) 7.12, 95% confidence interval (CI) 3.13-16.19; p < 0.001) and DBP (<60 mmHg) (HR 1.76, 95% CI 1.14-2.71; p = 0.011) [reference: 110 < SBP ≤120 mmHg; 70 < DBP ≤ 80 mmHg], respectively. Furthermore, primary outcome was higher in patients with higher SBP (>130 mmHg) (HR 1.51, 95% CI 1.12-2.03; p = 0.007) and DBP (>80 mmHg) (HR 1.61, 95% CI 1.20-2.18; p = 0.002), respectively. CONCLUSION: Maintaining a SBP from 90 to 130 mmHg and a DBP from 60 to 80 mmHg may be beneficial to patients with acute left ventricular MI in the long run.

Caspase-11-Gasdermin D-Mediated Pyroptosis Is Involved in the Pathogenesis of Atherosclerosis.

Background: Pyroptosis is a form of cell death triggered by proinflammatory signals. Recent studies have reported that oxidized phospholipids function as caspase-11 agonists to induce noncanonical inflammasome activation in immune cells. As the levels of oxidized phospholipids derived from ox-LDL are largely elevated in atherosclerotic lesions, this study sought to determine whether oxidized lipids trigger pyroptosis and subsequent inflammation in the pathogenesis of atherosclerosis. Methods and Results: In our current study, after integrating transcriptomic data available from the Gene Expression Omnibus with data from hyperlipidemic mice and ox-LDL-treated peritoneal macrophages, we discovered that caspase-4/11-gasdermin D-associated inflammatory signaling was significantly activated. Consistently, the mRNA expression of caspase-4 and gasdermin D was upregulated in peripheral blood mononuclear cells from patients with coronary heart disease. In particular, the expression of caspase-4 was closely associated with the severity of lesions in the coronary arteries. An in vivo study showed that caspase-11-gasdermin D activation occurred in response to a high-fat/high-cholesterol (HFHC) diet in ApoE-/- mice, while caspase-11 deletion largely attenuated the volume and macrophage infiltration of atherosclerotic lesions. An in vitro mechanistic study showed that caspase-11-mediated inflammation occurred partly via gasdermin D-mediated pyroptosis in macrophages. Suppressing gasdermin D in HFHC-fed ApoE-/- mice via delivery of an adeno-associated virus markedly decreased lesion volume and infiltrating macrophage numbers. Conclusion: Caspase-11-gasdermin D-mediated pyroptosis and the subsequent proinflammatory response in macrophages are involved in the pathogenesis of atherosclerosis. Therefore, targeting the caspase 11-gasdermin D may serve as an alternative strategy for the treatment of atherosclerosis.

A fluoride activated methylene blue releasing platform for imaging and antimicrobial photodynamic therapy of human dental plaque.

We report a F- activated methylene blue (MB) releasing platform for imaging and antimicrobial photodynamic therapy (aPDT). By utilizing this platform, one of the selected probes, FD-F3, displays a remarkable near-infrared fluorescence and absorption increase towards F- with good selectivity and low detection limit. This probe has been successfully applied for visualizing F- and performing F- activated aPDT in naturally grown human plaque biofilms.

Enhancement of nano-hydroxyapatite bonding to dentin through a collagen/calcium dual-affinitive peptide for dentinal tubule occlusion.

Calcium phosphate crystals, as the main component of dentin and enamel, have been widely used for the occlusion of dentinal tubules. However, the low bond strength and poor sealing effect limit their clinical practicality. In this study, a collagen/calcium dual-affinitive peptide E8DS (EEEEEEEEDSpESpSpEEDR) and nano-hydroxyapatite (nano-HAp) flocculi were developed to seal dentin tubules for reducing dentin hypersensitivity, whereas the E8DS peptides were pre-applied to improve the adhesion of occlusive hydroxyapatite coating on dentin collagen matrix for the long-lasting sealing effect and relief from hypersensitivity. Our study showed that E8DS peptides had a strong affinity with dentin collageneous matrix that almost 43.7% of initial E8DS peptides immobilized on exposed dentin samples remained detained after continuous washing by distilled water for four weeks at a rate of 1 mL/min. Nano-HAp flocculi were obtained by re-neutralization of HAp-HCl solution and then brushed onto the surfaces of pre-treated human dentin disks with E8DS peptides, which showed a perfect occlusion of exposed dentinal tubules, as compared with Nano-HAp only and a commercial desensitizer, Green Or. With only around 10-min E8DS peptide pre-treatment, the occlusive mineral layers remained intact against consecutively stirred washing in phosphate-buffered saline or coke for 15 min, and 6 min of tooth-brushing, which implied that our E8DS peptide could comparatively improve the durability of sealant-dentin interface bonds for long-lasting dentine desensitization.

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120.

Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation.

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.