Confocal Laser Scanning Microscopy Studies Research Articles

Comparative evaluation of different surface pretreatment methods on the depth of penetration of adhesive resin in sandwich technique: A confocal laser scanning microscopy study

Comparative evaluation of different surface pretreatment methods on the depth of penetration of adhesive resin in sandwich technique: A confocal laser scanning microscopy study

P-gp inhibition and enhanced oral bioavailability of amikacin Sulfate: A novel approach using Thiolated Chito-PEGylated Lipidic Hybrids

This study aimed to develop oral lipidic hybrids of amikacin sulfate (AMK), incorporating thiolated chitosan as a P-glycoprotein (P-gp) inhibitor to enhance intestinal absorptivity and bioavailability. Three formulations were designed: PEGylated Liposomes, Chitosan-functionalized PEGylated (Chito-PEGylated) Lipidic Hybrids, and Thiolated Chito-PEGylated Lipidic Hybrids. The physical characteristics of nanovesicles were assessed. Ex-vivo permeation and confocal laser scanning microscopy (CLSM) studies were conducted to evaluate the formulations’ potential to enhance AMK intestinal permeability. In-vivo pharmacokinetic studies in rats and histological/biochemical investigations assessed the safety profile and oral bioavailability. The AMK-loaded Thiolated Chito-PEGylated Lipidic Hybrids exhibited favorable physical characteristics, higher ex-vivo permeation parameters, and verified P-gp inhibition via CLSM. They demonstrated heightened oral bioavailability (68.62% absolute bioavailability) and a sufficient safety profile. Relative bioavailability was significantly higher (1556.3% and 448.79%) compared to PEGylated Liposomes and Chito-PEGylated Lipidic Hybrids, respectively, indicating remarkable oral AMK delivery with fewer doses, reduced side effects, and enhanced patient compliance.

Influence of root canal moisture on the penetration of TotalFill bioceramic sealer into the dentinal tubules: A confocal laser scanning microscopy study

Abstract Introduction: Bioceramic (BC) sealers exhibit multiple desirable biologic and physical properties. The aim of this study was to evaluate the effect of root canal moisture on the depth of TotalFill BC sealer penetration into dentinal tubules at different levels using a confocal laser scanning microscope (CLSM). Materials and Methods: The manuscript of this laboratory study has been written according to preferred reporting items for laboratory studies in endodontology 2021 guidelines. Forty single-rooted extracted human premolars were decoronated using a low-speed diamond disc, followed by root canal preparation using nickel–titanium files under copious irrigation. The samples were randomly allocated for obturation into four groups n = 10 based on the sealer and root canal moisture status. Group A: TotalFill BC sealer in wet canal, Group B: TotalFill BC sealer in dry canal, Group C: AH Plus sealer in wet canal, and Group D: AH Plus sealer in dry canal. The roots were sectioned at three different distances from the apex. The root segments were examined using CLSM to measure the extent of sealer penetration into the dentinal tubules. The data underwent analysis through a three-way mixed ANOVA test, followed by post hoc assessments (P < 0.05). Results: In wet canals, the tubular penetration of TotalFill BC sealer was notably greater than that of AH plus, with a statistically significant difference. However, AH Plus sealer had a statistically significant tubular penetration in the dry canals compared to the wet. Greater sealer penetration was observed at 5 mm from the apex, followed by 3 mm, and the least penetration depth was found at 1 mm from the apex. Conclusions: The presence of root canal moisture and distance from the apex had a significant impact on the penetration of TotalFill BC sealer into the dentinal tubules.

Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

AbstractOxygen (O2)-sensing matrices are promising tools for the live monitoring of extracellular O2 consumption levels in long-term cell cultures. In this study, ratiometric O2-sensing membranes were prepared by electrospinning, an easy, low-cost, scalable, and robust method for fabricating nanofibers. Poly(ε-caprolactone) and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, which was used as the O2-sensing probe, and rhodamine B isothiocyanate, which was used as the reference dye. The functionalized scaffolds were morphologically characterized by scanning electron microscopy, and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy, thermogravimetric analysis, and water contact angle measurement. The sensing capabilities were investigated by confocal laser scanning microscopy, performing photobleaching, reversibility, and calibration curve studies toward different dissolved O2 (DO) concentrations. Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5 to 20% and good stability under ratiometric imaging. In addition, the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines, namely metastatic melanoma cell line SK-MEL2, breast cancer cell line MCF-7, and pancreatic ductal adenocarcinoma cell line Panc-1, thus recreating a suitable biological environment in vitro. These O2-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O2 content during drug testing/validation and tissue regeneration processes. Graphic abstract

Synergistically engineered nanotransethosomes for co-delivery of methotrexate and baicalin for enhanced transdermal delivery against rheumatoid arthritis: formulation, characterisation and in vivo pharmacodynamic evaluation

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease that significantly impacts the quality of life of those affected. Owing to the complex pathophysiology of RA, it is not possible for any singular treatment to entirely impede the progression of the disease. Hence, the current study aimed to adopt a holistic and synergistic approach towards the management of RA by means of a co-delivery strategy involving methotrexate (MTH), a conventional slow-acting anti-rheumatic drug, and baicalin (BCN), a bioactive phytochemical using a transethosomal (TRS) gel formulation.Purpose: The present study aims to evaluate the potential benefits of administering MTH and BCN in nanoparticulate form, which may lead to improved stability and solubility, as well as enhanced penetration into the arthritic tissues of interest.Methods and results: The MTH-BCN-TRS that were synthesised exhibited small particle size of 151.3 nm and polydispersity index of 0.125, as well as a favourable zeta potential of −32.22 mV. Additional assessments were conducted, including a pharmacokinetic analysis, TEM, skin permeation analysis and confocal microscopy. According to the Confocal laser scanning microscopy (CLSM) study, the formulated MTH-BCN-TRS gel exhibited superior MTH and BCN permeation through the skin layers when compared to the MTH-BCN suspension gel. The MTT experiment on Raw 264.7 and SW982 cell lines revealed a considerable reduction (p < .05) in the IC50 value of the MTH-BCN-TRS formulation (9.2 mM and 43.2 mM, respectively) in comparison to the drug suspension. According to the findings of the in vivo study, it was found that the MTH-BCN-TRS gel exhibits significantly promising anti-arthritic properties when compared to the conventional diclofenac gel. This was demonstrated through histopathological studies and radiographic analysis. Furthermore, skin irritation investigation on Wistar albino rats confirmed that the formulated MTH-BCN-TRS is a safe option for topical treatment on the skin. The present study has confirmed that the formulated TRS vesicles are a valuable carrier for the transdermal delivery of MTH and BCN, which may be used for the management of rheumatoid arthritis.

Comparative Evaluation of Penetration of Various Nano-sized Intra-canal Medicaments: An In Vitro Confocal Laser Scanning Microscopic Study

ABSTRACT Background: To assess and contrast the depth of penetration of conventional calcium hydroxide, nanoparticle calcium hydroxide, silver nanoparticles, and combination of nanoparticle calcium hydroxide and silver nanoparticles into dentinal tubules using confocal laser scanning microscope. Materials and Methods: Eighty human single-rooted teeth were decoronated and instrumented up to Protaper Universal F3. Teeth were separated into four groups at random (n = 20) following chemomechanical preparation. Group 1: Control Conventional Calcium Hydroxide; Group 2: Nanoparticle Calcium Hydroxide; group 3: Silver Nanoparticles; group 4: Combination of Nanoparticle Calcium Hydroxide and Silver Nanoparticles. With the use of lentulo spirals, medications were injected into the canals, stirred up with ultrasound, and then kept at 37°C for 24 hours. A 1 mm thick slice was obtained at 5 mm from the root apex by sectioning the root perpendicular to the long axis of the tooth and then put on a glass slide to measure the depth of penetration using a confocal laser microscope. Statistical Analysis: To quantify the depth of penetration among the four groups, a one-way ANOVA was used and post hoc–Tukey’s test was used to compare between groups. Results: All of the groups were statistically significant, with group 4 showing the greatest depth of penetration, followed by group 2 and group 3. In contrast, all other groups in the intergroup comparison were statistically significant (P value 0.05) aside from the comparison of group 2 to group 3. Conclusion: Silver nanoparticles and nanoparticle calcium hydroxide together demonstrated greater penetration than calcium hydroxide, silver nanoparticles, and nanoparticle calcium hydroxide alone.

Shrimp Oil-Enriched Mayonnaise Prepared Using Fish Myofibrillar Protein as a Substitute for Egg Yolk: Physical, Rheological, and Sensory Properties

The effect of SO (shrimp oil) at various levels (5, 10, and 15%) on the stability of mayonnaise was investigated. Droplet size (d32 and d43), polydispersity index, and microstructure results showed an upsurge in droplet sizes with augmenting level of SO in mayonnaise (5 to 15%) (p &lt; 0.05). SO imparted a bright orange color to the mayonnaise as evidenced by increased a* and b* values with lower L* values (p &lt; 0.05). Moreover, the impact of a fish myofibrillar protein (FMP) substitution for egg yolk (0, 25, 50, 75%) in mayonnaise containing SO (5% and 10%) was also studied. Increasing the level of FMP substitution in SO-added mayonnaise showed a dilution effect and reduced a* and b* values (p &lt; 0.05). In addition, excessive FMP substitution up to 75% drastically increased centrifugal and thermal creaming indices, indicating lowered stability (p &lt; 0.05). Nevertheless, with the augmenting FMP substitutions, the viscosity, texture, and rheological properties in mayonnaise became lower (p &lt; 0.05). However, there were no differences in overall acceptability scores between 5% SO-added mayonnaise with 25% FMP substitution (SO5:FMP25) and 5% SO-added mayonnaise without FMP substitution (SO5:FMP0) (p &gt; 0.05). A confocal laser scanning microscopic (CLSM) study revealed a smaller droplet and less aggregation in the SO5:FMP0 sample, compared to SO5:FMP25. The incorporation of SO and FMP substitution yielded the resulting mayonnaise, which met the requirements of a healthy food since SO is rich in PUFA and the replacement of egg yolk by FMP can contribute several health benefits. The incorporation of SO as well as FMP as substitution for egg yolk therefore has potential in the development of functional foods.

Combined hydroxyphenyl-modified adhesive with the ion-pair strategy for long-lasting delivery of clonidine in single-layer matrix-type patch

Existing long-acting patches usually use controlled-release membranes to achieve constant rate delivery of the drug, which is complex and costly to prepare due to the 5-layer structure, so there is a necessity to develop a method to simplify the patch design and to control constant rate drug delivery. This study prepared a 7-day long-acting clonidine (CLO) patch using the ion-pair strategy based on hydroxyphenyl-modified pressure-sensitive adhesive (HP-PSA) and clarified the molecular mechanism of controlled delivery. Single factorial and central composite design (CCD) experiments were used to optimize the patch formulation, which was evaluated by a pharmacokinetic study. The controlled drug delivery mechanism was investigated by FTIR, 1H NMR, differential scanning calorimetry (DSC), confocal laser scanning microscopy (CLSM), molecular modeling, and rheology study. The optimized patch was formulated with 4.80% (w/w) CLO-AA (azelaic acid) as API and HP-PSA as PSA matrix and 95.00 μm thickness. The single-layer patch not only simplifies the preparation process but also has a zero-level skin penetration profile (r = 0.9592) in vitro. Compared with the commercial patch (AUC0-t = 7748.51 ± 662.76 h ng/ml, MRT0-t = 71.04 ± 9.30h), the pharmacokinetic behavior of the optimization group was semblable (AUC0-t = 8160.28 ± 694.04 h ng/ml, MRT0-t = 63.19 ± 12.38h). Mechanistic studies showed that ion-pair improved drug interaction with HP-PSA, leading to controlled drug release. Changes in drug physicochemical properties decreased skin permeability and increased drug-skin interaction, resulting in a constant delivery rate. In conclusion, the 7-day long-acting drug delivery of the CLO patch was achieved by the ion-pair strategy, which simplified the preparation process, and inspired a long-acting transdermal drug delivery system.

In vitro efficacy of Er:YAG laser-activated irrigation versus passive ultrasonic irrigation and sonic-powered irrigation for treating multispecies biofilms in artificial grooves and dentinal tubules: an SEM and CLSM study

BackgroundMultispecies biofilms located in the anatomical intricacies of the root canal system remain the greatest challenge in root canal disinfection. The efficacy of Er:YAG laser-activated irrigation techniques for treating multispecies biofilms in these hard-to-reach areas has not been proved. The objective of this laboratory study was to evaluate the effectiveness of two Er:YAG laser-activated irrigation techniques, namely, photon-induced photoacoustic streaming (PIPS) and shock wave-enhanced emission photoacoustic streaming (SWEEPS), in treating multispecies biofilms within apical artificial grooves and dentinal tubules, in comparison with conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), and sonic-powered irrigation (EDDY). Two types of multispecies root canal biofilm models were established in combination with two assessment methods using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) with the aim to obtain more meaningful results.MethodsNinety extracted human single-rooted premolars were chosen for two multispecies biofilm models. Each tooth was longitudinally split into two halves. In the first model, a deep narrow groove was created in the apical segment of the canal wall. After cultivating a mixed bacterial biofilm for 4 weeks, the split halves were reassembled and subjected to five irrigation techniques: CNI, PUI, EDD, PIPS, and SWEEPS. The residual biofilms inside and outside the groove in Model 1 were analyzed using SEM. For Model 2, the specimens were split longitudinally once more to evaluate the percentage of killed bacteria in the dentinal tubules across different canal sections (apical, middle, and coronal thirds) using CLSM. One-way analysis of variance and post hoc multiple comparisons were used to assess the antibiofilm efficacy of the 5 irrigation techniques.ResultsRobust biofilm growth was observed in all negative controls after 4 weeks. In Model 1, within each group, significantly fewer bacteria remained outside the groove than inside the groove (P < 0.05). SWEEPS, PIPS and EDDY had significantly greater biofilm removal efficacy than CNI and PUI, both from the outside and inside the groove (P < 0.05). Although SWEEPS was more effective than both PIPS and EDDY at removing biofilms inside the groove (P < 0.05), there were no significant differences among these methods outside the groove (P > 0.05). In Model 2, SWEEPS and EDDY exhibited superior bacterial killing efficacy within the dentinal tubules, followed by PIPS, PUI, and CNI (P < 0.05).ConclusionEr:YAG laser-activated irrigation techniques, along with EDDY, demonstrated significant antibiofilm efficacy in apical artificial grooves and dentinal tubules, areas that are typically challenging to access.

Analysis of the depth of penetration of an epoxy resin-based sealer following a final rinse of irrigants and use of activation systems: An in vitro study.

The objective of the study was to compare and evaluate the depth of penetration of an epoxy resin-based sealer following a final rinse of 17% ethylenediaminetetraacetic acid (EDTA) and 18% 1-hydroxyethylidene 1, 1-diphosphonate (HEDP), with diode laser and passive ultrasonic activation (PUI): an in vitro confocal laser scanning microscopy study. Fifty-two extracted human mandibular premolar teeth with single root and single canal were selected. They were disinfected in 0.1% thymol solution, cleaned of calculus and soft tissues, and stored in 0.1% thymol solution till use. All teeth were radiographed and selected as per the inclusion and exclusion criteria. The teeth were decoronated using a diamond disk under copious water spray to acquire a standardized root length of 14 mm. Working length was established by inserting a size 10-K file into each root canal until it is visible at the apical foramen and by subtracting 1 mm from the recorded length. Instrumentation of the root canal was done till master apical file size of F3 using ProTaper universal, rotary instruments. The canals were irrigated with 2 mL of 3% sodium hypochlorite between successive files. Teeth were randomly divided into four subgroups n = 12 according to the intervention. Passive ultrasonic irrigation and diode laser were used to activate the irrigants. Final irrigation was performed with distilled water. These specimens were examined using confocal laser scanning microscope (OLYMPUS FLUOVIEW FV 3000) for dentinal tubule penetration of the sealer. Two-way ANOVA test and Tukey's multiple post hoc test were used for statistical analysis. Highly significant difference was seen between the groups with EDTA and HEDP, with HEDP demonstrating the highest penetration. Among the activation techniques used in this study, PUI showed the highest penetration of the sealer. The least penetration was seen with diode laser activation and EDTA. The irrigation activation techniques significantly influence the penetration of sealer into root dentinal tubules. When penetration of sealer with different irrigation techniques and irrigants was evaluated, significant greater level of sealer penetration was attained with PUI activation of HEDP.

Impact of miconazole nitrate ferrying cationic and anionic nanoemulsion and gels on permeation profiles of across EpiDerm, artificial membrane, and skin: Instrumental evidences

Based on our previous report, the study was extended to investigate the impact of miconazole nitrate (MCN) loaded cationic/anionic nanoemulsions and nanoemulsion gels on permeation behaviour across artificial-membrane, EpiDerm, and rat skin. Nanoemulsions and gels were evaluated for size, charge, viscosity, size-distribution, pH, and percent entrapment efficiency (%EE). In vitro drug diffusion across artificial membrane and EpiDerm were conducted to get diffusion coefficients. Permeation profiles were studied using rat skin to investigate mechanistic insight of formulated mediated permeation followed by CLSM (confocal laser scanning microscopy), SEM (scanning electron microscopy), AFM (atomic force microscopy), and irritation studies. Results showed that MCNE11-Rh (probed cationic nanoemulsion at pH ∼ 7.2) and MNE11-Rh (probed anionic nanoemulsion at pH ∼ 7.2) showed size values of 158 nm and 145 nm, respectively whereas MCNE11-GR (probed cationic nanoemulsion gel at pH ∼ 6.8) and MNE11-GR (probed anionic nanoemulsion gel at pH ∼ 6.8) exhibited size values 257 nm and 243 nm, respectively. The %EE values were found to be as 91.5 % and 89.6 % for MCNE11-Rh and MNE11-Rh, respectively. The gels (∼6000 cP) elicited relatively high viscosity than nanoemulsions (∼3300 – 3500 cP). MCNE11-GR showed the highest values of permeation flux, diffusion rate, diffusion coefficient (D), and permeation coefficient (P) across artificial membrane, EpiDerm, and rat skin which may be attributed to three potential factors (cationic charge, composition, and hydration by the hydrophilic gel) working in tandem. Transepidermal water loss (TEWL) by the MCNE11-GR was maximum (14.4 g/m2h) than control (6.1 g/m2h) indicating augmented interaction of MCNE11-Rh with skin components. Conclusively, cationic nanoemulsion gel was promising carrier for enhanced permeation and the drug access to the dermal region to treat deep seated fungal infections.

A pilot study comparing the disinfecting effects of commercialized stable ClO2 solution (free of activation) with conventional H2O2 on dental unit waterlines in the dental practice setting.

Disinfection of dental unit waterlines (DUWLs) plays a key role in control and prevention of nosocomial infection in a dental clinic. The most conventional disinfectant is hydrogen peroxide (H2O2), while chlorine dioxide (ClO2) has been considered however was limited by the "activation" procedures. With the availability of commercialized stable ClO2 solution (free of activation), direct application of ClO2 in the dental practice became possible. This study was designed to compare the disinfecting effects of stable 5 ppm of ClO2 solution with conventional 0.24% of H2O2 on DUWLs in dental practice. Studies of colony-forming units (CFUs), confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM) were employed for evaluation. In CFUs studies, we found that the efficiency of ClO2 was no less than those of H2O2. In the morphological studies, the stronger disinfecting effects of ClO2 was verified by both CLSM and SEM studies for removal and prevention of biofilm. Importantly, ClO2 solution achieved a better disinfecting efficiency not only at the surface of bacterial biofilm, but also, it has penetrating effects, presented disinfecting effects from the surface to the bottom of the biofilm. This pilot study provided evidence regarding the efficiency of stable ClO2 solution on disinfection of DUWLs in the dental practice setting. Application of stable ClO2 solution in dental practice is therefore become possible.

New formulation technology to boost sun protection.

As every skin type worldwide is concerned by photoprotection, with consumers preferring cosmetic elegant and efficient sunscreen products, we aim at developing the most performant and desirable sun care products. We selected an interesting polymer, abbreviated AAHCP and designed scanning electron cryomicroscopy (cryoSEM), small angle and wide angle X-ray scattering and confocal laser scanning microscopy studies to understand its behaviour in solution and in simplex sun care formulations. This allowed us to develop innovative sunscreen formulation technology that was demonstrated by in vitro and in vivo photoprotection methods. Comprehensive photoprotection evaluations were made on the fully developed sun protection product. We observed the polymer oil structuring properties as well as its ability to form small and stable droplets in simplex emulsions. In vitro and in vivo sun protection factor (SPF) measurements demonstrated the sun protection boosting efficacy of AAHCP polymer in several emulsions or as a stand-alone emulsifier. This formulation technology also allowed to filtering system concentration optimization. Use-test performed on a fully developed AAHCP-based sunscreen validated its optimal performances as well as its ideal cosmetic features, with non-sticky, non-greasy perception and invisible skin result. For the first time, thanks to a new specific polymer creating a new type of emulsion, we succeed in reconciliate in a single sun care product maximal SPF efficacy, resistance to numerous stresses and optimal sensoriality.

Controlling the Morphological Features, Aspect Ratio and Emission Patterns of Supramolecular Copolymers by Restricted Dimensional Growth.

One of the bottlenecks associated with supramolecular polymerization of functional π-systems is the spontaneous assembly of monomers leading to one- or two-dimensional (1D or 2D) polymers without control over chain length and optical properties. In the case of supramolecular copolymerization of monomers that are structurally too diverse, preferential self-sorting occurs unless they are closely interacting donor-acceptor pairs. Herein, it is established that the spontaneous 1D polymerization of a phenyleneethynylene (PE) derivative and the 2D polymerization of a Bodipy derivative (BODIPY) can be controlled by copolymerizing them in different ratios, leading to unusual spindle-shaped structures with controlled aspect ratio, as evident by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) studies. For example, when the content of BODIPY is 50 % in the BODIPY-PE mixture, the 1D polymerization of PE is significantly restricted to form elongated spindle-like structures having an aspect ratio of 4-6. The addition of 75 % of BODIPY to PE resulted in circular spindles having an aspect ratio of 1-2.5, thereby completely restricting the 1D polymerization of PE monomers. Moreover, the resultant supramolecular copolymers exhibited morphology and aspect ratio dependent emission features as observed by the time-resolved emission studies.

Application of Box–Behnken design for the optimization of Plumbago zeylanica extract-loaded niosomes: preparation, characterization and in vitro antidiabetic and antioxidant activity

This study aimed to develop and evaluate a niosomal formulation loaded with Plumbago zeylanica extract (PZE). The solvent evaporation method was used to prepare niosomes and optimized using the Box–Behnken design. The optimized formulation (PZE-Ns-Opt) was characterized for drug release, DPPH assay, α-amylase inhibition assay, α-glucosidase inhibition assay, and confocal laser scanning microscopy (CLSM) study. PZE-Ns-Opt showed a vesicle size of 253.6 nm, PDI of 0.108, entrapment efficiency of 62.4%, and drug release of 84.01%. The CLSM image of the rat’s intestine suggested that the rhodamine red B-loaded PZE-Ns-Opts showed superior penetration compared to the control. Further, the antioxidant activity of the prepared formulation was exhibited as 89.46%±0.016 as compared to PZE (78.10%±0.005). In addition to this, α- amylase activity was inhibited by 95.11%±4.62, 85.88%±2.56, and 89.87%±3.65 by acarbose, PZE, and PZE-Ns-Opt, respectively. In comparison, the α-glucosidase activity was inhibited by 88.47%±1.04, 81.07%±0.50 and 85.78%±0.71 by acarbose, PZE, and PZE-Ns-Opt, respectively. To conclude, the development of PZE-Ns-Opt formulation and its characterization showed the establishment’s authenticity. The in vitro antidiabetic, antioxidant studies expressed the protective effect from oxidative stress, CLSM of rat’s intestine ability to penetrate to great extent and could be a promising candidate for the management of diabetes mellitus.

α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.

Antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin on Enterococcus faecalis: A confocal laser scanning microscopic study

Background: Sealers with antimicrobial properties play an important role in endodontic therapy success especially against Enterococcus faecalis infection found in failed root canal therapy. Addition of antibiotic agents to endodontic sealers may show significant increase in their antibacterial properties both against anaerobic and aerobic microbes. The purpose of the present study was to evaluate antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin against E. faecalis and post-root canal treatment viability of Enterococcus faecalis on the first and seventh day. Methods: A total of 60 extracted human mandibular premolar teeth were decoronated after initial decontamination with 1% NaOCl. Root length standardized to 12 mm. Canal instrumentation was done using ProTaper Universal file system till size F2 using 5.25% NaOCl. It was then infected with a pure strain of E. faecalis for a period of four days. Obturation was done using plain sealer, (n=30) and sealer-antibiotic combination, (n=30). Half of the teeth were sectioned at 24 hours (S, SA) and other half were sectioned seven days after obturation (S7, SA7). All samples were stained with SYTO9 and propidium iodide for imaging under Confocal Laser Scanning microscope. Statistical analysis was performed with the statistical software SPSS v. 17.0 (SPSS for Windows; SPSS Inc, Chicago, IL). Data was analysed using One Way ANOVA and post hoc Tukey test to determine statistical significance with p value &lt; 0.01 considered significant. Results: Statistically significant differences were observed in green to red ratio between group S (9.561976) and S7 (0.435418) (p &lt; 0.01). There was no difference found between SA (mean of green to red ratio, (0.70431) and SA7 (mean of green to red ratio, 0.85184). Conclusions: Antibiotics added to the sealer effectively eradicated of E. faecalis 24 hours post-obturation. However, after seven days, plain sealer was as effective as sealer-antibiotic combination.

Small Molecule AIEgens for Illuminating Sub-Cellular Endoplasmic Reticulum, Mitochondria, and Lysosomes.

Organelles are the working hubs of the cells. Hence, visualizing these organelles inside the cells is highly important for understanding their roles in pathological states and development of therapeutic strategies. Herein, we report the development of a novel highly substituted oxazoles with modular scaffolds (AIE-ER, AIE-Mito, and AIE-Lyso), which can home into endoplasmic reticulum (ER), mitochondria, and lysosomes inside the cells. These oxazoles showed remarkable aggregation-induced emission (AIE) property in water and in the solid state due to dual intramolecular H-bonding, which was confirmed by pH- and temperature-dependent fluorescence studies followed by molecular dynamics (MD) simulations and density functional theory (DFT) calculations. Confocal laser scanning microscopy studies revealed that AIE-ER, AIE-Mito, and AIE-Lyso efficiently homed into ER, mitochondria and lysosomes, respectively, in the HeLa cervical cancer cells and non-cancerous human retinal pigment epithelial RPE-1 cells within 3 h without showing any toxicity to the cells with high sub-cellular photostability. To the best of our knowledge, this is the first report of highly substituted oxazole-based small molecule AIEgens for organelle imaging. We anticipate these novel AIEgens have promise to image sub-cellular organelles in different diseased states as well as understanding the inter-organelle interactions towards the development of novel therapeutics.

Beta Caryophyllene-Loaded Nanostructured Lipid Carriers for Topical Management of Skin Disorders: Statistical Optimization, In Vitro and Dermatokinetic Evaluation.

This work aimed to overcome the disadvantages of the oral administration of beta-caryophyllene and boost efficiency by developing a nanostructured lipid carrier for topical administration of the drug in skin disorders. The heat emulsification method was utilized to produce beta-caryophyllene-loaded nanostructured lipid carriers. The newly created formulation was examined for its particle size, entrapment efficiency, and zeta potential after being improved using the Box-Behnken Design. The chosen formulation underwent tests to determine its ex vivo skin retention, dermatokinetic, in vitro release, antioxidant, and confocal laser scanning microscopy study. The findings of the characterization of the nanostructured lipid carriers demonstrated that the particles had a spherical form and a size of 210.86 nm (0.263 polydispersity index). The entrapment efficiency was determined to be 86.74%, and the zeta potential was measured to be -26.97 mV. The in vitro release investigation showed that nanostructure lipid carriers were capable of releasing regulated amounts of beta-caryophyllene for up to 24 hrs. In comparison to the traditional gel formulation, the ex vivo investigation demonstrated a 1.94-fold increase in the skin's capacity to retain the substance. According to the findings of the study, nanostructure lipid carriers loaded with beta-caryophyllene have the potential to be investigated for use as a topical administration method in skin disorders with enhanced skin retention and effectiveness.

Micromorphology and analysis of dentin surfaces after preparation with Er:YAG laser and application of self-etch adhesive system with 10-MDP. A confocal laser scanning microscope study.

The purpose of this study was to analyze and compare the micromorphology of the hybrid layer and the dentinal surface when the dentin was prepared with an Er:YAG laser or burs, and to find if there was any difference if the time of application of the self-etching primer on the dentin prepared with an Er:YAG laser was doubled.