Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold.

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

Characterization and in vitro bioactivity evaluation of polyvinyl alcohol incorporated electro spun chitosan/ fluor apatite nanofibrous scaffold for bone tissue engineering.

In this study, polyvinyl alcohol/chitosan/fluor apatite scaffolds with different concentrations of polyvinyl alcohol of 0.075 g ml-1 and 0.1 g ml-1, respectively named A and B fabricated by electrospinning method to use in tissue engineering. By examining the scaffolds by FE-SEM, an appetite layer formation which was on the scaffold surface was clearly observable. Increasing the concentration of polyvinyl alcohol from 0.075 g ml-1 to 0.1 g ml-1 in the nanofibrous scaffolds improved degradation characteristic with an optimized value of 26 ± 2 and 42 ± 1 % after 28 days, respectively. The mean diameter of fibers both scaffolds was 405 nm and 212 nm, respectively. Furthermore, the percentage of porosity in both scaffolds was calculated as 84% and 91%, separately. The level of hydrophilicity of the scaffolds was measured by the dynamic contact angle method. Moreover, the increase in the percentage of polyvinyl alcohol led to the decrease in the average contact angle in scaffold A and scaffold B from 90° to 70°, respectively. The results of bone marrow culture test with MG-63 (NCBI C555) cell on the surface of scaffolds demonstrated not cytotoxicity in the resulting scaffolds as well as a suitable substrate for the adhesion and growth of the cells. According to our findings, the electrospun fluorapatite-incorporated-chitosan/polyvinyl alcohol scaffold could provide a new nanocomposite for biomedical applications.

Intervención interdisciplinaria para la prevención primaria de la caries en lactantes de 0 a 18 meses / Interdisciplinary intervention for the primary prevention of dental caries in children under 18 months

Introducción: la caries es la enfermedad crónica más frecuente en la infancia. La presencia de caries en la dentición temporal es el principal factor de riesgo para desarrollar caries en la dentición definitiva. La mayoría de los factores de riesgo de la caries son modificables y pueden convertirse en elementos para la prevención y control de la enfermedad. Con el objetivo de reducir la incidencia de caries a la edad de 18 meses se diseña una intervención interdisciplinaria de prevención primaria dirigida a familias con niños que se visitan siguiendo el Protocol d’activitats preventives i de promoció de la salut a l’edat pediátrica (PAPPS). Material y métodos: ensayo clínico no aleatorizado, realizado en dos centros de asistencia primaria de Catalunya desde enero de 2019 hasta junio de 2022. En uno de los centros se diseñó e implementó una intervención educativa de prevención primaria de la caries con consejos y habilidades para las familias. En el otro centro se mantuvo el protocolo habitual de recomendaciones. Se evaluó y comparó la incidencia de caries en ambos grupos a la edad de 18 meses con un modelo de regresión logística estimado con el programa R. Resultados: la incidencia de caries a los 18 meses fue superior en los niños del grupo control (OR = 6,0; IC 95% 1,8-20,2), a pesar de que la valoración del riesgo de caries basada en el sistema llamado Caries Management by Risk Assessment (CAMBRA) indicó mayor riesgo de desarrollo de caries en los lactantes del grupo intervención. Conclusión: la intervención interdisciplinaria de prevención primaria de la caries incorporada en los programas de salud infantil reduce la incidencia de caries en los primeros años de vida. (AU)

Introduction: caries is the most common chronic disease in childhood. The presence of caries in the primary dentition is the main risk factor for developing caries in the permanent dentition. Most of the risk factors for caries are modifiable and can become elements for the prevention and control of the disease. With the goal of reducing the incidence of caries in children at age 18 months, we designed an interdisciplinary primary prevention intervention aimed at families with children who attended routine preventive visits within the PAPPS (“Protocol d’activitats preventives i de promoció de la salut a l’edat pediàtrica”) child health programme. Methodology: non-randomized clinical trial carried out in two primary care centres in Catalonia between January 2019 and June 2022. In one of the centres, an educational intervention for the primary prevention of caries was designed and implemented to provide families with guidance and skills. In the other centre, patients received standard care. The incidence of caries was assessed and compared in both groups at age 18 months by means of a logistic regression model fitted with the R software. Results: the incidence of caries at 18 months was higher in children in the control group (OR=6.0; 95% CI: 1.8-20.2), despite the fact that the caries risk assessment by means of the “Caries Management by Risk Assessment” (CAMBRA) protocol indicated a higher risk of caries in infants in the intervention group. Conclusion: the interdisciplinary primary caries prevention intervention integrated into the child health prevention and promotion programme achieved a reduction in the incidence of caries in early childhood. (AU)

A Comparative Study of the Effect of Tooth Mousse Plus Remin Pro and Fluor Protector Gel on Enamel Erosion: An In Vitro Study.

Background: The purpose of this in vitro study was to assess the effect of Tooth Mousse Plus, Remin Pro, and Fluor Protector Gel on enamel erosion, measuring mean weight loss after exposure to a demineralizing agent. Materials and methods: A total of 60 sound-extracted permanent incisors were sectioned and enamel specimens were randomly distributed to different groups. The initial weight of all specimens was registered. The samples were randomly divided into four groups (n = 30). Group I specimens were treated with tap water (control). Groups II, III, and IV were treated with Tooth Mousse, Remin Pro, and Fluor Protector Gel application. After that, specimens were placed all together in a plastic container with 6 mL of a soft drink and immersed for 8 minutes at room temperature, dried, and weighed. Specimens were weighed after each immersion period and mean weight loss was calculated. The data was analyzed for probability distribution using the Kolmogorov-Smirnov test. The intergroup comparison was done using a one-way analysis of variance (ANOVA) followed by post hoc analysis. Results: According to pairwise comparisons in post hoc analysis, the weight of specimens at baseline was significantly greater than the weight of specimens on day 12. The difference in the mean weight of the specimen from baseline to day 12 was 2.833 mg for group I, 2.367 mg for group II, 1.467 mg for group III, and 2.133 mg for group IV. Conclusion: Tooth Mousse Plus, Remin Pro, and Fluor Protector Gel have no significant effect on dental erosion. How to cite this article: Shukla K, Saxena A, Joshi J, et al. A Comparative Study of the Effect of Tooth Mousse Plus Remin Pro and Fluor Protector Gel on Enamel Erosion: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-1):S57-S62.

Simplifying Barley Leaf Rust Research: An Easy and Reproducible Infection Protocol for Puccinia hordei on a Small Laboratory Scale.

Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species. Key features Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation Plant age-independent protocol Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility No specialized equipment needed Includes simple spore harvesting method Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat) Protocol is also applicable for a detached leaf assay Graphical overview.

Plasmon-Enhanced Expansion Microscopy.

Expansion microscopy (ExM) is a rapidly emerging super-resolution microscopy technique that involves isotropic expansion of biological samples to improve spatial resolution. However, fluorescence signal dilution due to volumetric expansion is a hindrance to the widespread application of ExM. Here, we introduce plasmon-enhanced expansion microscopy (p-ExM) by harnessing an ultrabright fluorescent nanoconstruct, called plasmonic-fluor (PF), as a nanolabel. The unique structure of PFs renders nearly 15000-fold brighter fluorescence signal intensity and higher fluorescence retention following the ExM protocol (nearly 76%) compared to their conventional counterparts (<16% for IR-650). Individual PFs can be easily imaged using conventional fluorescence microscopes, making them excellent "digital" labels for ExM. We demonstrate that p-ExM enables improved tracing and decrypting of neural networks labeled with PFs, as evidenced by improved quantification of morphological markers (nearly a 2.5-fold increase in number of neurite terminal points). Overall, p-ExM complements the existing ExM techniques for probing structure-function relationships of various biological systems.

Fluorosis dental en adolescentes de instituciones educativas de Lima, Perú / Dental fluorosis in adolescents from Educational Institutions in Lima, Peru

Introducción: La fluorosis dental es una patología endémica causada por la ingestión excesiva de fluoruros que pueden producir una alteración durante el desarrollo del esmalte, y es considerado un importante problema de salud pública porque afecta la salud bucal y sistémica de la población. Objetivo: Este estudio tuvo como idea principal determinar la frecuencia y los niveles de fluorosis dental en escolares de 12 a 15 años pertenecientes a dos instituciones educativas de Lima - Perú. Método: Se realizó un estudio descriptivo de tipo transversal, la muestra estuvo conformada por 252 estudiantes, entre 12 y 15 años de dos centros educativos de Lima. El muestreo fue por selección sistemática de elementos muestrales. La fluorosis dental se evalúo mediante el Índice de Dean. Para determinar la frecuencia y los niveles de fluorosis dental, se realizó un examen clínico bucal y se desarrolló un cuestionario validado previamente. Resultados: La frecuencia de fluorosis dental fue de 44,8% (n=113) afectando más a los varones en un 27,39% (n=69) y a los adolescentes de 13 años (15,1%). En relación con los niveles de fluorosis, predominó el tipo "muy leve" (34,9%). También se encontró una asociación entre la presencia y el nivel de fluorosis con el número de aplicaciones de flúor (p<0,05). Además, se dijo que el índice comunitario de fluorosis de Dean fue de 0,43. Conclusiones: En la población escolar evaluada, la frecuencia de fluorosis fue de 44,8% y el nivel predominante de fluorosis fue muy leve. Además, el índice comunitario fue de 0,43, el cual mostró un nivel límite de importancia para la salud pública. Por lo que se puede considerar que el número de aplicaciones tópicas de flúor recibido por la población sin una planificación adecuada se podría convertir en un factor de riesgo para causar fluorosis dental.

Introduction: Dental fluorosis is an endemic pathology caused by the excessive ingestion of fluorides that can produce an alteration during the development of the enamel and is considered an important public health problema because it affects the oral and systemic health of the population. Objective: The objective of this study was to determine the frequency and levels of dental fluorosis in school children between 12 and 15 years of age belonging to two educational institutions in Lima, Peru. Method: A descriptive cross-sectional study was carried out, the sample consisted of 252 students between 12 and 15 years of age from two educational centers in Lima. Sampling was by systematic selection of sample elements. Dental fluorosis was evaluated using the Dean Index. To determine the frequency and levels of dental fluorosis a clinical oral examination was carried out and a previously validated questionnaire was developed. Results: The frequency of dental fluorosis was 44.8% (n=113) affecting more males by 27.39% (n=69) and adolescents aged 13 years (15.1%). In relation to the levels of fluorosis, the "very mild" type predominated (34.9%). An association was also found between the presence and level of fluorosis with the number of fluoride applications (p<0.05). In addition, Dean's community fluorosis index was reported to be 0.43. Conclusions: In the school population evaluated, the frequency of fluorosis was 44.8% and the predominant level of fluorosis was very mild. In addition, the community index was 0.43, which indicated a borderline level of public health importance. Therefore, it can be considered that the number of topical fluoride applications received by the population without adequate planning could become a risk factor for causing dental fluorosis.

Ammonium trifluoroborate-modified poly(ß-aminoesters): A case study for PET-guided in vivo pharmacokinetic studies of a non-viral gene delivery system.

Nucleic acid-based therapies have become a game-changing player in our way of conceiving pharmacology. Nevertheless, the inherent lability of the phosphodiester bond of the genetic material with respect to the blood nucleases severely hampers its delivery in naked form, therefore making it necessary to use delivery vectors. Among the potential non-viral vectors, polymeric materials such as the poly(ß-aminoesters) (PBAEs) stand out as promising gene carriers thanks to their ability to condense nucleic acids in the form of nanometric polyplexes. To keep advancing these systems into their translational preclinical phases, it would be highly valuable to gain accurate insights of their in vivo pharmacokinetic profile. We envisaged that positron emission tomography (PET)-guided imaging could provide us with both, an accurate assessment of the biodistribution of PBAE-derived polyplexes, as well shed light on their clearance process. In this sense, taking advantage of the efficient [19F]-to-[18F]­fluorine isotopic exchange presented by the ammonium trifluoroborate (AMBF3) group, we have designed and synthesized a new 18F-PET radiotracer based on the chemical modification of a linear poly(ß-aminoester). As proof of concept, the incorporation of the newly developed 18F-PBAE into a model nanoformulation was shown to be fully compatible with the formation of the polyplexes, their biophysical characterization, and all their in vitro and in vivo functional features. With this tool in hand, we were able to readily obtain key clues about the pharmacokinetic behavior of a series of oligopeptide-modified PBAEs (OM-PBAEs). The observations described in this study allow us to continue supporting these polymers as an outstanding non-viral gene delivery vector for future applications.

Estimating Fluor Emission Spectra Using Digital Image Analysis Compared to Spectrophotometer Measurements.

This paper describes a practical method for obtaining the spectra of lights emitted by a fluor in a liquid scintillator (LS) using a digital camera. The emission wavelength results obtained using a digital image were compared with those obtained using a fluorescence spectrophotometer. For general users, conventional spectrophotometers are expensive and difficult to access. Moreover, their experimental measurement setup and processes are highly complicated, and they require considerable care in handling. To overcome these limitations, a feasibility study was performed to obtain the emission spectrum through image analysis. Specifically, the emission spectrum of a fluor dissolved in a liquid scintillator was obtained using digital image analysis. An image processing method was employed to convert the light irradiated during camera exposure into wavelengths. Hue (H) and wavelength (W) are closely related. Thus, we obtained an H-W response curve in the 400~450 nm wavelength region, using a light-emitting diode. Another relevant advantage of the method described in this study is its non-invasiveness in sealed LS samples. Our results showed that this method has the potential to accurately investigate the emission wavelengths of fluor within acceptable uncertainties. We envision the use of this method to perform experiments in chemistry and physics laboratories in the future.

Preservation of Proteins in Human Plasma through Metal-Organic Framework Encapsulation.

Traditional cold chain systems of collection, transportation, and storage of biofluid specimens for eventual analysis pose a huge financial and environmental burden. These systems are impractical in pre-hospital and resource-limited settings, where refrigeration and electricity are not reliable or even available. Here, we develop an innovative technology using metal-organic frameworks (MOFs), a novel class of organic-inorganic hybrids with high thermal stability, as encapsulates for preserving the integrity of protein biomarkers in biofluids under ambient or non-refrigerated storage conditions. We encapsulate prostate-specific antigen (PSA) in whole patient plasma using hydrophilic zeolitic imidazolate framework-90 (ZIF-90) for preservation at 40 °C for 4 weeks and eventual on-demand reconstitution for antibody-based assays with recovery above 95% compared to storage at -20 °C. Without ZIF-90 encapsulation, only 10-30% of the PSA immunoactivity remained. Furthermore, we demonstrate encapsulation of multiple cancer biomarker proteins in whole patient plasma using ZIF-8 or ZIF-90 encapsulants for eventual on-demand reconstitution and analysis after 1 week at 40 °C. Overall, MOF encapsulation of patient biofluids is important as climate change may be affecting the stability and increase costs of maintaining biospecimen cold chain custody for the collection, transportation, and storage of biospecimens prior to analysis or for biobanking regardless of any countries' affluence.

Fluorosis dental en la primera infancia: estado del arte

La fluorosis dental se caracteriza por una hipomineralización de la estructura dental causada por ingesta excesiva de flúor sistémico. En la dentición decidua, su diagnóstico precoz es importante, dado que será un predictor para la aparición de lesiones en la dentición permanente. El objetivo de esta revisión fue describir la evidencia científica sobre la fluorosis dental en dentición decidua reportando su etiología y factores relacionados, prevalencia, diagnóstico y tratamiento. Se realizaron búsquedas electrónicas en las bases de datos Pub-Med/Medline, EbscoHost y ScienceDirect (noviembre/2020), utilizando las palabras clave "dental fluorosis", "deciduous teeth", "primary tooth", "primary teeth". El desarrollo de fluorosis dental en dentición decidua se relacionó con la ingesta de múltiples fuentes de flúor principalmente, agua potable, alimentos de la dieta, fórmulas infantiles, suplementos y uso de dentífricos fluorados en dosis inadecuadas. Algunos factores prenatales, como vivir en terrenos montañosos o de gran altitud y habitar en lugares cercanos a minas de combustión de carbón, también fueron asociados. La prevalencia de fluorosis dental reportada en los estudios varió entre 6,2 y 96,6 %, dependiendo principalmente de la concentración de flúor en agua potable. Para el diagnóstico de la fluorosis dental en la dentición decidua se deben considerar características como la localización, aspecto, extensión y color de la lesión, realizando diagnóstico diferencial con otro tipo de defectos en esmalte y dentina. Así mismo, el tratamiento de las lesiones dependerá de la severidad del defecto y condiciones individuales del paciente.

Dental fluorosis is characterized by a hypomineralization of the tooth structure caused by excessive intake of systemic fluoride. In the deciduous dentition, its early diagnosis is important since it will be a predictor for the appearance of lesions in the permanent dentition. The objective of this review was to describe the scientific evidence on dental fluorosis in deciduous dentition, reporting its etiology and related factors, prevalence, diagnosis and treatment. Electronic searches were conducted PubMed / Medline, EbscoHost and ScienceDirect (November / 2020) databases, using the keywords "dental fluorosis", "deciduous teeth", "primary tooth", "primary teeth". The development of Dental fluorosis in deciduous dentition was related to the intake of multiple sources of fluoride mainly; drinking water, diet foods, infant formulas, supplements and the use of luoridated toothpastes in inadequate doses. Some prenatal factors such as living in mountainous or high altitude terrain and living in places near coal-burning mines were also associated. The prevalence of dental fluorosis in early childhood reported in the studies varied between 6.2% and 96.6%, depending mainly on the concentration of fluoride in drinking water. For the diagnosis of dental fluorosis in the deciduous dentition, characteristics such as the location, appearance, extension and color of the lesion must be considered, making a differential diagnosis with other types of enamel and dentin defects. Evenly, the treatment of lesions will depend on the severity defects and individual patient conditions.

A Plasmonic Fluor-Lightened Microneedle Array Enables Ultrasensitive Multitarget Whole Blood Diagnosis of Anemia in A Paper Origami-Based Device.

This work reports a portable, origami-type paper device with a plasmonic fluor-labeled microneedle sensing module for the multiplexed quantification of anemia biomarkers in whole blood. Sequential steps, including serum separation, target enrichment, and multiplexed readout by a gel imager, are rapidly accomplished with the flexible and highly integrated device. The microneedle array enabled efficient sampling of trace targets from ng mL-1 to pg mL-1 level. Combined with the plasmonic fluor label, the signal is improved by ≈7.6 folds compared with the flat substrate-based assay. The device is applied to simultaneously quantify hemoglobin (Hb), ferritin, folic acid (FA), and vitamin B12 (VB12 ), which are four anemia biomarkers distributed in different environments with different concentration ranges. Featured by the small sample volume (150 µL), short assay time (20 min), low cost (2 $), robust stability, and user-friendliness, the device is promising for the rapid and accurate diagnosis of anemia in real practice.

Feasibility Study on the Discrimination of Fluor Concentration in the Liquid Scintillator Using PMT Waveform and Short-Pass Filter.

Neutrinos are difficult to detect because they weakly interact with matter, making their properties least known. The response of the neutrino detector depends on the optical properties of the liquid scintillator (LS). Monitoring any characteristic changes in the LS helps to understand the temporal variation of detector response. In this study, a detector filled with LS was used to study the characteristics of the neutrinos detector. We investigated a method to distinguish the concentrations of PPO and bis-MSB, which are fluors added to LS, through a photomultiplier tube (PMT) acting as an optical sensor. Conventionally, it is very challenging to discriminate the flour concentration dissolved in LS. We employed the information of pulse shape and PMT coupled with the short-pass filter. To date, no literature report on a measurement using such an experimental setup has been published. As the concentration of PPO was increased, changes in the pulse shape were observed. In addition, as the concentration of bis-MSB was increased, a decrease in the light yield was observed in the PMT equipped with the short-pass filter. This result suggests the feasibility of real-time monitoring of LS properties, which are correlated with the fluor concentration, using a PMT without extracting the LS samples from the detector during the data acquisition process.

Chemical, structural and cytotoxicity characterisation of experimental fluoride-doped calcium phosphates as promising remineralising materials for dental applications.

OBJECTIVES: This study aimed at evaluating the cytotoxicity, chemical and structural properties of experimental fluoride-doped calcium-phosphates as potential remineralising materials for dental applications. METHODS: Experimental calcium phosphates were formulated using ß-tricalcium phosphate, monocalcium phosphate monohydrate, calcium hydroxide, and different concentrations of calcium/sodium fluoride salts [(5 wt%: VSG5F), (10 wt%: VSG10F), (20 wt%: VSG20F)]. A fluoride-free calcium phosphate (VSG) was used as control. Each tested material was immersed in simulated body fluid (SBF), (24 h, 15 and 30 days) to assess their ability to crystallise into apatite-like. Cumulative fluoride release was assayed up to 45 days. Moreover, each powder was placed into a medium containing human dental pulp stem cells (200 mg/mL) and their cytotoxicity was analysed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (24 h, 48 h and 72 h incubation). These latter results were statistically analysed by ANOVA and Tukey's test (α = 0.05). RESULTS: All the experimental VSG-F materials produced fluoride-containing apatite-like crystals after SBF immersion. VSG20F presented prolonged release of fluoride ions into the storage media (45d). VSG, VSG10F and VSG20F showed a significant cytotoxicity at dilution of 1:1, while at 1:5, only VSG and VSG20F demonstrated a reduction in cell viability. At lower dilutions (1:10, 1:50 and 1:100) all specimens showed no significant toxicity to hDPSCs, but an increase in cell proliferation. SIGNIFICANCE: The experimental fluoride-doped calcium-phosphates are biocompatible and possess a clear ability to evoke fluoride-containing apatite-like crystallisation. Hence, they may be promising remineralising materials for dental applications.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats.

Análisis curricular de la enseñanza aprendizaje del flúor en la disciplina Estomatología General

Introducción: La enseñanza-aprendizaje de los métodos de prevención y control de la caries dental en estomatología requiere de una formación académica que implique el conocimiento y desarrollo de habilidades del uso del flúor. Objetivo: Analizar la contribución de los programas de las asignaturas de la disciplina Estomatología General del plan de estudio E de la carrera Estomatología a la sistematización en la enseñanza-aprendizaje del flúor. Métodos: Se realizó una investigación cualitativa mediante revisión de documentos curriculares de la carrera y programas de las asignaturas de la disciplina Estomatología General del plan de estudio E. Se analizaron los temas, contenidos, objetivos y sistemas de habilidades, orientaciones metodológicas y estrategias curriculares de los programas y su relación con la enseñanza aprendizaje del flúor. Resultados: Las ocho asignaturas que contribuyen al aprendizaje del flúor abarcan todos los años de la carrera, más de la mitad abordan de manera directa este contenido, en tres sus vínculos son indirectos y no se aborda dentro de los contenidos o habilidades en el programa. El sistema de conocimientos y sistema de habilidades, seguido de los objetivos, resultó ser donde más se plantea la enseñanza del flúor. Solo dos asignaturas lo abordan en las orientaciones metodológicas. Conclusiones: La estructura del diseño curricular posibilita el enfoque en sistema de la enseñanza-aprendizaje del flúor, sin embargo, no existe una estrategia de la disciplina que garantice la sistematización del conocimiento de las acciones preventivas y curativas.

Introduction: The teaching-learning process of methods for the prevention and control of dental caries in dentistry requires academic training that involves knowledge and development of skills in the use of fluoride. Objective: To analyze the contribution of the programs of the subjects of General Dentistry discipline in the Plan E of Dentistry career to the systematic teaching and learning process of the use of fluoride. Methods: A qualitative research was carried out by revising curricular documents of the studies and programs of the subjects of General Dentistry discipline of Plan E. Themes, contents, objectives and systems of abilities, methodological orientations and curricular strategies of the programs were analyzed as well as their relationship with the teaching-learning process of the use of fluoride. Results: The eight subjects that contribute to the leaning of the use of fluoride cover all the academic years of the dental studies. More than half of the subjects directly considers this topic in their programs. Three of the subjects has indirect approach of the topic in question, and it is not taught in the content or abilities in the program. The teaching of the use of fluoride was emphasized in the knowledge system and system of abilities, followed by the objective. It was only covered in two subjects, in the methodological orientations. Conclusions: The structure of the curricular design makes possible the system approach of the teaching-learning process of the use of fluoride. Unfortunately, there is no a strategy of the discipline that guarantees systematization of the knowledge of preventive and healing actions.

Effects of different toothpaste formulations on erosive tooth wear prevention: systematic review / Efeitos de diferentes dentifrícios na prevenção do desgaste dentário erosivo: revisão sistemática de literatura

Objective: erosive tooth wear is a multifactorial condition that results in loss of dental hard tissue, caused by a chemical and mechanical process. This paper aims to carry out a systematic review presenting the effects of different toothpaste formulations on the loss of dental enamel surface in vitro. Material and Methods: the searches were performed in the databases PubMed, Web of Science, LILACS and Scielo. Articles published from 2010 to 2020 were filtered, without language restriction. Articles that included abrasion and erosion protocols were searched, as they were more similar to clinical reality, since toothpaste is applied through tooth brushing. The searches with descriptors and free terms on the topic resulted in 992 articles, however only 12 were within the search criteria. Results: the selected studies appointed that association of conventional fluorides (NaF) with metallic fluorides can be a promising strategy for the reduction of surface loss by erosive tooth wear. Toothpastes containing sodium fluoride, as well as tin without chitosan, showed a reduction in surface loss, considered sufficient for individuals with medium exposure to acids. In children's toothpastes, the one containing sodium fluoride showed a reduction in surface loss when compared to non-fluoride dentifrices. Conclusion: different protocols may result in less or greater loss of enamel surface, and methodological differences should be considered. To clarify the effects of dentifrices on erosive tooth wear, other properties of dentifrices should be investigated. (AU)

Objetivo: o desgaste erosivo é uma condição multifatorial que resulta em perda de tecido duro dentário, causado por um processo químico e mecânico. Este trabalho tem como objetivo realizar uma revisão sistemática de literatura apresentando os efeitos de diferentes formulações de dentifrícios na perda de superfície de esmalte dentário in vitro. Material e Métodos: a busca foi realizada nas bases de dados PubMed, Web of Science, LILACS e Scielo. Foram filtrados artigos publicados no intervalo de 2010 a 2020, sem restrição de idioma. Buscou-se artigos que incluíssem protocolos de abrasão e erosão, por mais se assemelhar a realidade clínica, uma vez que o dentifrício é aplicado através da escovação dentária. A busca com descritores e termos livres sobre o tema encontrou 992 artigos, entretanto somente 12 estavam dentro dos critérios da pesquisa. Resultados: os estudos selecionados apontaram que associação dos fluoretos convencionais (NaF) com os metálicos pode ser uma estratégia promissora para a redução de perda de superfície pelo desgaste dentário erosivo. Dentifrícios contendo fluoreto de sódio, assim como estanho sem quitosana, apresentaram redução na perda de superfície, sendo considerada suficiente para indivíduos com exposição média aos ácidos. Em dentifrícios infantis, aquele que continha fluoreto de sódio apresentou uma redução de perda de superfície quando comparado com dentifrícios sem fluoreto. Conclusão:diferentes protocolos podem resultar em menor ou maior perda de superfície de esmalte e as diferenças (AU)

Control de la caries no restaurativa en incisivos centrales primarios, a los 36 meses de seguimiento / Controle cavitário não restaurador em incisivos centrais decíduos com 36 meses de acompanhamento / Non-restorative cavity control in deciduous central incisors at 36-month follow-up

La mínima intervención ha ido ganando cada vez más notoriedad en la Odontología, reflejando una creciente demanda por parte de los profesionales que buscan técnicas que promuevan tratamientos eficientes y que preserven la estructura dentaria. Objetivo: El presente estudio tuvo como objetivo promover el control de caries no restaurador (CCNR) en un paciente infantil, mediante una regularización de la cavidad de la lesión cariosa para permitir una mejor limpieza del área, junto con un protocolo complementario de fluoración. Presentación de caso: Paciente masculino de 4 años de edad con higiene bucal satisfactoria, sin embargo con acumulación de biopelícula entre los incisivos centrales superiores y sin síntomas ni quejas estéticas. Se alisaron las caras mesiales de los incisivos centrales con tiras abrasivas para eliminar zonas socavadas y facilitar el acceso para mejorar la higiene bucal, y se aplicó el barniz de flúor en 4 sesiones, con un intervalo de una semana entre cada aplicación. La madre de la paciente fue instruida sobre cuidados de higiene y alimentación. Durante 36 meses se realizó seguimiento clínico y radiográfico del paciente con retornos cada 12 meses. También se reevaluaron el índice de placa visible y los resultados de la guía de higiene y dieta. Conclusión; En este caso clínico, la fluorterapia asociada a la regularización superficial demostró ser una alternativa de tratamiento exitosa a largo plazo, permitiendo la preservación del tejido dentario y previniendo la progresión de las lesiones cariosas. El concepto ultraconservador puede ser una alternativa válida por su eficacia, sencillez, bajo coste y buena aceptación por parte de los niños y sus padres

A mínima intervenção vem ganhando cada vez mais notoriedade na Odontologia, refletindo uma crescente demanda de profissionais que buscam por técnicas que promovam tratamentos eficientes e preservem a estrutura dental. Objetivo: O presente estudo teve como objetivo promover um controle cavitário não restaurador (CCNR) em um paciente infantil, por meio de uma regularização da cavidade da lesão de cárie para permitir uma melhor higienização da área, associado a um protocolo complementar de flúor. Caso clinico: Paciente do sexo masculino, 4 anos de idade, com higiene bucal regular, entretanto com acúmulo de biofilme entre os incisivos centrais superiores e sem sintomatologia ou queixa estética. As superfícies mesiais dos incisivos centrais foram regularizadas com tiras abrasivas para remover áreas retentivas e facilitar o acesso para melhorar a higiene bucal. O verniz fluoretado foi aplicado em 4 sessões, com intervalo de uma semana entre cada aplicação. A mãe do paciente foi orientada quanto à higiene e cuidados com a dieta. Durante 36 meses, houve o acompanhamento clínico e radiográfico do paciente com retornos a cada 12 meses. O índice de placa visível e os resultados da orientação de higiene e dieta também foram reavaliados. Conclusão: Neste caso clínico, a regularização de superfícies associada a fluorterapia, mostrou-se uma alternativa de tratamento de sucesso a longo prazo, permitindo a preservação do tecido dentário e impedindo a progressão das lesões de cárie. O conceito ultraconservador pode ser uma alternativa válida devido à sua eficácia, simplicidade, baixo custo e boa aceitação pelas crianças e seus pais.

The minimal intervention has been gaining more notoriety in Dentistry, reflecting growing demand from professionals looking for techniques that promote efficient treatments and preserve the dental structure. Objective: The present study aimed to promote non-restorative cavity control (CCNR) in a child patient through regularization of the cavity of the carious lesion to allow better cleaning of the area, together with a complimentary fluoride protocol. Case presentation: Male patient, 4 years old, presenting adequate oral hygiene. However, biofilm accumulation occurs between the upper central incisors without symptoms or aesthetic complaints. Therefore, the mesial surfaces of the central incisors were smoothed with abrasive strips to remove undercut areas and facilitate access to improve oral hygiene. The fluoride varnish was applied in 4 sessions, with an interval of one week between each application. The patient's mother was instructed on hygiene and diet care. During 36 months, there was a clinical and radiographic follow-up of the patient with returns every 12 months. The visible plaque index and the hygiene and diet guidance results were also re-evaluated. Conclusion: In this clinical case, surface regularization associated fluortherapy proved to be a successful long-term treatment alternative, allowing the preservation of dental tissue and preventing the progression of caries lesions. The ultra-conservative concept can be a valid alternative due to its effectiveness, simplicity, low cost, and good acceptance by children and their parents.