Microemulsions Improve Topical Protoporphyrin IX (PpIX) Delivery for Photodynamic Therapy of Skin Cancer

Abstract We report here microemulsions (MEs) for topical delivery of protoporphyrin IX (PpIX) for Photodynamic Therapy (PDT) of skin cancers. Selected MEs consisting of Oil/Water (O/W) bicontinuous (BC) and Water/Oil (W/O) preparations were characterized as to pH, nanometric size, zeta potential, drug content, and viscosity. Sustained in vitro PpIX release was achieved from MEs 2A (O/W), 10B (BC) and 16B (W/O) through an artificial membrane for up to 24 h, characterizing MEs as drug delivery systems. None of these MEs showed permeation through the skin, demonstrating the required topical effect. After 4 h, in vitro retention of PpIX in the stratum corneum (SC) was higher from both ME 10B and control (PpIX at 60 µg/mL in PEG 300). However, in the Epidermis + Dermis ([Ep + D]), retention from ME 10B and ME 16B was ~40 times higher compared to control. Confocal Laser Scanning Microscopy (CLSM) showed higher fluorescence intensity in the SC for both control and ME 10B, whereas ME 10B fluorescence was higher in [Ep+D]. The results indicate that ME 10B is suitable for PpIX encapsulation, showing good characteristics and a localized effect for a potential delivery system for PDT-associated treatments of skin cancers.

Evaluation of different composite resin finishing and polishing protocols by confocal laser scan microscopy

Aim This study aimed to analyze the influence of finishing and polishing (F/P) protocols on resin surface through roughness (Ra) values and laser scan microscopy observations. Methods Forty-eight (n=48) resin specimens were sorted into four groups (n=12), according to the type of resin used: Filtek Z250 (Z250), Filtek Z350 (Z350), Filtek One Bulk Fill (BF), Filtek P60 (P60). The specimens were sorted into six groups according to the type of F/P system used (n=2/group): Control group, Diamond bur (KG Sorensen), Soflex Pop-On Discs (3M ESPE), Soflex Spiral (3M ESPE), Dura Gloss (American Burs), and Praxis (TDV). Results The highest roughness values (Ra) were attributed to BF group for all F/P systems, except for the Soft-Lex PopOn discs. The Soft-Lex PopOn, Spiral, and Praxis discs presented a better performance for the surface treatment of the tested composite resins. Regardless of the restorative material, the use of diamond bur or single-step abrasive rubber (Dura Gloss) were associated with the highest Ra values. Conclusion The effect of F/P systems on Ra is material-dependent and instrument or system-dependent.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Displasia epitelial corneal / Corneal epithelial dysplasia

Las neoplasias intraepiteliales córneo-conjuntival son lesiones premalignas, mal delimitadas, de crecimiento lento y escaso potencial de malignización. Solo el 9 por ciento progresa a carcinoma invasor de células escamosas. Posee varias formas de presentación y tiene múltiples dilataciones vasculares. La displasia epitelial corneal primaria es una forma poco frecuente de neoplasia intraepitelial córnea-epitelial. Se presenta un caso clínico confirmado por estudios anatomopatológicos. En el presente estudio se observó respuesta satisfactoria luego de un mes de tratamiento tópico con 5FU, sin efectos colaterales. El seguimiento durante tres años no ha mostrado signos de recidiva(AU)

Corneal-conjunctival intraepithelial neoplasms are premalignant, poorly demarcated, slow-growing lesions with low malignant potential. Only the 9 percent progresses to invasive squamous cell carcinoma. It appears in several forms and presents multiple vascular dilatations. Primary corneal epithelial dysplasia is a rare form of corneal-epithelial intraepithelial neoplasia. We present a clinical case, confirmed by anatomopathologic studies. In the present study we observed a satisfactory response after one month of topical treatment with 5FU, with no side effects. Follow-up for three years has shown no signs of recurrence(AU)

Cuerpos extraños intracorneales retenidos / Retained intracorneal foreign bodies

Los cuerpos extraños constituyen la segunda forma más común de traumatismos corneales. Pueden localizarse superficiales o intracorneales. Se realiza presentación de caso de paciente con cuerpos extraños intracorneales retenidos de 34 años de evolución secundario a trauma por onda expansiva, con el objetivo de mostrar los hallazgos clínicos y resultados por pruebas diagnósticas como la microscopía confocal corneal, frente de onda corneal por topografía y microscopía especular. La presencia de cuerpos extraños intracorneales en este paciente provocó respuestas en el tejido corneal en dependencia de su composición, nivel de profundidad; evidentes a pesar de tiempo de retención. La presencia de estos induce niveles altos de aberraciones corneales que dependen del grado de afectación(AU)

Foreign bodies are the second most common form of corneal trauma. They can be superficial or intracorneal. A case of a patient was presented, with retained intracorneal foreign bodies of 34 years of secondary evolution due to blast wave trauma. The objective is to show the clinical findings and results of diagnostic tests such as corneal confocal microscopy, corneal wavefront topography and specular microscopy. The presence of intracorneal foreign bodies in this patient provoked responses in the corneal tissue depending on its composition, depth level; evident in spite of retention time. The presence of intracorneal foreign bodies induces high levels of corneal aberrations depending on the degree of involvement(AU)

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Endotelitis por citomegalovirus y su diagnóstico mediante la microscopia confocal / Cytomegalovirus endotheliitis and its diagnosis by confocal microscopy

Objetivo: Describir las características del endotelio corneal por microscopia confocal en pacientes con lesiones basofílicas de inclusión como patognomónicas de endotelitis por citomegalovirus. Métodos: Se realizó un estudio observacional de casos clínicos en pacientes con diagnóstico de endotelitis por citomegalovirus, atendidos en la Consulta de Córnea del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", entre febrero del año 2010 y junio del 2018. La muestra incluyó 39 pacientes con diagnóstico clínico de endotelitis, a quienes se les realizó microscopia confocal con el equipo Confoscan 4 (Nidek Technologies). Resultados: De los 39 pacientes, 29 fueron del sexo masculino y 10 del femenino. Todos tenían entre 41 y 60 años de edad. En el 97,4 por ciento de los casos existió el antecedente de una conjuntivitis viral y un solo paciente fue positivo de HIV (2,6 por ciento). La mejor agudeza visual corregida de 0,3 o menos se mostró en el 100 por ciento de ellos antes del tratamiento, y después de este los 39 tenían entre 0,8 y 1,0. En el 100 por ciento de los casos se observaron cuerpos de inclusión basofílicos en el endotelio corneal, que fueron apreciables mediante la microscopia confocal. Conclusiones: Existe una relación entre la presencia de cuerpos de inclusión basofílicos en el endotelio corneal y las pruebas virológicas a citomegalovirus positivas, lo que puede permitir hacer PCR a casos que ya tienen confirmación mediante microscopia confocal de las características patognomónicas del endotelio corneal(AU)

Objective: Describe the characteristics of the corneal endothelium by confocal microscopy in patients with inclusion basophilic lesions as pathognomonic signs of cytomegalovirus endotheliitis. Methods: An observational study was conducted of clinical cases of patients diagnosed with cytomegalovirus endotheliitis attending the Cornea Service at Ramón Pando Ferrer Cuban Institute of Ophthalmology from February 2010 to June 2018. The study sample was 39 patients with a clinical diagnosis of endotheliitis who underwent confocal microscopy with a Confoscan 4 device (Nidek Technologies). Results: Of the 39 patients examined, 29 were male and 10 were female. All were aged 41-60 years. 97.4 percent had a history of viral conjunctivitis and only one was HIV positive (2.6 percent). Best corrected visual acuity was 0.3 or less in 100 percent before treatment, and 0.8 to 1.0 after treatment. Inclusion basophilic bodies visible by confocal microscopy were observed in the corneal endothelium of all patients. Conclusions: A relationship exists between the presence of inclusion basophilic bodies in the corneal endothelium and virological tests positive for cytomegalovirus, making it possible to perform PCR testing in cases with confocal microscopy confirmation of the pathognomonic characteristics of the corneal endothelium(AU)

Dentin bond strength evaluation between a conventional and universal adhesive using etch-and-rinse strategy

Aim: The aim of this study was to compare the microtensile bond strength (µTBS) and the characteristics of the adhesive interface of Scotchbond Universal - SU ­ etch-and-rise mode (3M ESPE) and Adper Scotchbond Multi-Purpose - MP (3M ESPE) to dentin over time. Methods: Class I cavity preparations were performed in 60 human molars that were randomly divided according to the dentin bonding system (DBS) used (n=30): (1) Acid conditioning + SU and (2) Acid conditioning + MP. For bonding strength (BS) analysis, 30 teeth (n = 15) were sectioned into sticks and submitted to the microtensile test in a universal testing machine after 24 hours and 12 months. The adhesive interface of the others 30 teeth was analyzed in a confocal microscope after 24 hours and 12 months. The data of µTBS were analyzed by two-way repeated measures ANOVA and Tukey's HSD (α = 0.05). Results: SU presented the lowest DBS compared to MP (p=0.000). Time did not influenced DBS for both adhesive systems (p=0.177). Confocal microscopy analysis showed no cracks between both adhesive systems tested. Conclusion: The results indicate that MP - µTBS showed a better performance compared to SU in total-etch mode

Effect of ethanol-conditioned dentine on sealer penetration into dentinal tubules: a confocal microscopy study

Aim: The aim of this study was to evaluate the effect of ethanol-conditioned dentin on endodontic sealer penetration into dentinal tubules by confocal laser scanning microscopy (CLSM). Methods: Forty human maxillary anterior teeth were instrumented and divided into four groups (n = 10) according to the drying methods: 1) wet: vacuum only, 2) paper points: vacuum + absorbent paper points, (3) 70% ethanol: 70% ethanol (1 min) + vacuum + absorbent paper points, and (4) 100% ethanol: 100% ethanol (1 min) + vacuum + absorbent paper points. All root canals were filled with resin-based endodontic sealer. Four sections from each third (cervical, middle, and apical) were examined by CLSM. Root canal wall perimeter infiltrated by sealer, maximum depth of sealer penetration, percentage of penetrated area, and fluorescence intensity of rhodamine B were measured. Statistical analysis was performed by analysis of variance and Tukey's tests (α = 0.05). Results: No statistical difference was found when percentage of root canal wall coverage infiltrated by sealer were compared. The groups in which ethanol solutions were used presented greater depth of sealer penetration, higher percentage of penetrated area, and higher fluorescence intensity of rhodamine B (p< 0.05) when compared with the wet and paper point groups. Overall, 100% ethanol produced better results than 70% ethanol, except for rhodamine B intensity (cervical third). In addition, the absorbent paper points drying method behaved better than did vacuum only group, except for rhodamine B intensity (apical third). Conclusion: Ethanol-conditioned dentin improved the penetration of resin-based sealer into dentinal tubules, especially at the concentration of 100%

Bowen's disease of the penile shaft presenting as a pigmented macule: dermoscopy, reflectance confocal microscopy and histopathological correlation,

Abstract The penile localization of pigmented Bowen's disease has been rarely reported and has been mostly related to human papillomavirus infection. Early diagnosis and treatment are important to prevent progression to invasive squamous cell carcinoma. However, diagnosis can be challenging because it may be difficult to distinguish from melanoma, even using dermoscopy. Reflectance confocal microscopy may be useful in suggesting the bedside diagnosis before the histopathological confirmation. A case of penile pigmented Bowen's disease is described along with its dermoscopy and reflectance confocal microscopy findings and their correlation with histopathology.

Liver structural injury in leptin-deficient (ob/ob) mice: lipogenesis, fibrogenesis, inflammation, and apoptosis / Lesión estructural del hígado en ratones con deficiencia de leptina (ob/ob): lipogénesis, fibrogénesis, inflamación y apoptosis

SUMMARY: Nonalcoholic fatty liver disease (NAFLD) might progress the steatosis to nonalcoholic steatohepatitis (NASH), reaching a cirrhosis state and possibly hepatocellular carcinoma. The liver of three-month-old C57BL/6J mice (wild-type, WT group, n=10) and leptin- deficient obese mice (ob/ob group, n=10) were studied, focusing on the mechanisms associated with the activation of the hepatic stellate cells (HSCs) and pro-fibrogenesis. The obese ob/ob animals' liver showed steatosis, increased lipogenesis gene expressions, inflammation, increased pro-inflammatory gene expressions, inflammatory infiltrate, and potential apoptosis linked to a high Caspase 3 expression. In ob/ob mice, liver sections were labeled in the fibrotic zones by anti-alpha-smooth muscle actin (alpha-SMA) and anti-Reelin, but not in the WT mice. Moreover, the alpha-SMA gene expression was higher in the ob/ob group's liver than the WT group. The pro-fibrogenic gene expressions were parallel to anti- alpha-SMA and anti-Reelin immunofluorescence, suggesting HSCs activation. In the ob/ob animals, there were increased gene expressions involved with lipogenesis (Peroxisome proliferator-activated receptor-gamma, Cell death-inducing DFFA-like effector-c, Sterol regulatory element-binding protein-1c, and Fatty acid synthase), pro-fibrogenesis (Transforming growth factor beta1, Smad proteins- 3, Yes-associated protein-1, Protein platelet-derived growth factor receptor beta), pro-inflammation (Tumor necrosis factor-alpha, and Interleukin-6), and apoptosis (Caspase 3). In conclusion, the results in obese ob/ob animals provide a clue to the events in humans. In a translational view, controlling these targets can help mitigate the hepatic effects of human obesity and NAFLD progression to NASH.

RESUMEN: La enfermedad del hígado graso no alcohólico (HGNA) puede progresar de la esteatosis a esteatohepatitis no alcohólica (ENA), alcanzando un estado de cirrosis y posiblemente carcinoma hepatocelular. Se estudió el hígado de ratones C57BL / 6J de tres meses de edad (tipo salvaje, grupo WT, n = 10) y ratones obesos con deficiencia de leptina (grupo ob/ob, n = 10), centrándose en los mecanismos asociados con la activación de las células estrelladas hepáticas (HSC) y profibrogénesis. El hígado de los animales obesos ob/ob mostró esteatosis, aumento de la expresión génica de la lipogénesis, inflamación, aumento de la expresión génica proinflamatoria, infiltrado inflamatorio y posible apoptosis ligada a una alta expresión de Caspasa 3. En ratones ob/ob, las sec- ciones de hígado se marcaron en las zonas fibróticas con anti-alfa- actina de músculo liso (alfa-SMA) y anti-Reelin, pero no en los ratones WT. Además, la expresión del gen alfa-SMA fue mayor en el hígado del grupo ob/ob que en el grupo WT. Las expresiones génicas profibrogénicas fueron paralelas a la inmunofluorescencia anti-alfa-SMA y anti-Reelin, lo que sugiere la activación de las HSC. En los animales ob/ob, hubo un aumento de las expresiones génicas involucradas con la lipogénesis (receptor activado por proliferador de peroxisoma gamma, efector c similar a DFFA inductor de muerte celular, proteína de unión al elemento regulador de esterol-1c y sintasa de ácidos grasos), pro-fibrogénesis (factor de crecimiento transformante beta 1, proteínas Smad-3, proteína-1 asociada a Yes, receptor beta del factor de crecimiento derivado de plaquetas de proteínas), proinflamación (factor de necrosis tumoral alfa e interleucina-6) y apoptosis (caspasa 3). ). En conclusión, los resultados en animales obesos ob/ob proporcionan una pista de los eventos en humanos. Desde un punto de vista traslacional, el control de estos objetivos puede ayudar a mitigar los efectos hepáticos de la obesidad humana y la progresión de HGNA a ENA.

Imagenología diferencial por microscopia confocal in vivo entre la epiteliopatía por acanthamoeba y la queratitis por herpes simple / In vivo confocal microscopy imaging for the differential diagnosis of Acanthamoeba epitheliopathy and herpes simplex keratitis

La queratitis por Acanthamoeba es una infección corneal de baja incidencia, condicionada por varios factores, pero con manifestación clínica variada y sintomatología típica. En su fase epitelial debe ser diferenciada de otras queratitis, específicamente de la queratitis por herpes simple, por la similitud entre ambas en cuanto a las características de la lesión corneal. La microscopia confocal in vivo constituye una alternativa diagnóstica. Es una biopsia fotográfica en cuyas imágenes podemos describir los quistes y trofozoítos de Acanthamoeba desde etapas iniciales, que nos ayudan a diferenciarla de otros tipos de queratitis e iniciar el tratamiento precoz. Se realizó una búsqueda de artículos publicados, con el objetivo de mostrar las imágenes por microscopia confocal de la fase epitelial de la infección corneal por Acanthamoeba y herpes simple. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

Acanthamoeba keratitis is a low-incidence corneal infection caused by several factors and characterized by a variety of clinical manifestations and typical symptoms. In its epithelial phase, it should be differentiated from other keratitis, particularly from herpes simplex keratitis, due to the similar characteristics of the corneal lesion. In vivo confocal microscopy is a diagnostic alternative consisting in a photographic biopsy showing images of Acanthamoeba cysts and trophozoites since their initial stages, thus allowing differentiation from other types of keratitis and the initiation of early treatment. A search was conducted of published papers with the purpose of showing confocal microscopy images of the epithelial phase of Acanthamoeba and herpes simplex corneal infection. Use was made of the platform Infomed, specifically the Virtual Health Library(AU)

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line / [Mudanças metabólicas induzidas por melitina em células da linhagem Madin-Darby Bovine Kidney]

In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10µg/mL). Flow cytometry analysis revealed that melittin at 2µg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2µg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5µg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.(AU)

Neste estudo, os efeitos tóxicos da melitina em células Madin-Darby Bovine Kidney (MDBK) foram analisados quanto à funcionalidade mitocondrial, por redução de MTT e citometria de fluxo, potencial de apoptose, necrose, produção de espécies reativas de oxigênio (ROS), peroxidação lipídica e fragmentação de DNA, utilizando-se citometria de fluxo e desestabilização da membrana celular, por microscopia confocal. A toxicidade apresentou características dose-dependentes e a atividade mitocondrial foi inibida até 78,24±3,59% (P<0,01, n = 6) em células MDBK expostas à melitina (10µg/mL). Análises por citometria de fluxo revelaram que a melitina a 2µg/mL apresentou o maior índice necrótico celular (P<0,05). A maior lipoperoxidação de membranas também foi na concentração de 2µg/mL de melitina (P<0,05), o que foi posteriormente confirmado por microscopia confocal. A maior produção de ROS aconteceu quando as células foram expostas a 2,5µg/mL de melitina (P<0,05), e essa concentração também aumentou a fragmentação de DNA (P<0,05). Houve uma significativa correlação positiva entre a lipoperoxidação de membranas e a produção de ROS (R=0,4158), funcionalidade mitocondrial (R=0,4149) e apoptose (R=0,4978). Portanto, o estresse oxidativo gerado pela melitina culminou na elevação de ROS intracelular, que inicia uma cascata de eventos tóxicos nas células MDBK.(AU)

Effects of ClpP protease on biofilm formation of Enterococcus faecalis

Abstract Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms. Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student's t-test or one-way analysis of variance (ANOVA). Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis. Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.

Advances in Noninvasive Methods for Screening and Evaluating Diabetic Peripheral Neuropathy / 中国医学科学院学报

Diabetic peripheral neuropathy(DPN),a chronic diabetic microvascular complication with a high incidence among diabetic patients,increases the risk of diabetic foot and amputation.Many methods are available for screening and evaluating DPN,including traditional 10 g monofilament,tuning fork and vibration perception,and tendon reflex tests,which should be combined with some nerve function score systems to improve the detection rate and accuracy for DPN.In recent years,a number of noninvasive new techniques have been developed for the evaluation of nerve injury,such as corneal confocal microscopy,quantitative sensory testing,current perception threshold test,sympathetic sudomotor function evaluation,and quantitative detection of skin advanced glycation end products.This paper reviews these noninvasive methods for screening and evaluating DPN to help clinicians detect and focus on DPN early.

Influence of EDTA and its Association with Benzalkonium Chloride on Enterococcus faecalis Adhesion to Dentin / Influencia del EDTA y su Asociación con Cloruro de Benzalconio en la Adhesión de Enterococcus faecalis a la Dentina

ABSTRACT: The aim of this in vitro study was to investigate the influence of ethylenediaminetetraacetic acid (EDTA) associated with the benzalkonium chloride (BAK) on the adhesion and formation of Enterococcus faecalis biofilms attached to coated dentin. Discs standard bovine dentin blocks were treated with the coating materials evaluated: Saline solution (control), 17 % EDTA, 17 % EDTA associated with 1 % BAK for 5 minutes and subsequently washed with saline solution. Afterwards, biofilms of E. faecalis (ATCC 29212) were grown on the surface of coated dentin blocks for time intervals of 1 hour and 7 days (n = 20) and were subsequently washed with phosphate-buffered saline (PBS). Bacterial viability and total biovolume were analyzed by confocal laser scanning microscopy (CLSM) using the Live/Dead technique. Nonparametric Kruskal-Wallis followed by Dunn tests were used to determine statistical differences (a = 5 %). The 17 % EDTA + 1 % BAK group showed significantly lower biovolume and bacterial viability values at the end of 1 hour (p < 0.05). After 7 days of contamination, the 17 % EDTA and 17 % EDTA + 1 % BAK groups showed similar results that differed statistically from those of the control group (p < 0.05). The saline solution group showed higher values. The use of BAK associated with EDTA on dentin blocks surfaces before exposure to contamination was able to interfere in the adhesion of E. faecalis to dentin. Also, dentin treatment by BAK associated with a chelating agent influences the secondary biofilm formation, which could have important effects on the long-term success of root canal treatment.

RESUMEN: El objetivo del estudio consistió en investigar in vitro, la influencia del ácido etilendiamino-tetraacético (EDTA) con cloruro de benzalconio (BAK) en la adhesión y formación de biopelículas de Enterococcus faecalis a la dentina. Discos de dentina bovina fueron tratadas con solución salina (control), 17 % de EDTA, 17% de EDTA asociado con 1 % de BAK durante 5 minutos y lavadas con solución salina. Las biopelículas de E. faecalis (ATCC 29212) se cultivaron sobre los discos de dentina durante intervalos de tiempo de 1 hora y 7 días (n = 20), lavados con solución salina tamponada con fosfato (PBS). La viabilidad bacteriana y el biovolumen total se analizaron mediante microscopía de barrido por láser (CLSM) utilizando la técnica Live / Dead. Se realizó prueba no paramétrica de Kruskal-Wallis, seguida por Dunn con una diferencia estadística (a = 5 %). El grupo de 17 % EDTA + 1 % BAK mostró valores significativamente menores de biovolumen y viabilidad bacteriana al final de 1 hora (p < 0,05). Después de 7 días de contaminación, los grupos de 17 % EDTA y 17 % EDTA + 1 % BAK mostraron resultados similares que diferían estadísticamente del grupo control (p < 0,05). La solución salina mostró valores más altos. La asociación de BAK con EDTA antes de la contaminación interfirió en la adhesión de E. faecalis. Además, el tratamiento de la dentina por BAK asociado con EDTA influye en la formación de biopelículas secundarias, lo que podría tener efectos importantes sobre el éxito a largo plazo del tratamiento del conducto radicular.

Dentinal tubule penetration of a calcium silicate-based root canal sealer using a specific calcium fluorophore

Abstract This study aimed to evaluate penetrability on dentinal tubule of a new bioceramic sealer through confocal laser scanning microscopy (CLSM). A specific fluorophore (Fluo-3) was mixed with the sealer. Forty distobuccal roots from maxillary molars were selected, and root canal preparation was carried out with Wave One Gold # 35.06 instruments. Roots were randomly assigned to 4 groups according to the filling procedures: Bioceramic/Lentulo (Sealer Plus BC); Bioceramic/EasyClean group, three activation of the sealer (3x20 s) with Easy Clean instrument; Bioceramic/Irrisonic: ultrasonic activation for 30 s; and AHplus/Lentulo: epoxy resin based sealer (AH Plus) was utilized with the same protocol as the BC/LE group. After 72 h, specimens were transversally sectioned at 2 and 7 mm from root apex and then analyzed through CLSM. Sealer penetration area on dentinal tubule was measured by Adobe Photoshop CC2018. Kruskal Wallis and Wilcoxon T tests were carried out. Penetrability results were similar for both sealers regardless of which technique was performed to activate them inside the root canal (p>0.05). It is reasonable to conclude that penetration of bioceramic and epoxy resin based sealers occurred unimpressively. The type of instrument used to activate bioceramic sealer did not affect penetrability. Fluo 3 should be recommended as the fluorophore to evaluate dentinal tubule penetration of bioceramic sealers.

Resumo Este estudo objetivou avaliar a penetração nos túbulos dentinários de um novo cimento biocerâmico utilizando microscopia confocal de varredura a laser (MCVL). Um fluoróforo específico (Fluo-3) foi misturado com o cimento. Quarenta raízes distovestibulares de molares superiores foram selecionados e o preparo do canal radicular foi realizado com instrumentos Wave One Gold #35.06. As raízes foram divididas randomicamente em quatro grupos de acordo com os procedimentos obturadores: Bioceramic/Lentulo: cimento biocerâmico (Sealer Plus BC); Bioceramic/EasyClean: três ativações do cimento com instrumento Easy Clean (3 x 20 s); Bioceramic/Irrisonic: ativação ultrassônica do cimento por 30 s e AHplus/Lentulo: cimento à base de resina epóxica (AH Plus) foi utilizado com o mesmo protocolo que o grupo Bioceramic/Lentulo. Após 72h, os espécimes foram seccionados transversalmente em 2 e 7 mm do ápice radicular e analisados com MCVL. A área de penetração nos túbulos dentinários foi mensurada com Adobe Photoshop CC2018. O teste de Kruskal Wallis e T de Wilcoxon foram realizados. Os resultados de penetração foram similares para ambos os cimentos independentemente de qual técnica foi utilizada para ativá-los no interior do canal radicular (p>0,05). É razoável concluir que a penetração de cimentos biocerâmicos e à base de resina epóxica ocorreram de forma pouco expressiva. O tipo de ativação do cimento biocerâmcio não afeta sua penetrabilidade nos túbulos dentinários. Fluo 3 deve ser recomendado como o fluoróforo para avaliar a penetração intratubular de cimentos biocerâmicos.

Evidence of autophagic vesicles in a patient with Lisch corneal dystrophy / Evidência de vesículas autofágicas em paciente com distrofia corneana de Lisch

ABSTRACT Lisch corneal dystrophy is a rare corneal disease characterized by the distinctive feature of highly vacuolated cells. Although this feature is important, the nature of these vacuoles within corneal cells remains unknown. Here, we sought to analyze corneal cells from a patient diagnosed with Lisch dystrophy to characterize the vacuoles within these cells. Analyses using histopathology examination, confocal microscopy, and transmission electron microscopy were all consistent with previous descriptions of Lisch cells. Importantly, the vacuoles within these cells appeared to be autophagosomes and autolysosomes, and could be stained with an anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody. Taken together, these findings indicate that the vacuoles we observed within superficial corneal cells of a patient with Lisch corneal dystrophy constituted autophagosomes and autolysosomes; this finding has not been previously reported and suggests a need for further analyses to define the role of autophagy in this ocular disease.

RESUMO A distrofia corneana de Lisch é uma doença rara, caracterizada principalmente pela presença de células altamente vacuoladas. Embora esta característica seja importante, a natureza desses vacúolos dentro das células da córnea permanece des conhecida. Aqui, procuramos analisar as células da córnea de um paciente diagnosticado com distrofia de Lisch para caracte rizar os vacúolos dentro dessas células. Análises utilizando exame histopatológico, microscopia confocal e microscopia eletrônica de transmissão foram todas consistentes com descrições previas de células de Lisch. Importante, os vacúolos dentro dessas células pareciam ser autofagossomos e autolisossomos, e po deriam ser corados com um anticorpo proteico 1A/1B-cadeia leve 3 (LC3) da proteína anti-microtúbulo associado a microtúbulos. Em conjunto, esses achados indicam que os vacúolos observados nas células superficiais da córnea de um paciente com distrofia corneana de Lisch constituíram autofagossomos e autolisossomos. Esse achado não foi relatado anteriormente e sugere a necessidade de mais análises para definir o papel da autofagia nessa doença ocular.

Microscopia confocal de la córnea en pacientes diabéticos / Confocal microscopy of the cornea in diabetic patients

RESUMEN Objetivo: Identificar las características morfológicas del epitelio, el estroma y el endotelio corneal, así como la densidad celular de este último mediante el empleo de la microscopia confocal de la córnea en pacientes diabéticos. Métodos: Se realizó un estudio descriptivo, comparativo, en 90 ojos; 60 de ellos pertenecientes a pacientes diabéticos (30 tipo 1 y 30 tipo 2) y 30 ojos a pacientes supuestamente sanos. El estudio se realizó en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer" entre enero del año 2012 y enero de 2017. Resultados: Predominó el sexo masculino con 66,7 por ciento en los pacientes con diabetes mellitus tipo 1; el sexo femenino en los pacientes con diabetes mellitus tipo 2 (60 por ciento) y aparentemente sanos (56,7 por ciento). En los pacientes con diabetes mellitus tipo 1 fueron más frecuentes las edades entre 45 y 54 años (33,3 por ciento) y entre 55 y 66 años en los pacientes con diabetes mellitus tipo 2 y aparentemente sanos con 60 y 40 por ciento respectivamente. La morfología del epitelio y el estroma corneal fue normal en el 86,7 y 87,3 por ciento respectivamente. Predominaron las alteraciones de la morfología endotelial en pacientes diabéticos tipo 1 (73,3 por ciento), así como el polimegatismo y el pleomorfismo (73,3 y 56,7 por ciento respectivamente) y la densidad celular más baja (2 222,76 células /mm2). Conclusiones: La ausencia de alteraciones morfológicas del epitelio y el estroma corneal y la presencia de polimegatismo y de pleomorfismo fueron los hallazgos más frecuentes(AU)

ABSTRACT Objective: Identify the morphological characteristics of the corneal epithelium, stroma and endothelium, as well as the cell density of the endothelium by means of confocal microscopy of the cornea in diabetic patients. Methods: A descriptive comparative study was conducted of 90 eyes: 60 from diabetic patients (30 type 1 and 30 type 2) and 30 from supposedly healthy patients, at Ramón Pando Ferrer Cuban Institute of Ophthalmology from January 2012 to January 2017. Results: A predominance was found of the male sex (66.7 percent) among patients with diabetes mellitus type 1 and of the female sex among patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (56.7 percent). The most common age ranges were 45-54 years for patients with diabetes mellitus type 1 (33.3 percent) and 55-66 years for patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (40 percent). Morphology of the corneal epithelium and stroma was normal in 86.7 percent and 87.3 percent, respectively. In type 1 diabetic patients there was a predominance of endothelial morphological alterations (73.3 percent), polymegethism and pleomorphism (73.3 percent and 56.7 percent, respectively) and the lowest cell density (2 222.76 cells /mm2). Conclusions: Absence of morphological alterations of the corneal epithelium and stroma, as well as the presence of polymegethism and pleomorphism were the most common findings(AU)