Abstract Extracellular vesicles (EV) are spherical structures limited by membranes and shed by several cell types. Specifically, outer membrane vesicles (OMVs) are nanometric particles constitutively produced by Gram-negative bacteria (GNB) under different environmental conditions. OMVs are biologically active; they are loaded with selected lipids, polysaccharides, proteins, and even different types of nucleic acids. OMVs from pathogenic oral bacteria play key roles in pathogen-host interactions, constituting a possible link between oral health and systemic disease. OMVs participate in adhesion, invasion, and damage to cells, as well as in modulating the host's immune response, biofilm formation, and promotion of virulence. The objective of this systematic review was to collect, analyze and synthesize the knowledge available on literature reviews on OMVs of the most studied pathogenic oral anaerobic GNB. This information was classified into the following categories: induction of vesiculation and biogenesis, its liberation from the parental cell, content, internalization by another host cell, and the interaction with the host cell. It was found that the most studied OMVs are those of Porphyromonas gingivalis and Bacteroides spp. and, to a lesser extent, Aggregatibacter spp., and Treponema spp. This systematic review provides a synthesis of the current knowledge regarding OMVs, with emphasis on the information available for periodontopathogens.

Resumen Las vesículas extracelulares (EV) son estructuras esféricas delimitadas por membranas y producidas por varios tipos de células. En particular, las vesículas de membrana externa (OMV) son partículas nanométricas producidas constitutivamente por bacterias Gram negativas (GNB) en diferentes condiciones ambientales. Las OMV son biológicamente activas; están cargadas de lípidos, polisacáridos, proteínas e incluso diferentes tipos de ácidos nucleicos. Las OMV de bacterias orales patógenas desempeñan papeles clave en las interacciones patógeno-hospedero, constituyendo un posible vínculo entre la salud oral y las enfermedades sistémicas. Las OMV participan en la adhesión, la invasión y el daño celular, así como en la modulación de la respuesta inmunitaria del hospedero, la formación de biopelículas y la promoción de la virulencia. El objetivo de esta revisión sistemática era recopilar, analizar y sintetizar los conocimientos disponibles sobre las OMV de las GNB anaerobias orales patógenas más estudiadas. Esta información se clasificó en las siguientes categorías: inducción de la vesiculación y biogénesis, su liberación de la célula parental, contenido, internalización por otra célula hospedadora e interacción con la célula hospedadora. Se observó que las OMV más estudiadas son las de Porphyromonas gingivalis y Bacteroides spp. y, en menor medida, Aggregatibacter spp. y Treponema spp. Esta revisión sistemática ofrece una síntesis de los conocimientos actuales sobre las OMV, haciendo hincapié en la información disponible para los periodontopatógenos.

ABSTRACT BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy. BACKGROUND (hTERT formation effect division OBJECTIVE tissue (cfDNA later SETTING Janeiro Brazil METHODS 1 postoperatively conversion reaction electrophoresis staining RESULTS CONCLUSION onethird third chemotherapy

Capillary electromigration techniques can be understood as the ones used to separate neutral compounds, solvated ions and ionized species, considering the differentiated migration among them when an electrical field is applied within a capillary column filled with a background electrolyte. Several existing capillary electrophoresis setups provide the possibility of developing reliable quantitative assays of inorganic ions, organic acids, fatty acids, amino acids, carbohydrates, nucleic acids, proteins, hormones, vitamins, macromolecules, and more. Therefore, given the chemical complexity of bodily fluid matrices, capillary electrophoresis (CE) and its variations present themselves as an advantageous strategy for enhancing traditional clinical diagnostic methods and for the development of new ones. The recent advances in the study of the human metabolome associated with technological improvements towards medical applications make CE a very useful and versatile technique for clinical laboratory assays. Within this context, this review has the purpose of presenting some traditional methods used for exams of blood, urine, saliva, feces, and sweat, and how CE can be implemented as a real alternative to enable faster, automated, and cost-effective analysis with a comprehensive perspective that shall result in better diagnostic possibilities for patients, being a powerful tool for helping physicians on the precision medicine achievements. compounds species electrolyte acids carbohydrates proteins hormones vitamins macromolecules more Therefore matrices (CE context blood urine saliva feces sweat faster automated costeffective cost effective patients achievements

Abstract Bacteria live in polymicrobial communities and constantly compete for resources. These organisms have evolved an array of antibacterial weapons to inhibit the growth or kill competitors. The arsenal comprises antibiotics, bacteriocins, and contact-dependent effectors that are either secreted in the medium or directly translocated into target cells. During bacterial antagonistic encounters, several cellular components important for life become a weak spot prone to an attack. Nucleic acids and the machinery responsible for their synthesis are well conserved across the tree of life. These molecules are part of the information flow in the central dogma of molecular biology and mediate long- and short-term storage for genetic information. The aim of this review is to summarize the diversity of antibacterial molecules that target nucleic acids during antagonistic interbacterial encounters and discuss their potential to promote the emergence antibiotic resistance. resources competitors antibiotics bacteriocins contactdependent contact dependent cells attack long shortterm short term resistance

Abstract Pressure-assisted thermal sterilization (PATS) is a new technology to inactivate bacterial spores and ensure food safety. Little has been known about the effects of PATS combining with ε-PL on the spore’s nucleic acid, enzymes and other key substances. This study aimed to investigate the inactivation effect of PATS combining with ε-PL on the spores of B. subtilis. The spores were treated with pressure 600 MPa at 25 °C, 65 °C and 75 °C, and ε-PL at 0.1% and 0.3%. After treatment, the survival rate of B. subtilis spores, leakage of nucleic acid and protein, the change in the cell membrane ATPase activity, the leakage of dipicolinic acid, and the damage on protein and nucleic acid of the spores were determined. The results showed that PATS combining with ε-PL inactivated more spores, and significantly increased the release of protein and nucleic acid compared to the control. ATPase activity reached the lowest value after the treatment of 600 MPa/75 °C combining with 0.3% ε-PL. The release of dipicolinic acid from the spores was increased by 600 MPa/75 °C combining with 0.3% ε-PL as compared with 600 MPa/75 °C treatment alone. FTIR analysis showed that a combination of PATS with ε-PL changed the spectral features of B. subtilis functional groups of proteins and nucleic acids. The PATS treatments when combined with ε-PL were found to shift the symmetric and antisymmetric stretching vibrational absorption peaks of phosphodiester group in nucleic acid molecules (P=O). This change suggested that the combined treatment denatured nucleic acid. The combined treatment also changed the protein from an ordered state to a disordered state, and decreased protein stability. The results improved our understanding on the principle of spore inactivation by PATS combining with ε-PL. Pressureassisted Pressure assisted (PATS safety εPL ε PL s substances B 60 2 C 6 7 01 0 1 0.1 03 3 determined control MPa75 MPa/7 0.3 εPL. PL. alone acids P=O. PO P=O . P O (P=O) stability 0. MPa7 MPa/ (P=O

Abstract Cellular metabolism is a constant source of reactive oxygen species (ROS). The production of hydrogen peroxide (H2O2) is particularly relevant, due to its role in cellular physiology. H2O2 can act as a classical intracellular signaling molecule in several processes like cell proliferation, hormone synthesis and secretion, immune cell regulation, angiogenesis, and apoptosis. However, H2O2 overproduction and accumulation, derived from increased mitochondrial electron transport chain activity, glucose autoxidation, and increased polyol flux, contribute to an imbalance in the redox state of the cells. High concentration of H2O2 induces cellular dysfunction through oxidation of macromolecules like proteins, lipids, carbohydrates and nucleic acids. H2O2-induced oxidative damage can contribute to the development and progression of degenerative diseases such as diabetes mellitus. Indeed, chronic hyperglycemia has been shown to cause an increased H2O2 concentration and oxidative damage that contribute to the pathogenesis and progression of diabetes complications.

Resumen En las reacciones bioquímicas del metabolismo donde participa el oxígeno molecular se presenta una constante formación de especies reactivas de oxígeno (ROS). Entre las diferentes ROS, destaca el peróxido de hidrógeno (H2O2) por su papel en la fisiología celular, ya que participa como segundo mensajero en múltiples procesos celulares como la proliferación celular, la síntesis y la secreción de hormonas, la regulación y la activación de células inmunes, la angiogénesis y los procesos apoptóticos. Sin embargo, un aumento exacerbado en la formación y la acumulación de H2O2, producido por el incremento en la actividad de la cadena transportadora de electrones en la mitocondria, la autooxidación de la glucosa y/o la activación de la vía del sorbitol, genera un desequilibrio en el estado de óxido-reducción de las células. Esta acumulación de H2O2 puede causar un daño celular a través de la oxidación de las proteínas, los lípidos, los carbohidratos y los ácidos nucleicos; al contribuir en el desarrollo y la progresión de enfermedades degenerativas y metabólicas como la diabetes mellitus. Al subir la concentración de la glucosa en la sangre, se presenta un incremento en la concentración del H2O2, que intensifica los procesos oxidantes que están ligados a la patogénesis, la progresión y las complicaciones de la diabetes mellitus.

Abstract Background RNA extraction is a step that precedes several molecular techniques. The fibrous tissue, more specifically the dura mater, has several limitations in routine protocols, and lacks optimization protocols to overcome these problems. Objective To test stock reagents and purification kits, optimizing commercial kit protocols for RNA extraction from the dura mater. Methods Dura mater samples were obtained from eight Wistar rats and maintained in two different stabilizers. The samples were purified using four different protocols, and the RNA was evaluated for the yield and purity in NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). Beta-actin gene was used for analyzing gene expression, since is one of the most used reference genes. Results The RNA preservation was similar in both stabilizers. The addition of an incubation step prior the purification protocols allowed better tissue digestion and RNA recovery. The RNA purified using the protocols membrane-based showed higher quality than liquid-liquid purification. This impact was observed in the 3-week evaluation using RT-qPCR. Conclusion Stabilizers are efficient for RNA preservation and membrane-based purification protocols are more suitable for RNA recovery from dura mater tissue, allowing the evaluation of gene expression in this type of tissue. Adaptations in the dura mater RNA extraction protocol differ from the pre-established protocols because it takes into account the peculiarity of fibrous tissue and low cellularity. In addition to providing a low-cost mechanism, based on techniques that are part of the laboratory routine, it is possible to improve the quality of the extracted material, ensuring greater efficiency in the use of subsequent techniques. problems kits stabilizers 200 Thermo Scientific Wilmington DE States. States . States) Betaactin Beta actin genes membranebased membrane liquidliquid liquid 3week week 3 RTqPCR. RTqPCR RT qPCR. qPCR RT-qPCR preestablished pre established cellularity lowcost cost mechanism material 20 2

Resumo Antecedentes A extração de RNA é uma etapa que antecede várias técnicas moleculares. O tecido fibroso, mais especificamente a dura-máter, apresenta várias limitações nos protocolos de rotina e carece de protocolos de otimização para superar estes problemas. Objetivo Testar reagentes de estoque e kits de purificação, otimizando protocolos de kits comerciais para extração de RNA da dura-máter. Métodos Amostras de dura-máter foram obtidas de oito ratos Wistar e mantidas em dois estabilizadores diferentes. As amostras foram purificadas em quatro protocolos diferentes e o RNA foi avaliado quanto ao rendimento e pureza no NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). O gene da beta-actina foi utilizado para analisar a expressão gênica, uma vez que é um dos genes de referência mais utilizados. Resultados A preservação do RNA foi semelhante em ambos os estabilizadores. A adição de uma etapa de incubação antes dos protocolos de purificação permitiu uma melhor digestão do tecido e recuperação de RNA. O RNA purificado pelos protocolos baseados em membrana apresentou qualidade superior ao da purificação líquido-líquido. Este impacto foi observado na avaliação de três semanas usando RT-qPCR. Conclusão Os estabilizadores são eficientes para preservação do RNA e os protocolos de purificação baseados em membrana são mais adequados para recuperação de RNA do tecido da dura-máter, permitindo a avaliação da expressão gênica neste tipo de tecido. As adaptações no protocolo de extração de RNA da dura-máter diferem dos protocolos preestabelecidos porque leva em consideração a peculiaridade do tecido fibroso e com baixa celularidade. Além de fornecer um mecanismo de baixo custo, baseado em técnicas que fazem parte da rotina laboratorial, é possível melhorar a qualidade do material extraído, garantindo maior eficácia no uso de técnicas subsequentes. moleculares duramáter, duramáter dura máter, máter problemas duramáter. máter. 200 Thermo Scientific Wilmington DE States. States . States) betaactina beta actina utilizados líquidolíquido. líquidolíquido líquido líquido. líquido-líquido RTqPCR. RTqPCR RT qPCR. qPCR RT-qPCR celularidade custo laboratorial extraído subsequentes 20 2

Abstract Therapeutics that inhibit enzymes, receptors, ion channels, and cotransporters have long been the mainstay of cardiovascular medicine. Now, oligonucleotide therapeutics offer a modern variation on this paradigm of protein inhibition. Rather than target a protein, however, small interfering ribonucleic acids and antisense oligonucleotides target the messenger RNA (mRNA) from which a protein is translated. Endogenous, cellular mechanisms enable the oligonucleotides to bind a selected sequence on a target mRNA, leading to its degradation. The catalytic nature of the process confers an advantage over the stoichiometric binding of traditional small molecule therapeutics to their respective protein targets. Advances in nucleic acid chemistry and delivery have enabled development of oligonucleotide therapeutics against a wide range of diseases, including hyperlipidemias and hereditary transthyretin-mediated amyloidosis with polyneuropathy. While most of these therapeutics were initially designed for rare diseases, recent clinical trials highlight the potential impact of oligonucleotides on more common forms of cardiovascular disease.

Resumen Introducción: El diagnóstico rápido y definitivo con identificación del patógeno es fundamental cuando hay una infección periprotésica. La secuenciación de próxima generación permite identificar el ADN en un germen determinado en poco tiempo. Hasta donde sabemos, no hay reportes sobre su empleo para el manejo de la infección periprotésica en Sudamérica. Nuestro objetivo fue demostrar la viabilidad diagnóstica de las muestras obtenidas de una serie de pacientes operados en Buenos Aires, Argentina, y analizadas con la técnica de secuenciación de próxima generación. Materiales y Métodos: Se analizó a una serie prospectiva de 20 pacientes sometidos a cirugía de revisión séptica y aséptica de cadera desde diciembre de 2019 hasta marzo de 2020. Se obtuvieron muestras intraoperatorias de líquido sinovial, tejido profundo y canal endomedular, que fueron enviadas para su análisis al laboratorio NexGen Microgen. Resultados: Se seleccionaron 17 pacientes, porque tenían una muestra apta para analizar. Los resultados se recibieron dentro de las 72 h de la cirugía. En un caso, el resultado de la secuenciación de próxima generación informó un germen distinto del identificado en los cultivos posoperatorios de partes blandas, esto permitió corregir la antibioticoterapia. En otro, esta técnica identificó Parabacteroides gordonii en una revisión aséptica, en otro, Morganella morganii, a partir de cultivos negativos en una revisión en un tiempo. Conclusión: Se demostró la viabilidad diagnóstica con la secuenciación de próxima generación, se pueden obtener resultados de microorganismos patógenos dentro de las 72 h posteriores a la cirugía en pacientes con infección periprotésica y cultivos negativos.

Abstract Introduction: Early diagnosis of a periprosthetic joint infection (PJI) and identification of the pathogen are paramount. Next-generation sequencing (NGS) can identify the nucleic acids in a given germ in a short period. To our knowledge, there are no reports of its use in the management of PJI in South America. Our objective was to demonstrate the diagnostic feasibility of the NGS technique on the samples obtained from a series of patients operated on in Buenos Aires, Argentina. Materials and Methods: A prospective series of 20 patients undergoing septic and aseptic hip revision surgery from December 2019 to March 2020 was analyzed. Intraoperative samples of synovial fluid, deep tissue, and intramedullary canal were obtained and sent to the NexGen Microgen laboratory (Texas, USA) for analysis. Results: Seventeen patients were finally eligible to present a sample suitable for analysis. In 100% of the samples, NGS results were obtained within 72 hours of surgery. In one case, the NGS result reported a germ different from the one identified in the postoperative soft tissue cultures, allowing antibiotic therapy to be corrected. In another case, NGS identified Parabacteroides gordonii in aseptic revision surgery. In another patient, the NGS identified Morganella morganii, in which conventional postoperative cultures were negative in single-stage revision surgery. Conclusion: In this study, we demonstrated the diagnostic feasibility of NGS, obtaining results within 72 hours immediately after surgery for pathogenic organisms in patients with PJI and negative cultures.

ABSTRACT Introduction: This paper focuses on the synthesis, physicochemical characterization, bioactivity evaluation, and innovative applications of new biohybrid nanomaterials. Sepiolite is a nanofibrous natural silicate that represents a very attractive nanocarrier for nucleic acids delivery into cells, among other innovative applications in life sciences and biotechnology. Methods: The physicochemical characterization and the bioactivity evaluation of biohybrids were performed using advanced techniques and procedures, including AFM, TEM, Confocal Microscopy, TLVM, FTIR, Z-potential measurements, UV-vis, FACS, Western blot, RT-qPCR, among others. Results: New biohybrids were obtained, based on the assembly of nucleic acids with sepiolite nanofibers. The mechanisms of cell internalization and response to sepiolite exposure were proposed. We demonstrated that sepiolite is a promising nanocarrier for the non-viral and stable transfer of plasmid DNA into bacteria, mammalian and human cells, with an optimized efficiency. Due to its ability to bind various biomolecules, sepiolite could be used as a nanocarrier for the simultaneous vectorization of diverse biological molecules, including proteins, nucleases and antibodies. In addition, we obtain new protocols for both, strongly improving bacterial transformation efficiency, and to extract plasmids from bacteria, in a rapid, convenient and inexpensive method that doesn't require competent cell preparation, representing an advantageous alternative to onerous commercial kits. As a conclusion, Because of the low cost of sepiolite, its low toxicity, its natural fluorescence, the ease and convenience of sepiolite/DNA synthesis methods, the possibilities of improving the transfection efficiency and additional enticing future developments, sepiolite represents a very attractive nanoplatform for potential applications in nanomedicine and nanobiotechnology.

RESUMEN Introducción: Este trabajo se centró en la síntesis, caracterización físicoquímica, evaluación de su bioactividad y aplicaciones biomédicas de nuevos nanomateriales biohíbridos. La sepiolita es un silicato natural nanofibroso que presenta atractivas características como nanoportador para la liberación de ácidos nucleicos en células, entre otras aplicaciones innovadoras en el área de las ciencias de la vida y de la biotecnología. Métodos: La caracterización físico-química y evaluación biológica de los biohíbridos fue realizada usando procedimientos y técnicas novedosas, incluyendo entre otras AFM, TEM, microscopía confocal, TLVM, FTIR, Potencial Zeta, UV-vis, FACS, Western-blot, RT- qPCR. Resultados: Se obtuvieron nuevos biohíbridos mediante ensamblado de ácidos nucleicos con nanofibras de sepiolita. Se propusieron sus mecanismos de internalización y respuesta celular. Demostramos que la sepiolita es un nanoportador prometedor para la transferencia estable no-viral de ADN plasmídico en bacterias, células de mamíferos y humanas, con eficiencia optimizada. Debido a su capacidad de anclar varias biomoléculas, la sepiolita constituye también un nanoportador para la vectorización simultánea de diferentes moléculas biológicas, incluyendo proteínas, nucleasas y anticuerpos. Adicionalmente, obtuvimos nuevos protocolos para aumentar significativamente la eficiencia de transformación bacterial, y para la extracción de ADN de bacterias, mediante métodos rápidos, seguros y económicos que no requieren la preparación de células competentes, representando una ventajosa alternativa a los costosos kits comerciales. En conclusión, la sepiolita es un producto de bajo costo, baja toxicidad y fluorescencia natural, con facilidad y conveniencia en métodos de síntesis de biohíbridos sepiolita/ADN, con posibilidad de aumentar su eficiencia de transfección, que junto a potenciales desarrollos futuros, representa una atractiva nanoplataforma para aplicaciones en nanomedicina y nanobiotecnología.

ABSTRACT Introduction: Collection media of clinical samples with the capacity to denature viruses reduce the risk of contagion during transportation and processing. Objective: To use the nucleic acids transport media (NATM) in nasopharyngeal swab samples collected for the diagnosis of SARS-CoV-2. Methods: An experimental study was conducted to demonstrate the medium capacity to inactivate viral infectivity. Zika virus (ZIKV), of biosafety level 2, was used as an enveloped virus model. The clinical performance of the NATM for the diagnosis of SARS-CoV-2 was evaluated. A ZIKV strain propagated in the Vero cell line was used and, prior to cells infection, ZIKV was in contact at different intervals (2; 15, and 30 min) with pure NATM; subsequently, serial dilutions (10-1-10-4) were performed. Viral inactivation was evaluated by RT-PCR in the supernatant and the collected cells when the propagation period was completed. CITOSWAB® VTM was used as reference to estimate the clinical performance of the NATM in 30 nasopharyngeal swabs collected for the diagnosis of SARS-CoV-2 infection. Results: ZIKV remained infectious at inoculum dilutions of ≥ 10-2, regardless of contact time. Clinical specificity and sensitivity of the NATM for the diagnosis of SARS-CoV-2 were 100%, respectively. Conclusions: Results suggest that ZIKV positive clinical samples at dilutions ≤ 10-1 of the NATM can be safely handled, which could potentially be applied to the molecular diagnosis of SARS-CoV-2.

RESUMEN Introducción: Los medios de colecta de muestras clínicas con capacidad de desnaturalizar virus reducen los riesgos de contagio durante el transporte y procesamiento. Objetivo: Emplear el medio de transporte de ácidos nucleicos (TAN) en muestras de exudado nasofaríngeo colectadas para el diagnóstico de SARS-CoV-2. Métodos: Se realizó un estudio experimental para demostrar la capacidad del medio de inactivar la infectividad viral. Se tomó como modelo de virus envuelto el virus Zika (VZk), cuyo nivel de bioseguridad es 2. Se evaluó el desempeño clínico del medio TAN para el diagnóstico de SARS-CoV-2. Se empleó una cepa del VZk propagada en la línea celular Vero y, previo a la infección de las células, el VZk se puso en contacto a intervalos de tiempo diferentes (2; 15 y 30 min) con el medio TAN puro; y luego se realizaron diluciones seriadas (10-1-10-4). La inactivación viral se evaluó por RT-PCR, en el sobrenadante y células colectadas, al culminar el periodo de propagación. El desempeño clínico del medio TAN se estimó tomando como referencia el CITOSWAB® VTM, en 30 exudados nasofaríngeos colectados para diagnóstico de la infección por SARS-CoV-2. Resultados: El VZk preservó su infectividad a diluciones del inóculo ≥ 10-2, independientemente del tiempo de contacto. La sensibilidad y especificidad clínica del medio TAN para el diagnóstico de SARS-CoV-2 fueron del 100 %, respectivamente. Conclusiones: Los resultados sugieren que muestras clínicas positivas a VZk en diluciones ≤ 10-1 del medio TAN pueden ser manipuladas de forma segura, lo que pudiera aplicarse potencialmente al diagnóstico molecular del SARS-CoV-2.

SUMMARY: Tissue clearing techniques are frequently used in the observation and description of anatomical structures and pathways without altering the three-dimensional layout of the anatomical specimen. Tissue optical clearing promotes preservation of three-dimensional structures, which allows the study of the internal anatomy in its original position and original spatial interaction. Among these techniques, Potassium Hydroxide (KOH) maceration clearing is one of the most widely used. However, the histological changes of tissue after KOH maceration have yet to be fully understood. Our aim is to describe the microscopical differences between macerated and normal tissue. To better understand said changes, two human fetuses with a gestation period of 16 to 28 weeks were cleared and processed for histological analysis. Microtome slides of the fetuses’ lower limbs were obtained and stained with Hematoxylin & Eosin, Periodic Acid Schiff (PAS), and Masson’s trichrome with the purpose of observing the histological and macromolecule composition changes in cleared tissue. Remarkable differences at a histological level regarding the composition of the cellular structures, since diaphanized tissues showed a predominance of extracellular matrix composed of collagen fibers with the absence of most of the nucleated cellular tissue. Phospholipid’s saponification, nucleic acids degradation and a change on proteins structural properties are the main factors inducing clearing. At the same time, molecular stability of collagen in alkaline conditions allows the specimen to maintain its shape after the process.

RESUMEN: Las técnicas de limpieza de tejido se utilizan con frecuencia en la observación y descripción de estructuras y vías anatómicas sin alterar el diseño tridimensional de la muestra anatómica. El aclaramiento óptico de tejidos promueve la preservación de estructuras tridimensionales, lo que permite el estudio de la anatomía interna en su posición original y la interacción espacial original. Entre estas técnicas, el aclarado por maceración con Hidróxido de Potasio (KOH) es una de las más utilizadas. Sin embargo, los cambios histológicos del tejido después de la maceración con KOH aún no se han entendido por completo. Nuestro objetivo es describir las diferencias microscópicas entre el tejido macerado y el normal. Para entender mejor dichos cambios, dos fetos humanos con un período de gestación de 16 a 28 semanas fueron aclarados y procesados para análisis histológicos. Se obtuvieron microtomos de las extremidades inferiores de los fetos y se tiñeron con hematoxilina y eosina, ácido peryódico de Schiff (PAS) y tricrómico de Masson con el fin de observar los cambios histológicos y de composición de macromoléculas en el tejido aclarado. Diferencias notables a nivel histológico en cuanto a la composición de las estructuras celulares, ya que los tejidos diafanizados mostraban un predominio de matriz extracelular compuesta por fibras de colágeno con ausencia de la mayor parte del tejido celular nucleado. La saponificación de los fosfolípidos, la degradación de los ácidos nucleicos y un cambio en las propiedades estructurales de las proteínas son los principales factores que inducen la depuración. Al mismo tiempo, la estabilidad molecular del colágeno en condiciones alcalinas permite que la muestra mantenga su forma después del proceso.

Resumen Técnicas moleculares requieren extracciones de ácidos nucleicos en cantidad y pureza adecuadas. Este trabajo describe un modelo lineal generalizado (GLM) de un factor ajustado con efectos fijos sobre el rendimiento de ácido nucleico (ng/μl) y la pureza (A260/A280 y A260/A230), para cinco métodos de extracción de ADN utilizando tarjetas FTA con sangre de cabra (Capra aegagrus hircus). Se ensayaron dos métodos comerciales basados en columnas de sílice (Invitrogen y Macherey Nagel; MN), método resina quelante (Chelex), método CTAB y el método de fenol-cloroformo-alcohol isoamílico (PCI). Adicionalmente, para MN, se evaluó una etapa de incubación con tampón PBS (Phosphate Buffered Saline) en alta temperatura previa a la lisis y una etapa de purificación posterior a la extracción utilizando un modelo de efecto fijo de dos factores con interacción. Las concentraciones de ADN y las proporciones de pureza fueron variables; la concentración más alta se obtuvo con el kit MN (170.45 ng/μl), pero con deficiencias en la pureza (0.32 de A260/A230, 0.34 de A260/A280). A pesar de esto, todos los métodos de extracción generaron productos PCR con cebadores específicos D-loop (ADNmt). El efecto combinado de las etapas de pre-incubación y post-purificación arrojó valores de pureza satisfactorios (1.89 para A260/A230 y 1.65 para A260/A280), así como relaciones de concentración (476.78 ng/μl) con baja variabilidad. En conclusión, la concentración y pureza del ADN de muestras de sangre mejora considerablemente cuando se usa un kit comercial en combinación con incubación previa a la lisis y purificación posterior a la extracción. Estos ácidos nucleicos se sugieren para uso en potenciales aplicaciones moleculares a posteriori.

Abstract Molecular techniques require extractions of nucleic acids in adequate quantity and purity. This work describes a generalized linear model (GLM) of an adjusted factor with fixed effects on nucleic acid yield (ng/μl) and purity (A260/A280 and A260/A230), for five methods of DNA extraction using FTA cards with goat (Capra aegagrus hircus) blood. Two commercial methods based on silica columns (Invitrogen and Macherey Nagel; MN), the chelating resin method (Chelex), the CTAB method and the phenol-chloroform-isoamyl alcohol (PCI) method were tested. Additionally, for MN, an incubation step with PBS (Phosphate Buffered Saline) buffer at high temperature prior to lysis and a purification step post extraction were evaluated using a fixed-effect model of two factors with interaction. DNA concentrations and purity ratios were variable; the highest concentration was obtained with the MN kit (170.45 ng/μl), but with deficiencies in purity (0.32 of A260/A230, 0.34 of A260/A280). Despite this, all extraction methods generated PCR products with specific D-loop primers (mtDNA). The combined effect of the pre-incubation and post-purification stages yielded satisfactory purity values (1.89 for A260/A230 and 1.65 for A260/A280), as well as concentration ratios (476.78 ng/μl) with low variability. In conclusion, the concentration and purity of DNA from blood samples is greatly improved when using a commercial kit in combination with pre-lysis incubation and post-extraction purification. These nucleic acids are suggested for use in potential molecular applications a posteriori.

Abstract Background Natural products represent important sources of antimicrobial compounds. Propolis and compounds from essential oils comprise good examples of such substances because of their inhibitory effects on bacterial spores, including bee pathogens. Methods Ethanol extracts of propolis (EEP) from Apis mellifera were prepared using different methods: double ultrasonication, double maceration and maceration associated with ultrasonication. Together with the antimicrobial peptides nisin and melittin, and compounds present in the essential oils of clove (Syzygium aromaticum) and cinnamon (Cinnamomum zeylanicum), assays were carried out on one Bacillus subtilis isolate and Paenibacillus alvei (ATCC 6344) against vegetative and sporulated forms, using the resazurin microtiter assay. Synergism with all the antimicrobials in association with tetracycline was verified by the time-kill curve method. Potassium and phosphate efflux, release of proteins and nucleic acids were investigated. Results EEPs showed the same MIC, 156.25 µg/mL against B. subtilis and 78.12 µg/mL against P. alvei. The peptides showed better activities against B. subtilis (MIC of 12 µg/mL for melittin and 37.50 µg/mL for nisin). Antimicrobials showed similar inhibitory effects, but cinnamaldehyde (39.06 µg/mL) showed the best action against P. alvei. Melittin and nisin showed the greatest capacity to reduce spores, regarding B. subtilis there was a 100% reduction at 6.25 and 0.78 µg/mL, respectively. Concerning P. alvei, the reduction was 93 and 98% at concentrations of 80 µg/mL of melittin and 15 µg/mL of nisin. EEPs showed the highest effects on the protein release against B. subtilis and P. alvei. Nucleic acid release, phosphate and potassium efflux assays indicated bacterial cell membrane damage. Synergism between antimicrobials and tetracycline was demonstrated against both bacteria. Conclusion All antimicrobials tested showed antibacterial activities against vegetative and sporulated forms of P. alvei and B. subtilis, especially nisin and melittin. Synergism with tetracycline and damage on bacterial cell membrane also occurred.