Combined sterilization and fabrication of drug-loaded scaffolds using supercritical CO2 technology

The access of biodegradable scaffolds to the clinical arena is constrained by the absence of a suitable sterilization technique for the processing of advanced polymeric materials. Sterilization with supercritical CO2 (scCO2) may circumvent some technological limitations (e.g., low temperature, no chemical residues on the material), although scCO2 can plasticize the polymer depending on the processing conditions used. In this latter case, the integration of the manufacturing and sterilization processes is of particular interest to obtain sterile and customized scaffolds in a single step. In this work, scCO2 was exploited as a concomitantly foaming and sterilizing agent for the first time, developing a one-step process for the production of vancomycin-loaded poly(ε-caprolactone) (PCL) bone scaffolds. The effect of the CO2 contact time on the sterility levels of the procedure was investigated, and the sterilization efficiency was evaluated against dry spores (Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus). Vancomycin-loaded PCL scaffolds had relevant sustained release profiles for the prophylaxis of infections at the grafted area, even those caused by methicillin-resistant Staphylococcus aureus (MRSA). The biological performance of the scaffolds was evaluated in vitro regarding human mesenchymal stem cells (hMSCs) attachment and growth. Finally, the biocompatibility and angiogenic response of the manufactured sterile scaffolds was assessed in ovo through chick chorioallantoic membrane (CAM) assaysThis research was funded by Xunta de Galicia [ED431C 2020/17], MICINN [PID2020-120010RB-I00], Consellería de Sanidade, Servizo Galego de Saúde, Axencia de Coñecemento en Saúde (ACIS, CT850A-G), Agencia Estatal de Investigación [AEI] and FEDER funds. V. Santos-Rosales acknowledges to Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2018/014]S

Antimicrobial activity of Latin American medicinal plant extracts

Resistance of pathogenic microorganisms to several drugs has increased due to the widespread use of antibiotics to treat infectious diseases. Hence, development of new antimicrobial drugs from plants is an area of active research in the search for medicinal, veterinary or agricultural industry use. For this work 19 plants species was collected, dried in the shade and oven. 20 g powder plant were macerated in cold either with dichloromethane (DCM) or methanol (MeOH) filtered, evaporated to yield determination expressed in grams per 100g of dry plant. Methods: Antibacterial activity assays of the DCM and MeOH extracts were performed by agar diffusion. Paper disks were then impregnated with 10 ul of extract (range: 340-614mg/disc for MeOH; 300-500mg/disc for DCM). Solvents of DCM or MeOH extracts were used as negative controls (NC) and Gentamicin and Cephazoline were used as positive control (PC). The microorganisms used for testing were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853. The inoculum was incorporated to Petri plates and paper disks with extracts were added, and incubated for 24 hours at 37°C. Results: All DCM extracts tested except Gentianella parviflora, Baccharis crispa, Bauhinia candicans, Terminalia langiflora and Picrosia australis showed activity against Staphylococcus aureus. The DCM extracts of the three species of Gaillardia showed to be as well active against Escherichia coli. Only the MeOH extracts of Baccharis crispa, Gentianella achalensis, Lippia turbinata, Lippia germinata, Terminalia australis and bicolor adesmia were active against Staphylococcus aureus. Conclusion: dichloromethane extracts of Gaillardia and Protousnea poepiggii species might be important sources for the isolation of compounds with antimicrobial activity with a potential use in the pharmaceutical industry.Facultad de Ciencias Exacta

Actividad antimicrobiana de germicidas halogenados frente a aislamientos hospitalarios

Antiseptics and disinfectants are extensively used in hospitals and health care settings for a variety of topical and hard-surface applications. In particular, they are essential part of infection control practices and in the prevention of nosocomial infections. Despite this, few is known about the mode of action of these biocides with respect to antibiotics. In general, the antimicrobial activity can be influenced by many factors such as formulation effects, presence of organic matter, synergy, temperature, dilution and test method. The widespread use of antiseptics and disinfectant products has prompted some speculation on the development of microbial resistance, in particular cross-resistance to antibiotics. The aim of this study was to evaluate microbiological resistance to halogenated compounds by studying the behaviour of the grampositive and gramnegative clinical isolates against halogenated biocides usually applied, with and without organic substance and applying distilled water, potable water and water of 300 ppm hardness as dilution means. The results indicate that the hospital microorganisms show a higher resistance to the biocides than the strain Staphylococcus aureus ATCC 6538, although the effective concentration in clean conditions was lesser than the recommended ones, for all the dilution means. In presence of organic matter the antimicrobial activity was reduced in accordance with the bactericidal concentration of each microorganisrn, due to the oxidant action of these disinfectantsLos antisépticos y desinfectantes se emplean en hospitales para una gran variedad de aplicaciones, tanto tópicas como sobre superficies. Estos compuestos son esenciales en el control y la prevención de las infecciones nosocomiales. A pesar de esto, se conoce bastante menos acerca del modo de acción de estos biocidas que de los antibióticos habitualmente empleados en terapéutica. La actividad antimicrobiana puede ser influenciada por muchos factores tales como la formulación, la presencia de materia orgánica, efectos de sinergia, temperatura, dilución e incluso del método de ensayo. El uso tan difundido de productos desinfectantes y antisépticos ha llevado a algunas especulaciones sobre el desarrollo de resistencia microbiana, y particularmente, resistencia cruzada con antibióticos. Con el propósito de estudiar la resistencia microbiana a germicidas halogenados de uso corriente en ,centros asistenciales, se evaluó el comportamiento de aislamientos hospitalarios tanto gram negativos como gram positivos frente a soluciones de hipoclorito de sodio, iodopovidona y tintura de iodo en presencia y ausencia de sustancias interferentes tales como materia orgánica y cationes. Los multados obtenidos indican que los microorganismos hospitalarios presentan mayor resistencia a los bioddas analizados con respecto al microorganismo de ref- erencia Staphylococcus aureus ATCC 6538. Sin embargo, en condiciones limpias este grupe de biocidas posee una alta eficacia, aún muy por debajo de las concentraciones de uso recomendadas, para todos los medios de dilución estudiados. Con la presencia de materia orgánica la actividad germicida disminuyó en todos los casos en relación directamente proporcional a la concentración, debido a la naturaleza predominantemente oxidante de estos compuestos

Supercritical CO2 technology for one-pot foaming and sterilization of polymeric scaffolds for bone regeneration

Sterilization is a quite challenging step in the development of novel polymeric scaffolds for regenerative medicine since conventional sterilization techniques may significantly alter their morphological and physicochemical properties. Supercritical (sc) sterilization, i.e. the use of scCO2 as a sterilizing agent, emerges as a promising sterilization method due to the mild operational conditions and excellent penetration capability. In this work, a scCO2 protocol was implemented for the one-pot preparation and sterilization of poly(-caprolactone) (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds. The sterilization conditions were established after screening against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) vegetative bacteria and spores of Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus. The transition from the sterilization conditions (140 bar, 39 °C) to the compressed foaming (60 bar, 26 °C) was performed through controlled depressurization (3.2 bar/min) and CO2 liquid flow. Controlled depressurization/pressurization cycles were subsequently applied. Using this scCO2 technology toolbox, sterile scaffolds of well-controlled pore architecture were obtained. This sterilization procedure successfully achieved not only SAL-6 against well-known resistant bacteria endospores but also improved the scaffold morphologies compared to standard gamma radiation sterilization proceduresThis work was supported by Xunta de Galicia [ED431F 2016/01, ED431C 2020/17], MCIUN [RTI2018-094131-A-I00], MINECO [SAF2017-83118-R], Consellería de Sanidade, Servizo Galego de Saúde, Axencia de Coñecemento e Saúde (ACIS, CT850A-G), Agencia Estatal de Investigación [AEI] and FEDER funds. V. Santos-Rosales acknowledges to Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2018/014]. C.A. García-González acknowledges to MINECO for a Ramón y Cajal Fellowship [RYC2014-15239]S

Antimicrobial activity of Latin American medicinal plant extracts

Resistance of pathogenic microorganisms to several drugs has increased due to the widespread use of antibiotics to treat infectious diseases. Hence, development of new antimicrobial drugs from plants is an area of active research in the search for medicinal, veterinary or agricultural industry use. For this work 19 plants species was collected, dried in the shade and oven. 20 g powder plant were macerated in cold either with dichloromethane (DCM) or methanol (MeOH) filtered, evaporated to yield determination expressed in grams per 100g of dry plant. Methods: Antibacterial activity assays of the DCM and MeOH extracts were performed by agar diffusion. Paper disks were then impregnated with 10 ul of extract (range: 340-614mg/disc for MeOH; 300-500mg/disc for DCM). Solvents of DCM or MeOH extracts were used as negative controls (NC) and Gentamicin and Cephazoline were used as positive control (PC). The microorganisms used for testing were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853. The inoculum was incorporated to Petri plates and paper disks with extracts were added, and incubated for 24 hours at 37°C. Results: All DCM extracts tested except Gentianella parviflora, Baccharis crispa, Bauhinia candicans, Terminalia langiflora and Picrosia australis showed activity against Staphylococcus aureus. The DCM extracts of the three species of Gaillardia showed to be as well active against Escherichia coli. Only the MeOH extracts of Baccharis crispa, Gentianella achalensis, Lippia turbinata, Lippia germinata, Terminalia australis and bicolor adesmia were active against Staphylococcus aureus. Conclusion: dichloromethane extracts of Gaillardia and Protousnea poepiggii species might be important sources for the isolation of compounds with antimicrobial activity with a potential use in the pharmaceutical industry.Facultad de Ciencias Exacta

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens

Background: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and hostpathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. Results: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immunerelated proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. Conclusion: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.This study was supported by a Consellería de Pesca e Asuntos Marítimos and the Dirección Xeral de I+D – Xunta de Galicia project (2004/CP480)S

RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG

La renovación de la palabra en el bicentenario de la Argentina : los colores de la mirada lingüística

El libro reúne trabajos en los que se exponen resultados de investigaciones presentadas por investigadores de Argentina, Chile, Brasil, España, Italia y Alemania en el XII Congreso de la Sociedad Argentina de Lingüística (SAL), Bicentenario: la renovación de la palabra, realizado en Mendoza, Argentina, entre el 6 y el 9 de abril de 2010. Las temáticas abordadas en los 167 capítulos muestran las grandes líneas de investigación que se desarrollan fundamentalmente en nuestro país, pero también en los otros países mencionados arriba, y señalan además las áreas que recién se inician, con poca tradición en nuestro país y que deberían fomentarse. Los trabajos aquí publicados se enmarcan dentro de las siguientes disciplinas y/o campos de investigación: Fonología, Sintaxis, Semántica y Pragmática, Lingüística Cognitiva, Análisis del Discurso, Psicolingüística, Adquisición de la Lengua, Sociolingüística y Dialectología, Didáctica de la lengua, Lingüística Aplicada, Lingüística Computacional, Historia de la Lengua y la Lingüística, Lenguas Aborígenes, Filosofía del Lenguaje, Lexicología y Terminología

Voice personalization and speaker de-identification in speech processing systems

This PhD dissertation is part of the research proyect SpeechTech4All, dedicated to the advanced research in the main speech technologies in all the official languages spoken in Spain, the speaker's emotion recognition, and the construction of experimental frameworks to show the realized work. It is a coordinated proyect with the Polytechnic University of Catalunya and the University of the Basque Country. Specifically, the PhD dissertation falls within in the work package 1 (voice generation) of the proyect. At present, the group of Vigo has his own unit selection based TTS system in Galician and Spanish (called Cotovía and available as open source in http://sourceforge.net/projects/cotovia/). Throughout this proyect, experiments with HMM-based speech techniques has been started. Thus, the lingüistic information provided by Cotovía has already been integrated in the HTS system (http://hts.sp.nitech.ac.jp/), and the HMMs corresponding to the different speakers employed in Cotovía have been generated. The purpose of the dissertation is the development of new algorithms and/or improvement of existing algorithms in order to obtain a synthetic speech of higher quality and/or adapt the speech quality to the requirements of the application in which it will be used (a tradeoff among memory, computational load and quality). With this aim, we will work on unit selection and HMM-based synthesis techniques, and also with hybrid models (combination of unit selection and HMM-based techniques). In addition, in order to provide the speech synthesis systems with a larger number of speakers and speaking styles, we will work on speaker adaptation techniques. We also contemplate the possibility of using the speech synthesis systems to analyze the robustness of speech identification systems.Esta tesis doctoral se enmarca dentro del proyecto de investigación SpeechTech4All, dedicado a la investigación avanzada en las principales tecnologías del habla en todas las lenguas oficiales habladas en España, al reconocimiento del estado emocional del hablante, y a la construcción de marcos experimentales que permitan mostrar el trabajo realizado. Se trata de un proyecto coordinado con la Universidad Politécnica de Cataluña y la Universidad del País Vasco. Concretamente, la tesis se encuadra dentro del paquete de trabajo 1 (generación de voz) del proyecto. En la actualidad, el grupo de Vigo dispone de su propio conversor texto-voz en gallego y castellano (denominado Cotovía y disponible como código abierto en http://sourceforge.net/projects/cotovia/), basado en técnicas de selección de unidades. A lo largo del presente proyecto se ha comenzado a trabajar en técnicas de síntesis de voz basadas en HMMs. Así, la información lingüística proporcionada por el conversor Cotovía ya ha sido integrada en el sistema HTS (http://hts.sp.nitech.ac.jp/), y se han generado los HMMs correspondientes a los distintos locutores empleados en Cotovía. El propósito de la tesis es el desarrollo de nuevos algoritmos y/o mejora de los algoritmos ya existentes con el objetivo de obtener una voz sintética de mayor calidad y/o adecuar la calidad de la voz a los requisitos de la aplicación en la que será utilizada (compromiso entre memoria, carga computacional y calidad). Con este objeto se trabajará en las técnicas de selección de unidades y síntesis por HMMs, así como con modelos híbridos (combinación de las técnicas de selección de unidades y de HMMs). Además, de cara a dotar a los sistemas de síntesis de voz de un mayor número de locutores y estilos de habla, se trabajará con técnicas de adaptación de locutor. También se contempla la posibilidad de utilizar sistemas de síntesis de voz para analizar la robustez de los sistemas de identificación por voz.Esta tese doutoral enmárcase dentro do proxecto de investigación SpeechTech4All, dedicado á investigación avanzada nas principais tecnoloxías da fala en tódalas linguas oficiais faladas en España, ó recoñecemento do estado emocional do falante, e á construción de marcos experimentais que permitan mostrar o traballo realizado. Trátase dun proxecto coordinado coa Universidade Politécnica de Cataluña e a Universidade do País Vasco. Concretamente, a tese encádrase dentro do paquete de traballo 1 (xeración de voz) do proxecto. Na actualidade, o grupo de Vigo dispón do seu propio convertedor texto-voz en galego e castelán (denominado Cotovía e dispoñible como código aberto en http://sourceforge.net/projects/cotovia/), baseado en técnicas de selección de unidades. Ó longo do presente proxecto comezouse a traballar en técnicas de síntese de voz baseadas en HMMs. Así, a información lingüística proporcionada polo convertedor Cotovía xa foi integrada no sistema HTS (http://hts.sp.nitech.ac.jp/), e xeráronse os HMMs correspondentes ós distintos locutores empregados en Cotovía. O propósito da tese é o desenvolvemento de novos algoritmos e/ou mellora dos algoritmos xa existentes co obxectivo de obter unha voz sintética de maior calidade e/ou adecuar a calidade da voz ós requisitos da aplicación na que será utilizada (compromiso entre memoria, carga computacional e calidade). Con este obxecto traballarase nas técnicas de selección de unidades e síntese por HMMs, así como con modelos híbridos (combinación das técnicas de selección de unidades e de HMMs). Ademais, de cara a dotar ós sistemas de síntese de voz dun maior número de locutores e estilos de fala, traballarase tamén con técnicas de adaptación de locutor. Tamén se contempla a posibilidade de utilizar sistemas de síntese de voz para analizar a robustez dos sistemas de identificación por voz.Gobierno de España|Ref. TEC2015-65345-PGobierno de España|Ref. TEC2015-67163-C2-1-RGobirno de España|Ref. TEC2012-38939-C03-01Gobierno de España|Ref. BES-2013-063708Xunta de Galicia | Ref. GRC2014/02

Behavior of gramnegative hospital microbial isolates against iodophor germicides

Las infecciones hospitalarias han adquirido una relevante importancia debido a la aparición de microorganismos resistentes a numerosos agentes biocidas. La flora residente hospitalaria se encuentra frecuentemente en ropas, alimentos, equipos biomédicos e incluso soluciones de desinfectantes, en un ambiente relativamente pequeño donde conviven los pacientes que en la mayoría de los casos tienen disminuidos sus mecanismos naturales de defensa, quedando fácilmente expuestos a las distintas y numerosas fuentes de infección. El riesgo de contraer infecciones se minimiza por medio del proceso de desinfección, cuya eficacia se encuentra influenciada por numerosos factores. Las bacterias gram negativas son algo más resistentes a la desinfección que las gram positivas y además, son agentes causales prevalentes de las infecciones hospitalarias. Por este motivo en el presente trabajo se evaluó el comportamiento de aislamientos hospitalarios correspondientes a gérmenes gram negativos frente a dos soluciones de iodóforos como iodopovidona al 10% y solución jabonosa de iodopovidona al 5%. Por medio de la concentración bactericida mínima y el coeficiente de fenol se seleccionaron aquellas cepas que evidenciaron mayor resistencia, que luego se estudiaron mediante el test de Kelsey-Sykes, por resultar más adecuado para evaluar los desinfectantes en el ámbito hospitalario. Este último se realizó usando agua dura como diluyente y suero al 20% para simular la presencia de materia orgánica. Los resultados obtenidos indican que las bacterias hospitalarias demuestran su resistencia con respecto a las cepas de referencia en las condiciones de ensayo establecidas, destacándose los géneros Klebsiella y Pseudomonas. En presencia de materia orgánica, se evidencia el carácter oxidante de la iodopovidona, ya que se inactiva notablemente.Hospital disinfection is a main factor in order to achieve a marked decrease in incidence and severity of infections. Emphasis on patient antisepsis and the elimination of the environmental source of pathogenic microorganisms are required. Frequently. iintrahospital infection causal microorganisms belong to Klebsiella and Pseudomonas genera. Patients exposed to them are usually immunodepressed ones, increasing morbidity and mortality rates. As a consequence. it is necessary to evaluate by simple methodologies the influence of disinfectants usually used in hospital environment over that flora. We used two iodophor germicides: iodopovidone at 10% and soap solution of iodopovidoiie at 5%. both of which were tittered by USP XXIII techhiques. The methodologies employed were bactericidal concentration, phenol coefficient, and Kelsey-Sykes test. Germicides were tested against reference microorganisms and hospital microbial isolates selected by antibiotic resistance. The Kelsey-Sykes technique was employed uinder a dilution condition of 300 ppm hard water, and in the presence of 20% serum in hard water. Under the assay conditions, the results obtanied demonstrated that the hospital isolates showed a higher resistance to the disinfectants than the referetice strains tested, particularly Pseudomoms and Klebsielh isolates. In presence of organic material, iodopovidone inactivates it markedly, showing its oxidant character.Colegio de Farmacéuticos de la Provincia de Buenos Aire