Real-time quantification of proteins secreted by artificial connective tissue made from uni- or multidirectional collagen I scaffolds and oral mucosa fibroblasts.

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC).

Initial Enterococcus faecalis-endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria-cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium-bacterium aggregation and bacterium-host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

Design, synthesis and cytotoxic evaluation of a selective serotonin reuptake inhibitor (SSRI) by virtual screening.

Depression is one of the most common mental illnesses, affecting almost 300 million people. According to the WHO, depression is one of the world's leading causes of disability and morbidity. People with this illness require both psychological and pharmaceutical treatment because severe depressive episodes often result in suicide. Selective serotonin reuptake inhibitors (SSRI) are widely used antidepressants that target the human serotonin transporter (hSERT). The crystallization of hSERT and the experimental data available allows cost and time-efficient computational tools like virtual screening (VS) to be utilized in the development of therapeutic agents. Here, we synthesized, characterized, and evaluated the biological activity of a novel SSRI analog of paroxetine, rationally designed by applying an artificial neural network-based QSAR model and a molecular docking analysis on hSERT. The analog N-substituted 18a showed higher affinity for the transporter (-10.2 kcal/mol), lower Ki value (1.19 nM) and a safer toxicological profile than paroxetine and was synthesized with a 71% yield. The in vitro cytotoxicity of the analog was evaluated using human glioblastoma (U87 MG), human neuroblastoma (SH SY5Y) and murine fibroblast (L929) cell lines. Also, the hemolytic ability of the compound was assessed on human erythrocytes. Results showed that analog 18a did not exhibit cytotoxic activity on the cell lines used and has no hemolytic activity at any of the concentrations tested, whereas with paroxetine, hemolysis was observed at 2.3, 1.29 y 0.67 mM. Based on these results, it is possible to suggest that analog 18a could be a promising new SSRI candidate for the treatment of this illness.

In silico design, synthesis and evaluation of a less toxic octinoxate alternative with suitable photoprotection properties.

Excessive UV exposure leads to several skin pathologies such as sunburns, photoaging and carcinogenesis. Currently, sunscreen use is the most important factor in protecting skin from photoinduced damage. Octinoxate is a commonly used UV filter, but its use has become controversial because it acts as an endocrine disruptor in both humans, and marine animals. Research has relied on biotechnology, structure activity relationship (SAR) studies and combinatorial chemistry to find new and less toxic UV filters. However, there are no current examples that describe the possible applications of in silico techniques for obtaining these compounds. Thus, this project sought to design an octinoxate analog that could be used as a less toxic, but equally effective, photoprotective alternative through ligand based virtual screening (LBVS). We designed 213 novel molecules based on the (E)-cinnamoyl moiety of octinoxate, but only 23 were found to be less toxic than the parent compound. Then, an artificial neural network (ANN) based model was built to predict the molar absorptivity of those 23 molecules, and the molecule that presented a similar molar absorptivity to that of octinoxate was chosen for synthesis (analog 4, 3-phenylpropyl (E)-3-(4-methoxyphenyl)acrylate). Synthesis for analog 4 resulted in a 90% yield, and its photoprotective properties, lipophilicity and cytotoxicity were then evaluated. Analog 4 absorbed UV radiation in the range of 250-340 nm, and it presented a molar absorptivity of 36,155 M - 1cm-1. Its lipophilicity was evaluated with RP-HPLC resulting in a logkw of 2.49 and its LC50 was greater than octinoxate's (67.41 nM vs. 45.67 nM). Therefore, results showed that ligand based virtual screening is an effective strategy for the development of new organic UV filters, because it guided the design of less toxic analogs and pinpointed the most likely analog to exhibit UV properties similar to those of octinoxate. In this case, analog 4 is a promising alternative to its parent compound since it proved to be more effective and less toxic.

Adjustable conduits for guided peripheral nerve regeneration prepared from bi-zonal unidirectional and multidirectional laminar scaffold of type I collagen.

Shortness of donor nerves has led to the development of nerve conduits that connect sectioned peripheral nerve stumps and help to prevent the formation of neuromas. Often, the standard diameters of these devices cannot be adapted at the time of surgery to the diameter of the nerve injured. In this work, scaffolds were developed to form filled nerve conduits with an inner matrix with unidirectional channels covered by a multidirectional pore zone. Collagen type I dispersions (5 mg/g and 8 mg/g) were sequentially frozen using different methods to obtain six laminar scaffolds (P1 to P5) formed by a unidirectional (U) pore/channel zone adjacent to a multidirectional (M) pore zone. The physicochemical and microstructural properties of the scaffolds were determined and compared, as well as their biodegradability, residual glutaraldehyde and cytocompatibility. Also, the Young's modulus of the conduits made by rolling up the bizonal scaffolds from the unidirectional to the multidirectional zone was determined. Based on these comparisons, the proliferation and differentiation of hASC were assessed only in the P3 scaffolds. The cells adhered, aligned in the same direction as the unidirectional porous fibers, proliferated, and differentiated into Schwann-like cells. Adjustable conduits made with the P3 scaffold were implanted in rats 10 mm sciatic nerve lesions to compare their performance with that of autologous sciatic nerve grafted lesions. The in vivo results demonstrated that the tested conduit can be adapted to the diameter of the nerve stumps to guide their growth and promote their regeneration.

[Efectos del aislamiento social, preventivo y obligatorio en pacientes con diabetes tipo 2].

Objetivo: Evaluar el impacto del aislamiento social, preventivo y obligatorio sobre al aumento de peso, el nivel de actividad física, la adherencia al tratamiento y la inmunización antigripal y antineumocócica en pacientes con diabetes tipo 2 (DM2). Método: Se realizó un seguimiento telefónico de pacientes con DM2 luego de al menos 100 días de comienzo del aislamiento. Se compararon dos regiones agrupadas en relación con el número de casos: región 1, más de 150 casos/100, 000 habitantes, y región 2, más de 150 casos/100,000 habitantes. Resultados: Se contactaron 454 pacientes con DM2. El 42% reportaron incremento de peso y el 7% refirió haber aumentado más de 5 kg. En relación con la actividad física, se observó un promedio más bajo en la región 1 (367.5 [0-5698] MET/sem) que en la región 2 (720 [0-7066] MET/sem) (p = 0.0009). La adherencia al tratamiento farmacológico disminuyó en forma global, pero aumentó en las zonas de mayor circulación viral. Por otra parte, hubo un incremento de vacunación antigripal y antineumocócica, alcanzando coberturas del 80% y el 70%, respectivamente. Conclusiones: En una población de alto riesgo cardiovascular y con una prevalencia de sobrepeso/obesidad elevada, el aislamiento social, preventivo y obligatorio se asoció con ganancia de peso y menos actividad física, lo que podría resultar deletéreo sobre la salud cardiovascular de los pacientes con DM2. Por otro lado, se observan algunos aspectos positivos, como el aumento de las inmunizaciones y el incremento de la adherencia en las zonas más afectadas.

Cardiovascular Risk Stratification in Patients with Metabolic Syndrome Without Diabetes or Cardiovascular Disease: Usefulness of Metabolic Syndrome Severity Score.

INTRODUCTION: The estimated cardiovascular risk determined by the different risk scores, could be heterogeneous in patients with metabolic syndrome without diabetes or vascular disease. This risk stratification could be improved by detecting subclinical carotid atheromatosis. AIMS: To estimate the cardiovascular risk measured by different scores in patients with metabolic syndrome and analyze its association with the presence of carotid plaque. METHODS: Non-diabetic patients with metabolic syndrome (Adult Treatment Panel III definition) without cardiovascular disease were enrolled. The Framingham score, the Reynolds score, the new score proposed by the 2013 ACC/AHA Guidelines and the Metabolic Syndrome Severity Calculator were calculated. Prevalence of carotid plaque was determined by ultrasound examination. A Receiver Operating Characteristic analysis was performed. RESULTS: A total of 238 patients were enrolled. Most patients were stratified as "low risk" by Framingham score (64%) and Reynolds score (70.1%). Using the 2013 ACC/AHA score, 45.3% of the population had a risk ≥7.5%. A significant correlation was found between classic scores but the agreement (concordance) was moderate. The correlation between classical scores and the Metabolic Syndrome Severity Calculator was poor. Overall, the prevalence of carotid plaque was 28.2%. The continuous metabolic syndrome score used in our study showed a good predictive power to detect carotid plaque (area under the curve 0.752). CONCLUSION: In this population, the calculated cardiovascular risk was heterogenic. The prevalence of carotid plaque was high. The Metabolic Syndrome Severity Calculator showed a good predictive power to detect carotid plaque.

Controlled release of an extract of Calendula officinalis flowers from a system based on the incorporation of gelatin-collagen microparticles into collagen I scaffolds: design and in vitro performance.

Aiming to develop biological skin dresses with improved performance in the treatment of skin wounds, acellular collagen I scaffolds were modified with polymeric microparticles and the subsequent loading of a hydroglycolic extract of Calendula officinalis flowers. Microparticles made of gelatin-collagen were produced by a water-in-oil emulsion/cross-linking method. Thereafter, these microparticles were mixed with collagen suspensions at three increasing concentrations and the resulting mixtures lyophilized to make microparticle-loaded porous collagen scaffolds. Resistance to enzymatic degradation, ability to associate with the C. officinalis extract, and the extract release profile of the three gelatin-collagen microparticle-scaffold prototypes were assessed in vitro and compared to collagen scaffolds without microparticles used as control. Data indicated that the incorporation of gelatin-collagen microparticles increased the resistance of the scaffolds to in vitro enzymatic degradation, as well as their association with the C. officinalis flower extract. In addition, a sharp decrease in cytotoxicity, as well as more prolonged release of the extract, was attained. Overall results support the potential of these systems to develop innovative dermal substitutes with improved features. Furthermore, the gelatin-collagen mixture represents a low-cost and scalable alternative with high clinical transferability, especially appealing in developing countries.

[Validity of peak flow record in asthmatic children residing in Mexico City]. / Validez en el registro del pico espiratorio máximo de niños asmáticos de la Ciudad de México.

OBJECTIVE: To determine the concordance between maximum peak expiratory flow records (PEFr) reported by the parents of asthmatic children and the electronic values stored by the AirWatch device (PEFe). MATERIAL AND METHODS: Records of PEF measurements between October 1998 and 1999 were obtained from 42 asthmatic children 5 to 15 years of age recruited at the Hospital Infantil de Mexico Federico Gomez, in Mexico City. Parents recorded the maximum value in the health diary. Spearman correlation was calculated between PEFe and PEFr and a mixed-effects logistic model was used. RESULTS: The correlation between PEFe and PEFr was r=0.96 (p<0.05) among children with a diagnosis of moderate or severe asthma and r=0.40 (p<0.05) among children diagnosed with mild asthma. Follow-up time, asthma severity, gender and age of the child and their interactions were predictors of the differences between PEFe and PEFr. CONCLUSIONS: Parents of children with moderate or severe asthma from 6 to 8 years of age report PEF values with greater accuracy during follow-up than others.

Morphology of hemocytes from the freshwater prawn Macrobrachium rosenbergii.

Using morphological criteria, we identified three types of blood cells in the freshwater prawn Macrobrachium rosenbergii. Hyaline hemocytes, the most abundant type, have few large cytoplasmic granules, a large nucleocytoplasmic ratio, and lyse spontaneously in the absence of anticoagulant. Granular hemocytes are heterogeneous in size and in density of their granules. They are phagocytic and readily spread on substrates. The third type of hemocytes, identified as undifferentiated hemocytes, are the least abundant. The hemocytes of this economically relevant crustacean are compared with blood cells of other decapods. J. Morphol. 234:147-153, 1997. © 1997 Wiley-Liss, Inc.

Validez en el registro del pico espiratorio máximo de niños asmáticos de la Ciudad de México / Validity of peak flow record in asthmatic children residing in Mexico City

OBJETIVO: Determinar la concordancia entre el pico espiratorio forzado (PEF) registrado por padres de niños asmáticos y el PEF electrónico que es el almacenado en el AirWatch (PEFe). MATERIAL Y MÉTODOS: Se obtuvo el PEF de 42 niños de 5 a 15 años de edad del Hospital Infantil de México Federico Gómez de la Ciudad de México entre octubre de 1998 y 1999. Los padres registraron el valor máximo del PEF en un diario de salud. Se calculó la correlación de Spearman entre el PEFe y el PEFr. Utilizamos un modelo logístico de efectos mixtos. RESULTADOS: La correlación del PEFe y el PEFr fue r= 0.96 (p<0.05) en niños con diagnóstico de asma moderada o severa y r= 0.40 (p< 0.05) en niños con asma leve. El tiempo de seguimiento y severidad del asma, el género y edad del niño y sus interacciones eran predictores de las diferencias entre el PEFe y el PEFr. CONCLUSIONES: Los padres de niños de 6 a 8 años con asma moderada o severa reportan mejor el PEF en el diario de salud a lo largo del seguimiento que los padres de otros grupos.

OBJECTIVE: To determine the concordance between maximum peak expiratory flow records (PEFr) reported by the parents of asthmatic children and the electronic values stored by the AirWatch device (PEFe). MATERIAL AND METHODS: Records of PEF measurements between October 1998 and 1999 were obtained from 42 asthmatic children 5 to 15 years of age recruited at the Hospital Infantil de Mexico Federico Gomez, in Mexico City. Parents recorded the maximum value in the health diary. Spearman correlation was calculated between PEFe and PEFr and a mixed-effects logistic model was used. RESULTS: The correlation between PEFe and PEFr was r=0.96 (p<0.05) among children with a diagnosis of moderate or severe asthma and r=0.40 (p<0.05) among children diagnosed with mild asthma. Follow-up time, asthma severity, gender and age of the child and their interactions were predictors of the differences between PEFe and PEFr. CONCLUSIONS: Parents of children with moderate or severe asthma from 6 to 8 years of age report PEF values with greater accuracy during follow-up than others.