Soro Bovino Fetal como fator de interferência na produção de citocinas por células-tronco de papila apical in vitro / Fetal bovine serum as an interference factor in cytokines production by stem cells from apical papilla in vitro

A endodontia e medicina regenerativa têm demonstrado grande interesse no potencial proliferativo e de diferenciação das Células-tronco de Papila Apical (SCAP), sendo que muitos estudos têm sido direcionados a avaliar as citocinas produzidas por tal população celular. No entanto, o suplemento mais comumente utilizado nos meios de cultura, o Soro Bovino Fetal (SBF), apresenta uma composição complexa e não totalmente conhecida, podendo interferir em diferentes fenômenos, sendo um deles a produção in vitro de citocinas pelas células. Portanto, este estudo teve como proposição: 1. avaliar a interferência do SBF na viabilidade celular das SCAP ativadas ou não por lipopolissacarídeo de Escherichia Coli (LPS) e 2. verificar a interferência de diferentes concentrações de SBF na produção das citocinas Interleucina (IL)-6, Fator de Crescimento Transformador (TGF)-1, Osteoprotegerina (OPG) e a quimiocina CCL2 no sobrenadante das SCAP ativadas por LPS. As células, previamente caracterizadas, obtidas do Biobanco da FOUSP, foram cultivadas em meio -MEM a 10% de SBF, plaqueadas e, após 24h, 48h, 72h, 7 e 14 dias de estímulo, foram então submetidas ao ensaio de MTT para a avaliação da viabilidade celular. A quantificação das citocinas foi realizada através do ensaio de imunoabsorção enzimática (ELISA), no tempo experimental de 24h. Os grupos foram organizados em triplicata de acordo com a concentração de SBF e presença ou não de LPS (1 g/mL). A análise estatística foi executada aplicando-se a análise de variância a dois critérios (two-way ANOVA) seguida de pós-teste de Tukey com nível de significância de 5%. Em 24h, as SCAP cultivadas em meio suplementado com qualquer concentração de SBF apresentaram maior metabolismo celular comparadas àquelas na ausência de soro. Para os tempos experimentais mais longos, de 7 e 14 dias, as SCAP ativadas por LPS mostraram um aumento significativo na viabilidade celular quanto cultivadas sob 10 e 15% de SBF. As duas concentrações testadas de SBF (1 e 10%) interferiram na produção de todas as citocinas avaliadas no presente estudo. Esse resultado enfatiza a importância de evitar a suplementação com SBF em estudos de detecção de citocinas envolvendo SCAP.

Human venous blood derivatives as fetal bovine serum substitute for fibroblast culture cells in a fibrin construct

Aim: Venous blood derivatives (VBDs) have been suggested as substitutes for Fetal Bovine Serum (FBS) to improve the clinical transition of cell-based therapies. The literature is not clear about which is the best VBDs substitute. The present study aimed to evaluate the influence of VBDs on cell viability and describe a new method to seed these cells in a 3D Platelet-Rich Fibrin (PRF). Methods: Blood was processed to obtain Platelet-Poor Plasma from PRF (P-PRF), Human Serum (HS), Platelet-Poor Plasma from PRP (P-PRP), activated-PRP (a-PRP), and Platelet lysate (PL). Cells were supplemented with each VBD at 10% and FBS at 10% was the control. Cell viability (fibroblast 3T3/NIH) test was evaluated with MTT assay in two ways: i) cell-seeded and expanded with VBD; ii) cell-seed with FBS and expanded with VBD. To seed the Fibrin construct, cells were suspended in PBS and dropped into the blood sample before performing Choukroun's protocol for PRF. Constructs were cultured for 7 days in VBD supplements and FBS. Histological and Immunohistochemical analysis with vimentin was performed. Cell viability was analyzed by one-way ANOVA. Results: VBD's production time was very heterogeneous. Cells expanded in HS and a-PRP has grown faster. VBD-supplemented culture media provided cell culture highly sensible to trypsin/EDTA 0.25%. Cells seeded and expanded with VBD presented viability comparable to FBS in HS, a-PRP, and P-PRP (p>0.05) and lower in P-PRF and PL groups (p<0.05). The viability of cell seed with FBS and expanded with VBD was similar between P-PRF, a-PRP, PL, and FBS (p>0.05) and lower in HS and P-PRP (p<0.005). PRF-seeded cells showed a positive expression of vimentin and were able to maintain all cells supplemented with VBD. Conclusion: VBD supplements were able to maintain fibroblast cells in 2D and 3D cultures. The new method of the fibrin-cell construct was efficient to insert the cells into the fibrin network

Preparation of vitexin albumin nanoparticles and its pharmacokinetic study / 中国中药杂志

This study aims to prepare vitexin albumin nanoparticles(VT-BSA-NPs) to alleviate the low bioavailability of vitexin(VT) in vivo due to its poor water solubility. VT micro powders were prepared by the antisolvent crystallization method, and the morphology, size, and physicochemical properties of VT micro powders were studied. The results showed that the VT micro powder had a particle size of(187.13±7.15) nm, an approximate spherical morphology, and a uniform size distribution. Compared with VT, the chemical structure of VT micro powders has not changed. VT-BSA-NPs were prepared from VT micro powders by desolvation-crosslinking curing method. The preparation process was screened by single factor test and orthogonal test, and the quality evaluation of the optimal prescription particle size, PDI, Zeta potential, EE, and morphology was performed. The results showed that the average particle size of VT-BSA-NPs was(124.33±0.47) nm; the PDI was 0.184±0.012; the Zeta potential was(-48.83±2.20) mV, and the encapsulation rate was 83.43%±0.39%, all of which met the formulation-related requirements. The morphological results showed that the VT-BSA-NPs were approximately spherical in appearance, regular in shape, and without adhesion on the surface. In vitro release results showed a significantly reduced release rate of VT-BSA-NPs compared with VT, indicating a good sustained release effect. LC-MS/MS was used to establish an analytical method for in vivo analysis of VT and study the plasma pharmacokinetics of VT-BSA-NPs in rats. The results showed that the specificity of the analytical method was good, and the extraction recovery was more than 90%. Compared with VT and VT micro powders, VT-BSA-NPs could significantly increase AUC, MRT, and t_(1/2), which was beneficial to improve the bioavailability of VT.

Semisolid formulations based on solid-in-oil-in-water systems containing proteins

Abstract In recent years, improvements have been made, through biotechnological processes, in the production and development of peptides capable of increasing collagen and elastin synthesis for anti-aging skin care. However, proteins have many limitations due to their structural, chemical and physical fragility to external aggressions, which may cause conformational changes, leading to loss of biological activity. Therefore, it is important to create delivery systems that protect these biomolecules from damage, allowing them to reach their target. This work aimed to develop a system able to carry bovine serum albumin (BSA), used as a model of a protein, and to incorporate this system in a semisolid formulation suitable for skin application. A microemulgel based on a solid-in-oil-in-water (S/O/W) microemulsion was prepared. Firstly, the association efficiency (AE) of lyophilized BSA-sucrose ester complex and the size of S/O nanodispersion were assessed; then, the characterization and stability evaluation of the final semisolid formulation through evaluation of pH, texture and rheological behavior were performed. The average value of AE was 54.74% ± 2.17. It was possible to develop an S/O/W microemulsion, which allowed the subsequent development of an S/O/W microemulgel that assured suitable pH, texture and rheological characteristics for skin application.

Action of benzalkonium chloride in different pH

Ammonium quaternary compounds are widely used in poultry and swine production as disinfectants in the control of pathogens. They act on gram-positive bacteria, gram-negative bacteria, enveloped fungi and viruses. However, in some conditions of pH and presence of organic matter can be inactivated. This study evaluated the action of ammonium quaternary compounds at 1:1,000 and 1:2,000 dilutions against Salmonella enterica serovarTyphimurium and Salmonella enterica serovar Enteritidis in the presence of three different organic matter simulators, fetal bovine serum, skim milk and whole milk concentration of 1, 3, 5, and 7% and at pH 6 and 9, with 15 min of contact. It was possible to verify that the organic matter simulators adjusted in the same conditions of contact time and percentage, in the in vitro tests, presented different results and the fetal bovine serum did not inactivate the disinfectant. However, the best result against S. Typhimurium and S. Enteritidis was obtained at pH 6 at the dilution of 1:1,000 in all organic matter simulators.

Supervivencia de células fibroblásticas humanas en ausencia de suplementación / Survival of Human Fibroblastic Cells in the Absence of Supplementation

Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares.

Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.

Cultivo de células epiteliales dentales: impacto del suero fetal bovino / Culture of dental epithelial cells: impact of fetal bovine serum

RESUMEN Objetivo. Describir la influencia del Suero Fetal Bovino (SFB) en la supervivencia, crecimiento y expresión de organelas celulares en las células epiteliales dentales de rata. Materiales y métodos. Cultivos de células epiteliales dentales de rata fueron llevados a cabo a 37°C en una atmosfera húmeda, en ausencia y a una concentración de 10% de SFB. Una evaluación morfológica fue realizada durante la proliferación y confluencia de las células en cultivo. Dobles marcajes por inmunofluorencia fueron efectuados haciendo uso de anticuerpos anti-actina, anti-TOMM20 y anti-LAMP1. Resultados. Se evidenciaron células epiteliales dentales circulares u ovoides con núcleos voluminosos durante la proliferación y confluencias de manera similar en las células cultivas en presencia y ausencia de SFB. La carencia de SFB impactó negativamente la proliferación de las células epiteliales. No fueron observadas alteraciones en la localización de los inmunomarcajes anti-actina, anti-TOMM20 y anti-LAMP1 en las dos condiciones de cultivos experimentales. Conclusiones. La supresión del SFB en el cultivo de células epiteliales dentales de rata disminuyó la supervivencia, proliferación y sugiere no tener un impacto sobre las organelas evaluadas.

ABSTRACT Objective. Describe the influence of Fetal bovine serum (FBS) on the survival, growth and expression of cellular organelles in rat dental epithelial cells. Material and methods. Cell cultures of rat dental epithelial cells were carried out at 37°C in a humid atmosphere, in the absence and at a concentration of 10% FBS. Morphological evaluation was performed during the proliferation and confluence of cell in culture. Double immunofluorescence labels were made using anti-Actin, anti-TOMM20A, and anti-LAMP1 antibodies. Results. Circular or ovoid dental epithelial cells with bulky nuclei were evidenced during proliferation and confluences in a similar manner in culturing cells in the presence and absence of FBS. The lack of FBS negatively impacts the proliferation of epithelial cells. No alterations were observed in the localization of the anti-actin, anti-TOMM20 and anti-LAMP1 immunomarkers in both conditions of experimental cultures. Conclusion. FBS suppression in rat dental epithelial cells decreased survival, proliferation and suggests not having an impact on the organelles evaluated.

Trichostrongylus colubriformis infection in Santa Inês lambs: impact on feed digestibility, blood markers, and nitrogen balance / Infecção por Trichostrongylus colubriformis em cordeiros Santa Inês: impacto na digestibilidade dos alimentos, marcadores sanguíneos e balanço de nitrogênio

Abstract This study aimed to evaluate the effects of Trichostrongylus colubriformis infection on the hemato-biochemical parameters, feed digestibility, and nitrogen balance in Santa Inês lambs. Eighteen three-month-old Santa Ines castrated male lambs (16.9 ± 1.43 kg of body weight) were randomly distributed in two experimental treatments: infected with T. colubriformis (I, n = 9) and uninfected (U, n = 9). The I group received a total of 45,000 L3 larvae of T. colubriformis (5,000 infective larvae, three times per week, for three weeks). During the experimental period, blood, feed digestibility, and nitrogen balance were evaluated. The I lambs showed a reduction in erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, and total proteins, as well as an increase in platelets and eosinophils compared to those in the U group (p < 0.05). With the exception of total protein content, these values were within the normal range for the species. Furthermore, lower dry matter and organic matter digestibility were observed in the I lambs (p = 0.08). The present findings highlight that T. colubriformis infection has the potential to impair some hemato-biochemical parameters as well as feed digestibility in lambs, which could affect their productivity.

Resumo O objetivo deste estudo foi avaliar os efeitos da infecção por Trichostrongylus colubriformis nos parâmetros hemato-bioquímicos, digestibilidade aparente dos nutrientes e balanço de nitrogênio de cordeiros Santa Inês. Dezoito cordeiros Santa Inês, de três meses de idade e castrados (16,9 ± 1,43 kg de peso corporal), foram distribuídos aleatoriamente em dois tratamentos experimentais: infectados com T. colubriformis (I, n = 9) e não infectados (U, n = 9). O grupo I recebeu um total de 45.000 larvas L3 de T. colubriformis (5.000 larvas infectantes, três vezes por semana, durante três semanas). Durante o período experimental, foram avaliadas as variáveis sanguíneas, digestibilidade e balanço de nitrogênio. Os cordeiros I apresentaram redução de eritrócitos, hemoglobina, hematócrito, volume corpuscular médio e proteínas totais, além de aumento de plaquetas e eosinófilos, quando comparados ao grupo U (p< 0,05). Contudo, exceto para proteínas totais, os valores estavam dentro do intervalo normal para a espécie. Além disso, a digestibilidade da matéria seca e da matéria orgânica foi menor no grupo I de cordeiros (p = 0,08). Os presentes achados destacam que a infecção por T. colubriformis teve potencial para prejudicar alguns parâmetros hemato-bioquímicos, bem como a digestibilidade aparente da dieta, o que poderia afetar a produtividade dos cordeiros.

Biological activity of synthesized 5-{1-[(4-chlorophenyl)sulfonyl]piperidin-4-yl}-2-mercapto-1, 3, 4-oxadiazole derivatives demonstrated by in silico and BSA binding studies

We synthesized a series of compounds bearing pharmacologically important 1,3,4-oxadiazole and piperidine moieties. Spectral data analysis by 1H-NMR, 13C-NMR, IR and EI-MS was used to elucidate the structures of the synthesized molecules. Docking studies explained the different types of interaction of the compounds with amino acids, while bovine serum albumin (BSA) binding interactions showed their pharmacological effectiveness. Antibacterial screening of these compounds demonstrated moderate to strong activity against Salmonella typhi and Bacillus subtilis but only weak to moderate activity against the other three bacterial strains tested. Seven compounds were the most active members as acetyl cholinesterase inhibitors. All the compounds presented displayed strong inhibitory activity against urease. Compounds 7l, 7m, 7n, 7o, 7p, 7r, 7u, 7v, 7x and 7v were highly active, with respective IC50 values of 2.14±0.003, 0.63±0.001, 2.17±0.006, 1.13±0.003, 1.21±0.005, 6.28±0.003, 2.39±0.005, 2.15±0.002, 2.26±0.003 and 2.14±0.002 µM, compared to thiourea, used as the reference standard (IC50 = 21.25±0.15 µM). These new urease inhibitors could replace existing drugs after their evaluation in comprehensive in vivo studies.

Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein

Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactide-co-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.

Novel ractopamine-protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Various macromolecules in in vitro growth medium influence growth, maturation, and parthenogenetic development of pig oocytes derived from small antral follicles / 대한수의학회지

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF (113.0–114.8 µm) were significantly larger than that of no IVG-SAF (111.8 µm). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

Study on interaction between ginsenosides Rg_1,Rb_1 and Ro and bovine serum albumin / 中国中药杂志

Small molecules with physiological or pharmacological activities need to interact with biological macromolecules in order to function in the body. As the protein with the highest proportion of plasma protein,serum albumin is the main protein binding to various endogenous or exogenous small molecules. Serum albumin interacts with small molecules in a reversible non-covalent manner and transports small molecules to target sites. Bovine serum albumin( BSA) is an ideal target protein for drug research because of its low cost and high homology with human serum albumin. The research on the interaction between drugs and BSA has become a hotspot in the fields of pharmacy,medicine,biology and chemistry. In this research,molecular docking method was used to study the interaction between three small ginsenosides with high pharmacological value( Rg_1,Rb_1,Ro) and bovine serum albumin( BSA),and the binding mode information of three ginsenosides interacting with BSA was obtained. The results of molecular docking showed that ginsenosides and amino acid residues in the active pocket of proteins could be combined by hydrophobic action,hydrogen bonding and electrostatic action. The interaction between small ginsenosides and bovine serum albumin is not the only form,and their interaction has many forms of force. The interaction between these molecules and various weak forces is the key factor for the stability of the complex. The results of this study can provide the structural information of computer simulation for the determination of the interaction patterns between active components and proteins of ginseng.

Preparation, characterization, and antiproliferative activities of biotin-decorated docetaxel-loaded bovine serum albumin nanoparticles

ABSTRACT The aim of the present study was to characterize biotin-decorated docetaxel-loaded bovine serum albumin nanoparticles (DTX-BIO-BSA-NPs) and evaluate their antiproliferative activity in vitro. The particle size of prepared DTX-BIO-BSA-NPs was found to be always lower than 200 nm, with sizes of 166.9, 160.3, 159.0, 176.1 and 184.8 nm and the zeta potential was -29.51, -28.54, -36.54, -36.08 and -27.56 mV after redissolution with water for 0, 1, 2, 4 and 8 hours, respectively. The polydispersity index (PDI) was stable in the range of 0.170 - 0.178. In the in vitro drug-release study, the DTX-BIO-BSA-NPs targeted a human breast cancer cell line MCF-7 effectively. The x-ray diffraction spectrum and DSC curve of DTX-BIO-BSA-NPs suggested that docetaxel was in an amorphous or disordered crystalline phase in DTX-BIO-BSA-NPs. In vitro cytotoxicity results showed that DTX-BIO-BSA-NPs inhibits proliferation of MCF-7, SGC7901, LS-174T and A549 cells in a concentration-dependent manner after exposure to DTX-BIO-BSA-NPs for 48 hours. Taken together, these results indicate that DTX-BIO-BSA-NPs may have potential as an alternative delivery system for parenteral administration of docetaxel.

Estudo comparativo entre a produção de fosfolipases extracelulares e proteinases do gênero Candida isoladas a partir de infecções de cavidade oral / Comparative study between extracellular phospholipase and proteinase production in clinically important of the genus Candida isolated in oral candidiasis patients

Introdução: A habilidade da Candida spp. em produzir enzimas proteolíticas, tais como fosfolipase e proteinases, tem um papel importante na patogenicidade destas leveduras. Objetivo: Determinar as espécies causadoras das infecções orais por Candida spp., além de investigar a atividade in vitro das fosfolipases e proteinases em isolados clínicos do gênero Candida, provenientes de pacientes com candidíase oral. Material e método: Isolados de Candida spp., pertencentes à Coleção de Cultivos Fúngicos do Laboratório de Microbiologia e Patologia Oral do Departamento de Odontologia da Faculdade da Serra Gaúcha, foram analisados. Produção de fosfolipases foi analisada utilizando-se Ágar gema de ovo. Liberação de proteinases foi medida utilizando-se extrato de levedura adicionado à albumina bovina. Resultado: Um total de 35 isolados clínicos do gênero Candida foi testado. C. albicans foi a espécie predominante (77%). Os demais isolados identificados foram: C. parapsilosis (20%) e C. tropicalis (2%). Ao comparar a atividade de fosfolipase do grupo C. albicans com o grupo Candida não-albicans, foi encontrada diferença significativa (P=0,04). Não foi encontrada diferença significativa entre a C. albicans e a C. não-albicans, para a produção de proteinase. A liberação de proteinase foi significativamente maior quando comparada à produção de fosfolipase para o gênero Candida (P=0,04). Diferença estatisticamente significativa foi encontrada quando a atividade de fosfolipase e proteinase da C. albicans foi comparada à atividade das espécies de C. não-albicans (P=0,02). Conclusão: Diferentes quantificações de fosfolipase extracelular e atividade de proteinase têm sido atribuídas aos isolados clínicos de C. albicans quando comparados a outras espécies de Candida.

Introduction: The ability of the genus Candida to produce secretory enzimes such as proteinase and phospholipases to play an important role in the patogenicity of these yeasts. Objective: To determine the Candida species isolates from oral cavity infections and to investigate in vitro phospholipase and protease activities in clinically important of the genus Candida isolated in oral candidiasis patients. Material and method: Candida species isolated of the Fungal Culture Collection of Oral Microbiology and Pathology Testing Service Laboratory of the Dentistry Departament of Faculdade da Serra Gaúcha were evaluated. The phospholipases detection was assayed using the egg yolk agar plate method. Determination of protease production was performed in agar plates containing bovine serum albumine and yeasts extract. Result: A total of 35 isolates of the genus Candida were tested. C. albicans was the species predominant (77% of isolates), followed by C. parapsilosis (20%) and C. tropicalis (2%). When compared the phospholipase activity of the C. albicans group with the non-albicans Candida species types group was observed significant difference among of this groups (P=0.04). No statiscally significant difference between the C.albicans and non-albicans Candida species types when was compared to proteinase production. Proteinase production was higher and statiscally significant when compared to phospholipase activity in the genus Candida isolates (P=0.04). Statiscally significant differences were found between phosphoplipases and proteases activity for C. albicans when compared to non-albicans Candida species types (P=0.02). Conclusion: Differences in the secretion rates of phospholipase extracellular and proteases activity have been attributed to clinical strains of C. albicans when compared to others Candida species types.

Antibody response in silver catfish (Rhamdia quelen) immunized with a model antigen associated with different adjuvants

Adjuvants are essential to boost the immune response to inoculated antigen and play a central role in vaccine development. In this study, we investigated the efficacy of several adjuvants in the production of anti-bovine serum albumin (BSA) antibodies in silver catfish. Two hundred and seventy juvenile silver catfish (60–80 g) of both sexes were intraperitoneally vaccinated with BSA (200 µg/fish) alone or mixed to the following adjuvants: Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), aluminum hydroxide (AlOH), Montanide, four types of cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) and three concentrations of β-glucan, and the immune enhancing property was evaluated by measuring anti-BSA antibodies in blood samples at biweekly intervals. Our results demonstrated that CpGs ODNs and β-glucan were as effective as classical adjuvants (FCA, FIA, AlOH and Montanide) in promoting anti-BSA antibodies and that the kinetics of antibody production induced by all adjuvants used in our study had a similar trend to that observed in other fish species, with a peak at 28 days post-vaccination. These results may be useful for the selection of adjuvants for vaccine formulation intended for silver catfish and for the development of vaccine and vaccination strategies to other fish species.

Biomodifying effect of epigallocatechin-3-gallate on dentine substrate splicing surface / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of epigallocatechin-3-gallate (EGCG) on biomodification of demineralized dentine substrate, in its permeability, hydrophobicity, and inhibition ability to collagen enzymatic degradation.</p><p><b>METHODS</b>The dentine substrates were treated with simulated pulpal pressure created by mixtures of 0.02%, 0.1% EGCG/bovine serum albumin (BSA) in acidic environment (pH4.4) for 48 h. A fluid-transport model was used to measure the fluid permeability through demineralized dentine substrate. Positive replicas of dentine substrate were fabricated before and after being subjected to acidic environment for scanning electron microscope (SEM) examination. The blank group contained no EGCG and the positive group were treated with Gluma desensitizer. Static contact angle measurements on demineralized dentin and 0.1% EGCG primed dentin were performed by contact angle analyzer. The priming time were 60 s, 120 s, 0.5 h, 1 h. Dentine specimens bonded with Adper single bond 2 were subjected to 100 mg/L collagenase and observed under SEM. Resin-bonded specimens (with 0.02%, 0.1%, 0.5% EGCG priming, or without EGCG priming) were created for micro-tensile bond strength evaluation (MTBS). Resin-bonded specimens after thermol cycling were created for MTBS evaluation.</p><p><b>RESULTS</b>The fluid permeability in the blank control group increased ([151.3±22.3]%), the fluid permeability in 0.1% EGCG/BSA group decreased ([23.7±6.3]%). Compared to the blank control group, the contact angle of 120 s, 0.5 h, 1 h groups increased by 31.0%, 53.5%, 57.8% in deep dentin and 37.4%, 59.3%, 62.4% in shallow dentin. The SEM examination showed that 0.1% and 0.5% EGCG priming for 120 s significantly increased dentin collagen's resistance to collagenase. The immediate MTBS of 0.1% and 0.5% EGCG groups were (29.4±4.8) and (19.8± 4.9) MPa. After thermol cycling, the MTBS of 0.1% and 0.5% EGCG groups were (19.9±5.1) and (15.3± 6.3) MPa.</p><p><b>CONCLUSIONS</b>Under acidic environment (pH4.4), the 0.1% EGCG can reduce dentine permeability under acidic environment. The 0.1% EGCG can increase hydrophobicity of dentin substrate, and strengthen dentin substrate's resistance to collagenase hydrolysis, thus increased the resin-dentin bonding durability.</p>

Inhibition of advanced glycation end product formation by burdock root extract / 한국영양학회지

PURPOSE: Diabetic complications are a major concern to manage progression of diabetes. Production of advanced glycation end products (AGEs) due to high blood glucose is one of the mechanisms leading to diabetic complications. Multiple pharmacologic AGE inhibitory agents are currently under development, but clinical applications are still limited due to safety issues. Thus, it is necessary to identify a safe anti-glycation agent. It is known that burdock roots have antioxidant, anti-inflammatory, and anti-cancer activities. The objective of the present study was to investigate the inhibitory role of burdock roots on the formation of high glucose-induced glycation of bovine serum albumin (BSA). METHODS: In this study, glycation of BSA by glucose, galactose, or fructose at 37℃ for 3 weeks was assessed based on levels of α-dicarbonyl compounds (early-stage glycation products), fructosamine (intermediate products of glycation), and fluorescent AGEs (late-stage glycation products). In order to compare the inhibitory actions of burdock root extract in AGE formation, aminoguanidine (AG), a pharmacological AGE inhibitor, was used as a positive control. RESULTS: BSA glycation by glucose, fructose, and galatose was dose- and time-dependently produced. Burdock root extract at a concentration of 4 mg/mL almost completely inhibited glucose-induced BSA glycation. The results demonstrate that burdock root extract inhibited AGE formation with an IC₅₀ value of 1.534 mg/mL, and inhibitory activity was found to be more effective than the standard anti-glycation agent aminoguanidine. This study identified a novel function of burdock root as a potential anti-glycation agent. CONCLUSION: Our findings suggest that burdock root could be beneficial for preventing diabetic complications.

Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody / Estudo da nova estratégia de revestimento para stents coronários: revestimento simultâneo de VEGF e anticorpo anti-CD34

Abstract Introduction: Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD), with a major limitation of in-stent restenosis (ISR). The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF) and anti-CD34 antibody on 316L stainless steel. Methods: Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V)10 group. Eight sheets from the D-(H-V)10 group were coated with anti-CD34 antibody and termed as the D-(H-V)10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody. Results: The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V)10 and D-(H-V)10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V)10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody. Conclusion: Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy. .

Resumo Introdução: O stent coronário intravascular tem sido utilizado no tratamento de doença arterial coronária, com uma maior limitação de restenose intra-stent (RIS). O aço inoxidável 316 tem sido amplamente utilizado para stents. Neste estudo, foi desenvolvido um novo método de revestimento para reduzir a RIS para revestir simultaneamente o fator de crescimento endotelial vascular (VEGF) e anti-CD34 em aço inoxidável 316L. Métodos: Placas de aço inoxidável 316L redondas no grupo DH foram polimerizadas com compostos gerados a partir da reacção de condensação de dopamina e heparina utilizando N- (3-dimetilaminopropil) -N'-etilcarbodiimida (EDC) e N-hidroxissuccinimida (NHS). Dezesseis folhas a partir do grupo DH foram ainda imersas em 1 ug/ml de VEGF 165 e 3 mg/ml de heparina sódica, um após outro por 10 vezes, sendo denominado como o grupo D-(HV)10. Oito folhas de D-(HV)10 foram revestidas com anticorpo anti-CD34 e denominado como grupo D-(HV)10-A. Testes de imunofluorescência e ELISA foram usados para avaliar se os discos de aço inoxidável 316L foram revestidos com sucesso com VEGF e anticorpo anti-CD34. Resultados: Os resultados dos testes de imunofluorescência e ELISA mostraram que o VEGF pôde ser detectado nos grupos D-(HV)10 e D-(HV)10-A, evidenciando que as chapas de aço foram cobertas com VEGF com sucesso. O anticorpo anti-CD34 podia apenas ser observado no grupo D-(HV)10-A, o único grupo revestido com anticorpo CD34. Ambos os resultados sugerem que as chapas de aço inoxidável 316L foram revestidas com sucesso com VEGF e anticorpo anti-CD34. Conclusão: Nosso estudo desenvolveu um método para revestir simultaneamente VEGF e anti-CD34 de aço inoxidável. Esta pesquisa tem um papel fundamental para a nova estratégia de revestimento. .

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.