Cell behavior on silica-hydroxyapatite coaxial composite.

Progress in the manufacture of scaffolds in tissue engineering lies in the successful combination of materials such as bioceramics having properties as porosity, biocompatibility, water retention, protein adsorption, mechanical strength and biomineralization. Hydroxyapatite (HA) is a ceramic material with lots of potential in tissue regeneration, however, its structural characteristics need to be improved for better performance. In this study, silica-hydroxyapatite (SiO2-HA) non-woven ceramic electrospunned membranes were prepared through the sol-gel method. Infrared spectra, scanning electron microscopy and XRD confirmed the structure and composition of composite. The obtained SiO2-HA polymeric fibers had approximately 230±20 nm in diameter and were then sintered at 800°C average diameter decreased to 110±17 nm. Three configurations of the membranes were obtained and tested in vitro, showing that the composite of SiO2-HA fibers showed a high percentage of viability on a fibroblast cell line. It is concluded that the fibers of SiO2-HA set in a coaxial configuration may be helpful to develop materials for bone regeneration.

Epithelial Growth Factor-Anchored on Polycaprolactone/6-deoxy-6-amino-ß-cyclodextrin Nanofibers: In Vitro and In Vivo Evaluation.

Polycaprolactone (PCL) is a well-known FDA approved biomaterial for tissue engineering. However, its hydrophobic properties limit its use for skin wound healing which makes its functionalization necessary. In this work, we present the fabrication and evaluation of PCL nanofibers by the electrospinning technique, as well as PCL functionalized with 6-deoxy-6-amino-ß-cyclodextrin (aminated nanofibers). Afterwards, epithelial growth factor (EGF) was anchored onto hydrophilic PCL/deoxy-6-amino-ß-cyclodextrin. The characterization of the three electrospun fibers was made by means of field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR); Confocal-Raman Spectroscopy were used for elucidated the chemical structure, the hydrophilicity was determined by Contact Angle (CA). In vitro cell proliferation test was made by seeding embryonic fibroblast cell line (3T3) onto the electrospun mats and in vivo studies in a murine model were conducted to prove its effectivity as skin wound healing material. The in vitro studies showed that aminated nanofibers without and with EGF had 100 and 150% more cell proliferation of 3T3 cells against the PCL alone, respectively. In vivo results showed that skin wound healing in a murine model was accelerated by the incorporation of the EGF. In addition, the EGF had favorable effects in epidermal cell proliferation. The study demonstrates that a protein of high biological interest like EGF can be attached covalently to the surface of a synthetic material enriched with amino groups. This kind of biomaterial has a great potential for applications in skin regeneration and wound healing.

Crystal structure of a C-type lysozyme from Litopenaeus vanamei exhibiting a high binding constant to its chitotriose inhibitor.

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (-) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P61 and the latter in P212121 with two monomers in the asymmetric unit. Since the enzyme crystallized at a pH where lysozyme activity is deficient, the ligand could not be observed in the P61 structure; therefore, we performed a docking simulation with chitotriose to compare with the hen egg lysozyme crystallized in the presence of the ligand. Remarkably, additional amino acids in LvL caused an increase in the length of α-helix H4 (residues 97-103) that is directly related to ligand recognition. The Ka for chitotriose (4.1 × 105 M-1), as determined by Isothermal Titration Calorimetry, was one order of magnitude higher than those for lysozymes from hen and duck eggs. Our results revealed new interactions of chitiotriose with residues in helix H4.

In silico Study of the Pharmacologic Properties and Cytotoxicity Pathways in Cancer Cells of Various Indolylquinone Analogues of Perezone.

Several indolylquinone analogues of perezone, a natural sesquiterpene quinone, were characterized in this work by theoretical methods. In addition, some physicochemical, toxicological and metabolic properties were predicted using bioinformatics software. The predicted physicochemical properties are in agreement with the solubility and cLogP values, the penetration across the cell membrane, and absorption values, as well as with a possible apoptosis-activated mechanism of cytotoxic action. The toxicological predictions suggest no mutagenic, tumorigenic or reproductive effects of the four target molecules. Complementarily, the results of a performed docking study show high scoring values and hydrogen bonding values in agreement with the cytotoxicity IC50 value ranking, i.e: indolylmenadione > indolylperezone > indolylplumbagine > indolylisoperezone. Consequently, it is possible to suggest an appropriate apoptotic pathway for each compound. Finally, potential metabolic pathways of the molecules were proposed.

Polyphenolic Compounds and Digestive Enzymes: In Vitro Non-Covalent Interactions.

The digestive enzymes-polyphenolic compounds (PCs) interactions behind the inhibition of these enzymes have not been completely studied. The existing studies have mainly analyzed polyphenolic extracts and reported inhibition percentages of catalytic activities determined by UV-Vis spectroscopy techniques. Recently, pure PCs and new methods such as isothermal titration calorimetry and circular dichroism have been applied to describe these interactions. The present review focuses on PCs structural characteristics behind the inhibition of digestive enzymes, and progress of the used methods. Some characteristics such as molecular weight, number and position of substitution, and glycosylation of flavonoids seem to be related to the inhibitory effect of PCs; also, this effect seems to be different for carbohydrate-hydrolyzing enzymes and proteases. The digestive enzyme-PCs molecular interactions have shown that non-covalent binding, mostly by van der Waals forces, hydrogen binding, hydrophobic binding, and other electrostatic forces regulate them. These interactions were mainly associated to non-competitive type inhibitions of the enzymatic activities. The present review emphasizes on the digestive enzymes such as α-glycosidase (AG), α-amylase (PA), lipase (PL), pepsin (PE), trypsin (TP), and chymotrypsin (CT). Existing studies conducted in vitro allow one to elucidate the characteristics of the structure-function relationships, where differences between the structures of PCs might be the reason for different in vivo effects.

DNA Methylation of Cellular Retinoic Acid-Binding Proteins in Cervical Cancer.

This study determined the methylation status of cellular retinoic acid-binding protein (CRABP) gene promoters and associated them with demographic characteristics, habits, and the presence of human papilloma virus (HPV) in patients with cervical cancer (CC), low and high squamous intraepithelial lesions, and no intraepithelial lesion. Women (n = 158) were selected from the Colposcopy Clinic of Sanitary Jurisdiction II in Ciudad Juarez, Chihuahua, Mexico. Demographic characteristics and habit information were collected. Cervical biopsy and endocervical scraping were used to determine methylation in promoter regions by methylation-specific polymerase chain reaction technique. We found hemi-methylation patterns in the promoter regions of CRABP1 and CRABP2; there was 28.5% hemi-methylation in CRABP1 and 7.0% in that of CRABP2. Methylation in CRABP1 was associated with age (≥35 years, P = 0.002), family history of cancer (P = 0.032), the presence of HPV-16 (P = 0.013), and no alcohol intake (P = 0.035). These epigenetic changes could be involved in the CC process, and CRABP1 has the potential to be a predictive molecular marker of retinoid therapy response.

Chitosan/Mimosa tenuiflora films as potential cellular patch for skin regeneration.

Bio-composites films were prepared by casting and drying of aqueous solutions containing different weight ratios of chitosan and bark of Mimosa tenuiflora. The physico-chemical and functional properties of the films were characterized by scanning electron microscopy, dynamical mechanical analysis, wettability, cytotoxicity and in vitro antibacterial activities. The morphology studies confirmed that the presence of Mimosa tenuiflora change the surface of films. Moreover, the incorporation of Mimosa tenuiflora improved the thermal stability of the films, as it was indicated by the changes in the glass temperatures obtained. Water-uptake ability changed in relation to polymeric composition of film. This property increased by the addition of Mimosa tenuiflora to the film. Improved antibacterial properties were measured against Escherichia Coli and Micrococcus lysodeikticus or luteus. Finally, cytotoxicity was studied by MTT assay and the films were non-toxic. These preliminary results provide a cheap way to prepare chitosan/Mimosa tenuiflora films for wound healing and skin regeneration.

[Not Available]. / Deficiencia de micronutrientes en la dieta del paciente con lesiones precancerosas del cérvix de una clínica de colposcopía en Ciudad Juárez, México.

Introducción: las lesiones intraepiteliales escamosas (LIE) son un estado de transición hacia el cáncer cervicouterino (CaCu) y un déficit de micronutrientes puede acelerar este proceso. Por ello, determinar la existencia de este déficit y conocer qué factores se asocian permitiría una posible prevención en esta población de riesgo.Objetivo: determinar la presencia de alguna deficiencia de micronutrientes involucrados en el proceso anticancerígeno y asociar este déficit con hábitos y factores demográficos en pacientes con LIE de Ciudad Juárez, Chihuahua, México.Métodos:en un estudio transversal analítico fueron seleccionadas 102 pacientes con LIE. Se realizó una encuesta dietaría (recordatorio de 24 horas) para estimar la ingesta de micronutrientes. La deficiencia fue determinada con un consumo < 75% de la ingesta diaria recomendada o sugerida (IDR o IDS) en México. Algunos hábitos y factores demográficos fueron obtenidos mediante la entrevista con la paciente. Se realizó un modelo de regresión logística para asociar la presencia de deficiencia con factores que afectan a la ingesta o incrementan el requerimiento de micronutrientes.Resultados:el retinol, ácido fólico, zinc, vitaminas C y E, considerados como micronutrientes en el proceso anticancerígeno del CaCu, se encontraron por debajo del 75% de la IDR. Aquellas mujeres con sobrepeso, obesidad y amas de casa se asociaron significativamente con la deficiencia de micronutrientes.Conclusión: el sobrepeso, la obesidad y la ocupación han sido asociados para presentar deficiencias de micronutrientes en este estudio. Estas variables convergen en una posible inseguridad alimentaria, la cual podría asociarse al incremento de incidencia de CaCu en México.

Deficiencia de micronutrientes en la dieta del paciente con lesiones precancerosas del cérvix de una clínica de colposcopía en Ciudad Juárez, México / Micronutrient deficiency in the diet of the patient with precancerous cervix lesions on a colposcopy clinic at Ciudad Juárez, Mexico

Introducción: las lesiones intraepiteliales escamosas (LIE) son un estado de transición hacia el cáncer cervicouterino (CaCu) y un déficit de cronutrientes puede acelerar este proceso. Por ello, determinar la existencia de este déficit y conocer qué factores se asocian permitiría una posible prevención en esta población de riesgo. Objetivo: determinar la presencia de alguna deficiencia de micronutrientes involucrados en el proceso anticancerígeno y asociar este déficit con hábitos y factores demográficos en pacientes con LIE de Ciudad Juárez, Chihuahua, México. Métodos: en un estudio transversal analítico fueron seleccionadas 102 pacientes con LIE. Se realizó una encuesta dietaría (recordatorio de 24 horas) para estimar la ingesta de micronutrientes. La deficiencia fue determinada con un consumo < 75% de la ingesta diaria recomendada o sugerida (IDR o IDS) en México. Algunos hábitos y factores demográficos fueron obtenidos mediante la entrevista con la paciente. Se realizó un modelo de regresión logística para asociar la presencia de deficiencia con factores que afectan a la ingesta o incrementan el requerimiento de micronutrientes. Resultados: el retinol, ácido fólico, zinc, vitaminas C y E, considerados como micronutrientes en el proceso anticancerígeno del CaCu, se encontraron por debajo del 75% de la IDR. Aquellas mujeres con sobrepeso, obesidad y amas de casa se asociaron significativamente con la deficiencia de micronutrientes. Conclusión: el sobrepeso, la obesidad y la ocupación han sido asociados para presentar deficiencias de micronutrientes en este estudio. Estas variables convergen en una posible inseguridad alimentaria, la cual podría asociarse al incremento de incidencia de CaCu en México (AU)

Introduction: Squamous Intraepithelial Lesions (SIL) is a state of transition to cervical cancer (CC), and micronutrient deficiencies can speed up this process. Therefore, determining the existence of this deficit and know what factors are associated would allow for possible prevention in this population at risk. Objective: To determine the presence of some micronutrient deficiencies involved in the anti-carcinogenic process, also associate this deficit with habits and demographic factors in patients with SIL in Ciudad Juarez, Chihuahua, Mexico. Methods: An analytic cross-sectional study, 102 patients were selected with SIL. A dietary survey (24-hour recall) was performed to estimate the intake of micronutrients. The deficiency was determined when the consumption was less than 75% of the Recommended Dietary Allowances (RDA) or suggested in Mexico. Some habits and demographic factors were obtained by interview with the patient. A logistic regression was performed to associate the presence of deficiencies with factors that affecting the intake or increase the requirement of micronutrients. Results: Retinol, folic acid, zinc, vitamins C and E, considered micronutrients in the anti-carcinogenic process CC, were less than 75% of the RDA. Women with overweight, obesity and housewives, were significantly associated with micronutrient deficiencies. Conclusion: Overweight, obesity and occupation have been associated to present micronutrient deficiencies in this study. These variables converge on a possible food insecurity, which could be associated with increased incidence of CC in Mexico (AU)

Polycaprolactone/Amino-ß-Cyclodextrin Inclusion Complex Prepared by an Electrospinning Technique.

Electrospun scaffolds of neat poly-ε-caprolactone (PCL), poly-ε-caprolactone/ß-cyclodextrin inclusion complex (PCL/ß-CD) and poly-ε-caprolactone amino derivative inclusion complex (PCL/ß-CD-NH2) were prepared by the electrospinning technique. The obtained mats were analyzed by a theoretical model using the Hartree⁻Fock method with an STO-3G basis set, and characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), differential scanning calorimetry (DSC), confocal-Raman spectroscopy, proton nuclear magnetic resonance (¹HNMR) and contact angle measure (CA). Different mixtures of solvents, such as dimethylformamide (DMF)-tetrahydrofuran (THF), dichlormethane (DCM)-dimethyl sulfoxide (DMSO) and 2,2,2-Trifluoroethanol (TFE), were tested in the fiber preparation. The results indicate that electrospun nanofibers have a pseudorotaxane structure and when it was prepared using a 2,2,2-Trifluoroethanol (TFE) as solvent, the nanofibers were electrospun well and, with the other solvents, fibers present defects such as molten fibers and bead-like defects into the fiber structure. This work provides insights into the design of PCL/ß-CD-NH2 based scaffolds that could have applications in the biomedical field.

Krüppel-like factor 5 as potential molecular marker in cervical cancer and the KLF family profile expression.

Cervical cancer (CC) as other cancer types, presents molecular deregulations, such as the alterations of transcription factors. Krüppel-like factors (KLF) are a family of transcriptional regulators. They are involved in diverse cellular processes, such as proliferation, apoptosis, and angiogenesis among others. Here, we analyzed the expression of all 17 KLF members at messenger RNA (mRNA) level, and protein expression of the two most commonly altered KLF5 and KLF6 in cervical tissues. Fifty-nine cervical tissues ranging from normal tissue to CC were evaluated for KLF1-17 mRNA expression by end-point RT-PCR and KLF5 by qRT-PCR. For KLF5 and KLF6 protein analysis, a tissue microarray was constructed containing the 59 cases and subjected for immunohistochemistry assay and KLF6 IVS1-27G>A single nucleotide polymorphism by direct DNA sequencing. KLF2-16 expressions were present in normal tissue, whereas all 17 were present in Low-Grade Squamous Intraepithelial Lesion, High-Grade-SIL and CC, unrelated to presence of human papillomavirus (HPV). KLF5 mRNA expression gradually increased throughout the subgroups and overexpressed in CC (p=0.01). KLF5 and KLF6 proteins were immunodetected in all samples. For the KLF6 SNP analysis, 80% of the CC population analyzed presented GG genotype and the remaining 20% presented GA genotype (p=0.491). Our present data show that KLFs expression could not be related to HPV presence, at least at transcriptional level, and KLF5 mRNA overexpression could represent a potential molecular marker for CC; KLF6 SNP has no relation to increased risk of CC.

Molecular cloning of a beta-glucan pattern-recognition lipoprotein from the white shrimp Penaeus (Litopenaeus) vannamei: correlations between the deduced amino acid sequence and the native protein structure.

The hemolymph pattern-recognition beta-glucan binding protein from the white shrimp Penaeus (Litopenaeus) vannamei is also a high density lipoprotein (betaGBP-HDL) involved in innate immunity. The betaGBP-HDL full length cDNA sequence determined was 6.3 kb long, and contains a long 3'UTR region with a polyadenylation signal and a poly-A+ tail. The open reading frame is 1454 amino acids long and the N-terminal residue of the mature protein is localized in position 198 of the ORF. Comparison of the betaGBP-HDL amino acid sequence against GenBank detected only significant similarity to betaGBP from the crayfish Pacifastacus leniusculus. betaGBP-HDL is expressed in hepatopancreas, muscle, pleopods and gills, but not in hemocytes as determined by RT-PCR. We discuss the analysis of the deduced primary sequence in terms of the predicted secondary structure, glucanase-like and RGD motives relevant to its dual roles in defence and lipid transport.

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei).

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.

Migration of bisphenol A (BPA) from epoxy can coatings to jalapeño peppers and an acid food simulant.

Effects of heat processing, storage time, and temperature on migration of bisphenol A (BPA) from an epoxy type can coating to an acid food simulant and jalapeño peppers were determined. Commercial jalapeño pepper cans (8 oz, dimensions 211 x 300) were stored at 25 degrees C for 40, 70, and 160 days. A solution of 3% acetic acid was canned in 211 x 300 cans from the same batch used for jalapeño peppers. Heat processing was applied to two-thirds of the cans, and the remaining cans were not heat processed. Cans were stored at 25 and 35 degrees C for 0, 40, 70, and 160 days. Results showed that there is a minimal effect of heat treatment. An effect of storage time on migration of BPA during the first 40 days at 25 degrees C was observed. An increase on migration of BPA was observed with storage time at 35 degrees C. The highest level of migration was 15.33 microg/kg of BPA at 160 days at 35 degrees C. A correction factor of approximately 0.4 was calculated for migration under simulating conditions of storage compared to the real ones. The highest level of BPA found in jalapeño peppers cans, surveyed from three supermarkets, was 5.59 +/- 2.43 microg/kg. Migration of BPA, performed according to the European and Mercosur conditions, was 65.45 +/- 5.29 microg/kg. All the migration values found in this study were below those legislation limits (3 mg/kg).

Biophysical evidence of lipid and carbohydrate binding activities of shrimp high density lipoprotein/beta glucan binding protein.

Crustacean High Density Lipoprotein/beta-Glucan Binding Protein (HDL/BGBP) has been studied due to its role in nutrition and immune response via activation of the defense cells (hemocytes) upon binding 1,3-D-beta-glucan carbohydrates. In this study, HDL/BGBP was found to be composed mainly of beta sheets, as determined by circular dichroism. Lipoprotein aggregation resulted when HDL/BGBP interacted with phospolipid vesicles, laminaribiose (1,3-beta-glucan disaccharide) or heparin. HDL/BGBP has similar dissociation constants for laminaribiose (K(d)=22 mM) or heparin (K(d)=46 mM) as determined by 90 degrees light scattering.