Self-assembly study of type I collagen extracted from male Wistar Hannover rat tail tendons.

BACKGROUND: Collagen, the most abundant protein in the animal kingdom, represents a promising biomaterial for regenerative medicine applications due to its structural diversity and self-assembling complexity. Despite collagen's widely known structural and functional features, the thermodynamics behind its fibrillogenic self-assembling process is still to be fully understood. In this work we report on a series of spectroscopic, mechanical, morphological and thermodynamic characterizations of high purity type I collagen (with a D-pattern of 65 nm) extracted from Wistar Hannover rat tail. Our herein reported results can be of help to elucidate differences in self-assembly states of proteins using ITC to improve the design of energy responsive and dynamic materials for applications in tissue engineering and regenerative medicine. METHODS: Herein we report the systematic study on the self-assembling fibrillogenesis mechanism of type I collagen, we provide morphological and thermodynamic evidence associated to different self-assembly events using ITC titrations. We provide thorough characterization of the effect of pH, effect of salts and protein conformation on self-assembled collagen samples via several complementary biophysical techniques, including circular dichroism (CD), Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA). RESULTS: Emphasis was made on the use of isothermal titration calorimetry (ITC) for the thermodynamic monitoring of fibrillogenesis stages of the protein. An overall self-assembly enthalpy value of 3.27 ± 0.85 J/mol was found. Different stages of the self-assembly mechanism were identified, initial stages take place at pH values lower than the protein isoelectric point (pI), however, higher energy release events were recorded at collagen's pI. Denatured collagen employed as a control exhibited higher energy absorption at its pI, suggesting different energy exchange mechanisms as a consequence of different aggregation routes.

Nonirritant and Cytocompatible Tinospora cordifolia Nanoparticles for Topical Antioxidant Treatments.

Tinospora cordifolia extract contains antioxidants such as polyphenols, and thus, it has been used as a natural phytochemical antioxidant therapeutic agent. Many of these compounds are insoluble or only partially soluble in water. In this study, we produced a novel aqueous nanoparticle formulation, with an average particle size of 182.9 ± 3.8 nm, to improve the dispersion of the bioactive compounds in water and to increment its bioavailability. The nanoparticles are composed of polyphenols, alkaloids, and glycosides. We studied the effect of this nanoparticle formulation on mouse 3T3 fibroblast cell viability and New Zealand rabbit dermal irritability tests. Concentrations of 2.5, 25, and 250 µg/mL resulted in similar cell viability to cells in culture media. An intermediate concentration of 12.45 mg/ml was used for the acute dermal irritability test. There were no severe alterations that compromised animal health. These results represent a precedent for application of such nanoparticles derived from plant stems, such as Tinospora cordifolia, in biomedicine and in antiaging cosmetic treatments.

Increased Fibroblast Metabolic Activity of Collagen Scaffolds via the Addition of Propolis Nanoparticles.

Propolis natural extracts have been used since ancient times due to their antioxidant, anti-inflammatory, antiviral, and antimicrobial activities. In this study, we produced scaffolds of type I collagen, extracted from Wistar Hanover rat tail tendons, and impregnated them with propolis nanoparticles (NPs) for applications in regenerative medicine. Our results show that the impregnation of propolis NPs to collagen scaffolds affected the collagen denaturation temperature and tensile strength. The changes in structural collagen self-assembly due to contact with organic nanoparticles were shown for the first time. The fibril collagen secondary structure was preserved, and the D-pattern gap increased to 135 ± 28 nm, without losing the microfiber structure. We also show that the properties of the collagen scaffolds depended on the concentration of propolis NPs. A concentration of 100 µg/mL of propolis NPs with 1 mg of collagen, with a hydrodynamic diameter of 173 nm, was found to be an optimal concentration to enhance 3T3 fibroblast cell metabolic activity and cell proliferation. The expected outcome from this research is both scientifically and socially relevant since the home scaffold using natural nanoparticles can be produced using a simple method and could be widely used for local medical care in developing communities.

Proteogenomic analysis of the Clostridium difficile exoproteome reveals a correlation between phylogenetic distribution and virulence potential.

C. difficile induces antibiotic-associated diarrhea due to the action of two secreted toxins, TcdA and TcdB. A considerable range of virulence among C. difficile strains has been widely reported. During a hospital outbreak, 46 isolates were collected that belonged to different genotypes. Of those, the majority corresponded to two virulent strains, the globally distributed Sequence Type 1 (ST1)_North American Pulsotype 1 (NAP1) and the endemic ST54_NAPCR1 genotypes, respectively. Whereas the virulence of the latter has been attributed to increased secretion of toxins and production of a highly cytotoxic TcdB, these characteristics do not explain the increased lethality of the former. We undertook a proteomic comparative approach of the isolates participating in the outbreak to look for proteins present in the exoproteome of the ST1_NAP1and ST54_NAPCR1 strains. We used a low virulent ST2_NAP4 strain isolated also in the outbreak as control. Dendrograms constructed using the exoproteomes of the strains were very similar to those created using genomic information, suggesting an association between secreted proteins and relative virulence of the strains. By 2D electrophoresis and mass spectrometry it was found that approximately half of the proteins are shared among strains of different genotypes. From the identified proteins, the surface-located SlpA draw our attention due to its detection in ST54_NAPCR1 exoproteomes. Biochemical analysis indicated that the processing of SlpA is different in the ST54_NAPCR1 strain and confirmed that this strain secretes more SlpA than its counterparts. Furthermore, SlpA from the ST54_NAPCR1 strain exerted an increased proinflammatory activity. Altogether, these results indicate that the exoproteome composition correlates with the C. difficile genotype and suggest that particular proteins secreted by some strains could synergize with the effects of TcdA and TcdB increasing their virulence.

Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities.

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.

Toxicidad subcrónica del extracto acuoso de las hojas y los brotes florales de Stachytarpheta jamaicensis (L.) Vahl. (Verbenaceae) / Subchronic toxicity of the aqueous extract from the leaves and floral buds of Stachytarpheta jamaicensis (L.) Vahl. (Verbenaceae)

Se determinó la toxicidad de las hojas y los brotes florales de la especie botánica medicinal Stachytarpheta jamaicensis (L.) Vahl. El material botánico (hojas y brotes florales) fue recolectado en febrero de 1996 por el señor Víctor Rojas del Instituto Nacional de Biodiversidad (número de testigo 400 VR) en Santa Rosa, Guanacaste en las siguientes coordenadas: 10°50´20´´N-85°37´30´´W y una altitud de 290 msnm. Los extractos acuosos, preparados por decocción, de las hojas y los brotes florales de Stachytarpheta jamaicensis, se administraron por vía oral a ratones NGP machos y hembras. El extracto de las hojas se administró durante 90 d y el de los brotes florales durante 60 d. Ambos extractos fueron administrados durante 5 d consecutivos por semana. Los grupos control recibieron 0,5 mL de agua destilada por animal. El extracto acuoso de las hojas causó 40 (por ciento) de mortalidad tanto en machos como en hembras. Se evidenciaron signos de toxicidad con la administración del extracto de los brotes florales sin producir mortalidad. Todos los signos registrados sugieren un efecto general depresor sobre el sistema nervioso central, por lo que se plantea la necesidad de realizar pruebas biológicas dirigidas a la validación de un posible efecto sedante-tranquilizante de esta planta y la realización de curvas dosis-efecto de la toxicidad de esta especie

Actividad farmacológica del extracto acuoso de la madera de Quassia amara (Simarubaceae) en ratas y ratones albinos / Pharmacological activity of the aqueous wood extract from Quassia amara (Simarubaceae)on albino rats and mice

All the assays were done with an aqueous preparation of dry wood from Quassia amara imarubaceae). For the hippocratic assay, 12 female SDN rats were used, with an average weight of 144 g and separated in three groups of four individuals each. The dose used were 500 mg/kg and 1,000 mg/kg and the control group received 0.5 ml of distilled water. The extract administration and the observation of the animals were done daily during nine days. Acute toxicity of the preparation was studied with 25 male NGP mice with an average weight of 20.13 g, in groups of five individuals per dose. The oral administration was carry out with the following doses: 250, 500, 750 and 1,000 mg/kg, the control group received 0.5 ml of distilled water. No sign of acute toxicity was observed at any dose. For the toxicity analysis via intraperitoneal injection 15 male NGP mice were assigned to five groups (5 animals each) with doses of 500 and 1,000 mg/kg and a control group with 0.5 ml of distilled water. The group with the dose of 500 mg/kg, presented acute toxicity signs with a 24 hr recovery, and the 1,000 mg/kg dose was lethal to a 100 percent within 24 hr. The measuring of the peristaltic activity (movement of the intestinal content) were performed on 30 NGP male mice with an average weight of 22 g assigned to three groups of ten individuals each. One dose of 500 mg/kg and 1,000 mg/kg were orally administrated to each experimental group and 0.5 ml of distilled water to the control group. The marker used was activated carbon, orally supplied to every mice 30 min after the administration of the aqueous extract. The animals are decapitated and the measurement of the carbon motion in the small intestine was done after 30 min. Both dose increased the intestinal movement compared to the control group, but only the 1,000 mg/kg dose showed a statistically significant difference (p < or = 0.05)