Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation.

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Creation of a vascular inducing device using mesenchymal stem cells to induce angiogenesis.

Conventional treatments of peripheral vascular disease and coronary artery disease have partial success but are still limited. Methods to deliver angiogenic factors into ischemic areas using gene, protein and cell therapies are faced with difficult issues such a delivery, effective concentration and duration of action. Tissue engineering offers the possibility of creating a functional self-contained three-dimensional (3D) unit that works as a coordinated biological pump that can secrete a whole range of angiogenic factors. We report a tissue engineering approach using decellularized micro-fragments and mesenchymal stem cells (MSCs) to create a vascular inducing device (VID). Proteomic analysis of the decellularized micro-fragments and of the VIDs reveals a large number of extracellular-matrix (ECM) proteins. Moreover, the VIDs were found to transcribe and secrete a whole repertoire of angiogenic factors in a sustained manner. Furthermore, preliminary results of implantation VIDs into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice indicate formation of vascular network at the site within a week. We propose that those VIDs could serve as a safe, localized, simple and powerful method for the treatment of certain types of vascular diseases.

Paracrine Signaling from a Three-Dimensional Model of Bladder Carcinoma and from Normal Bladder Switch the Phenotype of Stromal Fibroblasts.

We present a three-dimensional model based on acellular scaffolds to recreate bladder carcinoma in vitro that closely describes the in vivo behavior of carcinoma cells. The integrity of the basement membrane and protein composition of the bladder scaffolds were examined by Laminin immunostaining and LC-MS/MS. Human primary bladder carcinoma cells were then grown on standard monolayer cultures and also seeded on the bladder scaffolds. Apparently, carcinoma cells adhered to the scaffold basement membrane and created a contiguous one-layer epithelium (engineered micro-carcinomas (EMCs)). Surprisingly, the gene expression pattern displayed by EMCs was similar to the profile expressed by the carcinoma cells cultured on plastic. However, the pattern of secreted growth factors was significantly different, as VEGF, FGF, and PIGF were secreted at higher levels by EMCs. We found that only the combination of factors secreted by EMCs, but not the carcinoma cells grown on plastic dishes, was able to induce either the pro-inflammatory phenotype or the myofibroblast phenotype depending on the concentration of the secreted factors. We found that the pro-inflammatory phenotype could be reversed. We propose a unique platform that allows one to decipher the paracrine signaling of bladder carcinoma and how this molecular signaling can switch the phenotypes of fibroblasts.

Angiogenic potential of mesenchymal stem cells derived from patients with diabetes seeded on decellularized micro fragments.

Mesenchymal stem cells (MSCs) are a potential source of angiogenic factors which may promote wound healing in poorly vascularized diabetic foot ulcers. We demonstrate that MSCs of patients with diabetic foot ulcers seeded on decellularized micro-fragments transcribe and secrete angiogenic factors in amounts comparable to MSCs derived from healthy individuals.

An in vivo Like Micro-Carcinoma Model.

We here present a novel micro-system which allows to reconstitute an in vivo lung carcinoma where the various constituting epithelial and/or stromal structural and/or cellular components can be incorporated at will. In contrast to various "organs on a chip" the model is based on the observation that in nature, epithelial cells are always supported by a connective tissue or stroma. The model is based on acellular micro-scaffolds of microscopic dimensions which enable seeded cells to obtain gases and nutrients through diffusion thus avoiding the need for vascularization. As a proof of concept, we show that in this model, Calu-3 cells can form a well-organized, continuous, polarized, one-layer epithelium lining the stromal derived alveolar cavities, and express a different pattern of tumor-related genes than when grown as standard monolayer cultures on plastic culture dishes. To our knowledge, this model, introduces for the first time a system where the function of carcinogenic cells can be tested in vitro in an environment that closely mimics the natural in vivo situation.

Identification of genes responsive to the application of ethanol on sugarcane leaves.

The control of gene expression in precise time and space is a desirable attribute of chemically inducible systems. Ethanol is a chemical inducer with favourable features, such as being inexpensive and easy to apply. The aim of this study was to identify ethanol-responsive genes in sugarcane. The cDNA macroarray technique was adopted to identify transcript changes in sugarcane leaves (Saccharum spp. cv SP80-3280) exposed to ethanol. The expression profiles of sugarcane genes were analysed using nylon filters containing 3,575 cDNA clones from the leaf roll library of the SUCEST project. Seventy expressed sequence tags (ESTs) presented altered expression patterns, including ESTs corresponding to genes related to transcriptional and translational processes, abiotic stress and others. Several genes of unknown function were also identified. Among the 48 ESTs up-regulated by ethanol, an abiotic stress-responsive protein and an unknown function gene presented rapid induction by ethanol. The macroarray data of selected ethanol-responsive EST were confirmed by RNA-blot hybridisation. The expression profile of the 48 up-regulated genes was compared in two other cultivars: SP89-1115 and SP90-3414. Surprisingly, no gene showed a similar expression profile in the three cultivars. This result suggests that sugarcane plants have a high diversity in their responses to ethanol.

Influencia de la ansiedad y depresión sobre la discapacidad relacionada con la migraña / Influence of anxiety and depression on migraine-related disability

Introducción: existe una fuerte asociación entre la migraña y los trastornos del estado de ánimo, que podría derivar en mayor discapacidad y peor pronóstico para estos pacientes. Objetivo: evaluar el impacto de la sintomatología de ansiedad y depresión sobre el grado de discapacidad en pacientes con migraña. Materiales y Métodos: se seleccionaron 50 personas con migraña (25 con aura y 25 sin aura) y 25 controles sanos pareados por edad y sexo. Se midieron los síntomas de depresión y ansiedad utilizando las escalas de Zung. En los pacientes con migraña se midió el grado de discapacidad utilizando el Headache Impact Test (HIT-6). La asociación entre la puntuación de las escalas de Zung y HIT-6 se analizó empleando una regresión linear. Resultados: el diagnóstico de migraña se relacionó significativamente con mayores puntuaciones en la escala de depresión (39,2±10 vs. 31,9±6,8, p=0,002) y ansiedad (36,7±10,2 vs. 28,5±6,7, p=0,0001) de Zung. En los pacientes con migraña, se encontró una asociación significativa entre la puntuación de las escalas de sintomatología depresiva (OR, 3,53; IC95%, 1,33-5,72, p=0,002) y de ansiedad (OR, 3,47; IC95%, 1,33-5,61, p=0,002) con el grado de discapacidad (HIT-6). Dichas asociaciones se mantuvieron luego de ajustarlas por edad, sexo y la frecuencia de episodios de migraña. Conclusiones: la migraña se encuentra relacionada con una mayor presencia de sintomatología de ansiedad y depresión. Esta asociación tiene un impacto negativo sobre la funcionalidad de las personas con esta patología.

Introduction: there is a strong association between migraine and mood disorders, which could lead to greater disability and poorer prognosis in these patients. Objective: to evaluate the impact of symptoms of anxiety and depression on the degree of disability in patients with migraine. Materials and Methods: we selected 50 people with migraine (25 with aura and 25 without aura) and 25 healthy controls matched for age and sex. We measured symptoms of depression and anxiety using Zung scales. In patients with migraine was measured the degree of disability using the Headache Impact Test (HIT-6). The association between score and Zung scales HIT-6 was analyzed using linear regression. Results: the diagnosis of migraine was significantly associated with higher scores on the depression scale (39.2 ± 10 vs 31.9 ± 6.8, p = 0.002) and anxiety (36.7 ± 10.2 vs 28.5 ± 6.7, p = 0.0001) of Zung. In patients with migraine, we found a significant association between the score of the scales of depressive symptoms (OR, 3.53, CI 95% 1.33 to 5.72, p = 0.002) and anxiety (OR, 3, 47, CI 95%, 1.33 to 5.61, p = 0.002) with the degree of disability (HIT-6). These associations remained after adjusting for age, sex and frequency of migraine episodes. Conclusions: migraine is associated with increased presence of symptoms of anxiety and depression. This association has a negative impact on the functionality of people with this condition.

Estudio de correlación entre marcadores de inflamación y grosor íntima media carotídeo, en pacientes con ataque cerebrovascular isquémico / An analytical cross-sectional study to determine the association between intimae media

Introducción. El estudio de factores emergentes de riesgo para enfermedad cerebrovascular ha cobrado importancia, especialmente el de las infecciones y los estados inflamatorios. Nuestro grupo ha postulado que en nuestra población dietas inapropiadas y la baja infraestructura sanitaria, puede generar un estado de inflamación crónica. Objetivo. Determinar la asociación entre el grosor de íntima media carotideo (GIM) y los niveles de marcadores inflamación. Métodos. Estudio transversal analítico de 34 pacientes con enfermedad cerebrovascular isquémica y 34 sujetos sanos pareados por edad, género y procedencia geográfica. La población de estudio es una submuestra del estudio multicéntrico nacional de factores de riesgo para enfermedad cerebrovascular isquémica (FREC-VI). Se tomaron muestras sanguíneas para determinación de factor de necrosis tumoral alpha, interleucina 6 y proteína C reactiva (FNT, IL6, PCR), fibrinógeno y eco doppler color carotídeo. Resultados. Los niveles de PCR e IL6 se encontraron significativamente más elevados en los sujetos casos comparados con los controles, p=0.001 y p=0.0001 respectivamente. El fibrinógeno y el FNT no mostraron diferencias significativas. No se encontraron diferencias en los valores del grosor íntima media en las arterias carótidas comunes e internas. El OR para PCR fue de 8.39 (IC95 por ciento:1.38-50.65), para IL6 de 1.45 (IC95 por ciento:1.07-1.95), y para FNT 1.11 (IC95 por ciento: 0.99-1.23). La PCR y la IL6 presentaron correlaciones significativas con el grosor íntima media de la carótida externa derecha. Conclusiones. La IL6 y la PCR son los marcadores inflamatorios asociados al ataque cerebrovascular isquémico. Los niveles elevados de PCR e IL6 se correlacionan directamente con el grosor íntima media de la arteria carótida externa derecha. No se encontró asociación del FNT y el fibrinógeno con GIM

Gerontologia : uma visao psicossocial da velhice / Gerontology - A Psychological View of Old Age

Neste artigo sao feitas reflexoes sobre a velhice, ultimo periodo de vida do individuo, periodo este que termina com a morte