Formation of gel and solid phases in acrylic cuvettes upon exposure to DMSO, oxygen and light: implications for fluorescence spectroscopy.

We here report the formation of a turbid-gel phase in acrylic cuvettes upon exposure to pure Dimethyl Sulfoxide (DMSO) at room temperature. The observed phenomenon occurred over a 10 h to 14 h incubation in the presence of environmental oxygen. After the turbid gel was removed from the cuvette, it became a white solid exhibiting unique emission behavior. The formation of the turbid-gel phase was accelerated upon exposure to UV 295 LED pulses of light from 6 h to 8 h. Surprisingly, subsequent exposure of the white solid to a few microliters of pure DMSO and vortexing resulted in its transformation into a transparent gel state in just a few minutes, eventually acquiring transparent and liquid properties. Additionally, the white-solid phase can load other molecules, such as Resveratrol and Quercetin, leading to shifts in the respective emission spectra compared with the same molecule in liquid and pure DMSO. These novel findings highlight the interaction between UV photons, oxygen, DMSO and Acrylic, and potentially distort fluorescence spectroscopy experiments.

Interfacial Aß fibril formation is modulated by the disorder-order state of the lipids: The concept of the physical environment as amyloid inductor in biomembranes.

The behavior of amphiphilic molecules such as lipids, peptides and their mixtures at the air/water interface allow us to evaluate and visualize the arrangement formed in a confined and controlled surface area. We have studied the surface properties of the zwitterionic DPPC lipid and Aß(1-40) amyloid peptide in mixed films at different temperatures (from 15 to 40 °C). In this range of temperature the surface properties of pure Aß(1-40) peptide remained unchanged, whereas DPPC undergoes its characteristic liquid-expanded â†’ liquid-condensed bidimensional phase transition that depends on the temperature and lateral pressure. This particular property of DPPC makes it possible to dynamically study the influence of the lipid phase state on amyloid structure formation at the interface in a continuous, isothermal and abrupt change on the environmental condition. As the mixed film is compressed the fibril-like structure of Aß(1-40) is triggered specifically in the liquid-expanded region, independently of temperature, and it is selectively excluded from the well-visible liquid condensed domains of DPPC. The Aß amyloid fibers were visualized by using BAM and AFM and they were Thio T positive. In mixed DPPC/Aß(1-40) films the condensed domains (in between 11 mN/m to 20 mN/m) become irregular probably due to the fibril-like structures is imposing additional lateral stress sequestering lipid molecules in the surrounding liquid-expanded phase to self-organize into amyloids.

Aß Amyloid Fibers Drastically Alter the Topography and Mechanical Properties of Lipid Membranes.

Alzheimer's disease (AD) is related to the fibrillation of the Aß peptides at neuronal membranes, a process that depends on the lipid composition and may impart different physical states to the membrane. In the present work, we study the properties of the Aß peptide when mixed with a zwitterionic lipid (DMPC), using the Langmuir monolayer technique as an approach to control membrane physical conditions. First, we build on previous characterizations of pure Aß monolayers and observe that, in addition to high shear, these films present a pronounced compressional hysteresis. When Aß is assembled with DMPC in a binary film, the resulting membranes become heterogeneous, with a peptide-enriched phase distributed in a network-like pattern, and they exhibit a lateral transition that depends on the Aß content. At lower peptide proportions, the films segregate into two well-defined phases: one consisting of lipids and another enriched with peptides. The reflectivity of these phases differs from that obtained for pure Aß films. Thus, the formed fibers effectively cover most of the interface area and remain stable at higher pressures (from 20 to 30 mN m-1 depending on Aß content) compared to pure peptide films (17 mN m-1). Furthermore, such structures induce a compressional hysteresis in the film, similar to that of pure peptide films (which is nonexistent in the pure lipid monolayer), even at low peptide proportions. We claim that the mechanical properties at the interface are governed by the size of the fibril-like structures. Based on the low molar fractions and surface packing at which these phenomena were observed, we postulate that as a consequence of peptide intermolecular interactions, Aß may have drastic effects on the molecular arrangement and mechanical properties of a lipid membrane.

Embriopatía diabética y trastornos en el desarrollo reproductivo materno en ratas con diabetes pregestacional / Diabetic embryopathy and disorders in maternal reproductive development in rats with pregestational diabetes

Resumen OBJETIVOS: Determinar la repercusión de la diabetes pregestacional, con hiperglucemias moderadas, en el rendimiento reproductivo de la rata, crecimiento, desarrollo y morfología embrionaria en ratas Wistar. MATERIALES Y MÉTODOS: Estudio longitudinal, prospectivo y experimental efectuado en la Unidad de Investigaciones Biomédicas de la Universidad de Ciencias Médicas de Villa Clara, Cuba, en un modelo de diabetes moderada inducida neonatalmente a crías hembras de ratas Wistar de dos días de nacidas mediante la administración subcutánea de 100 mg/kg de peso corporal de estreptozotocina en una única dosis. A los 120 días de nacidas, las ratas de ambos grupos de experimentación (diabético y control) se aparearon con machos sanos. Se determinaron el peso y la glucemia durante la gestación y a los 11.5 días se practicó la cesárea. Se analizaron las variables del rendimiento reproductivo materno y de crecimiento, desarrollo y morfología externa en los embriones. Acorde con los desenlaces se utilizaron pruebas no paramétricas para el análisis de las variables cuantitativas y la prueba de χ2 para las variables cualitativas. RESULTADOS: La hiperglucemia moderada pregestacional provocó modificaciones en la ganancia de peso de la madre, la cantidad de reabsorciones, sitios de implantación, pérdidas preimplantación y eficiencia de implantación, así como en la morfología, talla y cantidad de somitas en los embriones. CONCLUSIONES: La diabetes moderada pregestacional alteró el rendimiento reproductivo materno y el crecimiento y desarrollo intrauterino de la descendencia en etapa embrionaria. La embriopatía diabética se manifestó, además, con malformaciones del sistema nervioso central.

Abstract OBJECTIVES: Diabetes mellitus is one of the most frequent disorders of pregnancy with adverse consequences for the mother and a high risk of diabetic embryopathy in the offspring. The objective of the research was to determine the effect of pregestational diabetes with moderate hyperglycemia on maternal reproductive performance, growth, development and embryonic morphology in Wistar rats. MATERIALS AND METHODS: Longitudinal, prospective and experimental study carried out at the Biomedical Research Unit of the University of Medical Sciences of Villa Clara, Cuba. A model of neonatally induced moderate diabetes was used in female Wistar rat pups two days old, by subcutaneous administration of 100 mg/kg of body weight of streptozotocin in a single dose. At 120 days after birth, rats from both experimental groups (diabetic and control) were mated with healthy males. Weight and glycemia were determined during pregnancy and at 11,5 days the cesarean section was performed. The variables of maternal reproductive performance and of growth, development and external morphology in the embryos were analyzed. According to the results, non-parametric tests were used for the analysis of the quantitative variables and the Chi-square test for the qualitative variables. RESULTS: Moderate pregestational diabetes caused changes in maternal weight gain, number of resorptions, implantation sites, preimplantation loss, and implantation efficiency, as well as in morphology, size, and number of somites in embryos. CONCLUSIONS: Moderate pregestational diabetes altered maternal reproductive performance and intrauterine growth and development of embryonic offspring. Diabetic embryopathy was also manifested by malformations of the central nervous system.

Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface.

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted. Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.

Gangliosides smelt nanostructured amyloid Aß(1-40) fibrils in a membrane lipid environment.

Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aß(1-40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aß(1-40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aß(1-40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes. Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy. We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).

Stitching together a nm thick peptide-based semiconductor sheet using UV light.

Langmuir monolayer allows for a two-dimensional nano-scale organization of amphiphilic molecules. We have adapted this technique to measure lateral and transverse conductivity in confined peptide nanosheets for the first time. We reported that two retro-isomers amphipathic peptides form stable monolayers showing a semiconductor-like behavior. Both peptides exhibit the same hydrophobicity and surface stability. They differ in the lateral conductivity and current-voltage due to the asymmetric peptide bond backbone orientation at the interface. Both peptides contain several tyrosines allowing the lateral crosslinking in neighboring molecules induced by UVB. UVB-light induces changes in the lateral conductivity and current-voltage behavior as well as monolayer heterogeneity monitored by Brewster Angle Microscopy. The semiconductor properties depend on the peptide bond backbone orientation and tyrosine crosslinking. Our results indicate that one may design extended nano-sheets with particular electric properties, reminiscent of semiconductors. We propose to exploit such properties for biosensing and neural interfaces.

Mass spectrometric and kinetics characterization of modified species of Growth Hormone Releasing Hexapeptide generated under thermal stress in different pH and buffers.

Growth Hormone Releasing Peptide-6 (GHRP-6) is a promising molecule (H-His1-d-Trp- Ala-Trp-d-Phe-Lys6-NH2) for the treatment of several diseases. Studies on the degradation pathways of this molecule under stressed conditions are needed to develop appropriate formulations. Degradation products (DPs) of GHRP-6, generated by heating in the dark at 60 °C with pH ranging from 3.0 to 8.0 and in presence of common buffers, were isolated by RP-HPLC and characterized by ESI-MS/MS. C-terminal deamidation of GHRP-6 was generated preferentially at pH 3.0 and 8.0. Hydrolysis and head-to-tail cyclization were favored at pH ranging from 6.0 to 7.0 in phosphate containing buffers. A DP with +12 Da molecular mass was presumably originated by the reaction with formaldehyde derived from some of the additives and/or elastomeric closures. Certain DPs derived from the acylation reaction of the tri- and di-carboxylic buffering species were favored at pH 3.0-6.0 and indicate that buffer components, including those "Generally Recognized as Safe", may potentially introduce chemical modifications and product heterogeneity. Nano LC-MS/MS analysis revealed GHRP-6 was also detected as a low-abundance species with Trp oxidized to 5-hydroxy, kynurenine, and N-formylkynurenine. The kinetics for the formation of the major degradation products was also studied by RP-HPLC.

Aß-Amyloid Fibrils Are Self-Triggered by the Interfacial Lipid Environment and Low Peptide Content.

We studied the surface properties of Aß(1-40) amyloid peptides mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) (liquid state) or 1,2-disteraoyl-phosphatidylcholine (DSPC) (solid state) phospholipids by using nanostructured lipid/peptide films (Langmuir monolayers). Pure Aß(1-40) amyloid peptides form insoluble monolayers without forming fibril-like structures. In a lipid environment [phospholipid/Aß(1-40) peptide mixtures], we observed that both miscibility and stability of the films depend on the peptide content. At low Aß(1-40) amyloid peptide proportion (from 2.5 to 10% of peptide area proportion), we observed the formation of a fibril-like structure when mixed only with POPC lipids. The stability acquired by these mixed films is within 20-35 mN·m-1 compatible with the equivalent surface pressure postulated for natural biomembranes. Fibrils are clearly evidenced directly from the monolayers by using Brewster angle microscopy. The so-called nanostructured fibrils are thioflavin T positive when observed by fluorescence microscopy. The amyloid fibril network at the surface was also evidenced by atomic force microscopy when the films are transferred onto a mica support. Aß(1-40) amyloid mixed with the solid DSPC lipid showed an immiscible behavior in all peptide proportions without fibril formation. We postulated that the amyloid fibrillogenesis at the membrane can be dynamically nano-self-triggered at the surface by the quality of the interfacial environment, that is, the physical state of the water-lipid interface and the relative content of amyloid protein present at the interface.

Experimental diabetes impairs maternal reproductive performance in pregnant Wistar rats and their offspring.

The aim of this study was to determine the effect of mild hyperglycemia on metabolism during pregnancy, the maternal reproductive performance, and the characteristics of the offspring in neonatal mild diabetic-induced Wistar rats. The experimental diabetes model was generated by neonatal streptozotocin administration (100 mg of streptozotocin/Kg bw/sc) in female Wistar rats. At adulthood, the control and diabetic group were mated. At the 20th day of gestation, a maternal and fetal blood sample were collected for biochemical measurement. The maternal livers, fetal livers, and placenta were removed for oxidative stress measurements. Maternal reproductive outcomes and fetal and placental morphometric measurements were analyzed. The fetuses were classified as small, appropriate, and large for pregnancy age, and examined for the presence of external anomalies. The diabetic group showed mild hyperglycemia, altered glucose tolerance, increased total cholesterol, triglycerides, and hemoglobin A1c during pregnancy. At the 20th day of gestation the diabetic mothers presented increased reabsorptions and embryonic losses before and after implantation, reduced corpora lutea number, litter size, implantation sites, live fetuses, and decreased efficiency of implantation rate. Similarly, the offspring showed reduced fetal, craniofacial, and placental dimensions, in addition to a higher proportion of small fetuses for pregnancy age. Mild hyperglycemia during pregnancy did not generate marked oxidative stress in the mother, and in fetal liver and placenta decreased antioxidant activity was evident by significant consumption of reduced glutathione. Mild diabetes led to a negative impact on maternal reproductive performance and characteristics of the offspring. This experimental model reproduced maternal and fetal outcomes of pregnant rats presenting controlled diabetes. ABBREVIATIONS: bw: body weight; sc: subcutaneous; DM: diabetes mellitus; STZ: streptozotocin; OGTT: oral glucose tolerance test; ITT: insulin tolerance test; GSH: glutathione; MDA: malondialdehyde; AOPPs: advanced oxidation protein products; TBARs: thiobarbituric acid reaction; SPA: small for pregancy age; APA: appropriate for pregnancy age; LPG: large for pregnancy age; ROS: reactive oxygen species.

Concentration of Zinc, Copper, Iron, Calcium, and Magnesium in the Serum, Tissues, and Urine of Streptozotocin-Induced Mild Diabetic Rat Model.

The present study aimed to investigate, in the streptozotocin-induced mild diabetic rat model, the zinc (Zn), copper (Cu), iron (Fe), calcium (Ca), and magnesium (Mg) concentration in serum, liver, and kidney tissues, and urine samples from adult Wistar rats treated neonatally with streptozotocin (STZ). Diabetes was induced by subcutaneous administration of streptozotocin (100 mg/Kg) in female Wistar rats of 2 days old (STZ, n = 10). Control group (CG, n = 10) received only sodium-citrate buffer. The mineral concentrations were measured by atomic absorption spectrophotometry. The validity and accuracy were checked by conventional methods. STZ neonatal injection successfully leaded to mild diabetes in the adult rats. Serum concentrations of Zn, Cu, Fe, Ca, and Mg showed no changes (p > 0.05) due to diabetes. The Zn, Fe, Ca, and Mg concentrations in liver and kidney tissues were not different (p > 0.05) between STZ and CG. The mean values of Cu were higher (p < 0.05) in liver and kidney samples from STZ as compared to CG. Urine minerals concentrations (Zn, Cu, Fe and Ca) in STZ-rats group were lower (p < 0.05) than CG. However, the content of all evaluated minerals in the excreted urine were higher (p < 0.01) in STZ-rats during a 24 h collection period. Urinary excretion of Zn, Cu, Fe, Ca, and Mg was strongly correlated with urinary volume during the 24 h period (r > 0.7; p < 0.001). Observed changes in mineral metabolism of STZ-induced mild diabetes model could be due to the endocrine imbalance associated with the diabetic condition.

Proyecto Vedado: salud-electrónica en la atención primaria de salud: diseño y resultados iniciales / Vedado Project: electronic health in primary health care. Design and initial results

Se expone brevemente el diseño, justificación, resultados iniciales y perspectivos, y dificultades del portafolio de proyectos “Vedado” de salud-electrónica (salud-e) en la atención primaria de salud y medicina general integral, entre los años 2002 y 2006. Se describen los resultados obtenidos con un sistema de información gerencial de atención primaria, un sistema tele electrocardiográfico de 2da. y 3ra. opinión, unos sitios web del Policlínico Vedado, un tutorial en multimedia de anatomía en atención primaria, y una universidad virtual desde este propio nivel de atención. Se enuncian los resultados perspectivos de una red electrónica y centro colaborador en investigación a distancia y en línea del sistema nacional de salud a partir de la atención primaria, del uso de asistentes personales digitales y tecnología inalámbrica, así como de una historia clínica electrónica única desde el consultorio del Médico de Familia al resto del sistema. Se concluye que los resultados obtenidos han comenzado lentamente a mejorar la eficiencia de este nivel de atención y nuestra especialidad. Se vislumbra que la continuación de estos proyectos y extensión de sus resultados originará un salto de calidad en los servicios de la Medicina General Integral en las áreas de salud, sobre todo, las rurales y más aisladas del país. Se recomienda formalizar un centro y red de investigación en salud-e desde la atención primaria, con presupuesto para proyectos de investigación, incluir técnicas de tele-cirugía y cirugía de mínimo acceso, sobre todo en áreas rurales, así como también facilitar la investigación en colaboración en salud-e y los entrenamientos e intercambios académicos en los países de avanzada.

Proyecto Vedado: salud-electrónica en la atención primaria de salud: diseño y resultados iniciales / Vedado Project: electronic health in primary health care. Design and initial results

Se expone brevemente el diseño, justificación, resultados iniciales y perspectivos, y dificultades del portafolio de proyectos “Vedado” de salud-electrónica (salud-e) en la atención primaria de salud y medicina general integral, entre los años 2002 y 2006. Se describen los resultados obtenidos con un sistema de información gerencial de atención primaria, un sistema tele electrocardiográfico de 2da. y 3ra. opinión, unos sitios web del Policlínico Vedado, un tutorial en multimedia de anatomía en atención primaria, y una universidad virtual desde este propio nivel de atención. Se enuncian los resultados perspectivos de una red electrónica y centro colaborador en investigación a distancia y en línea del sistema nacional de salud a partir de la atención primaria, del uso de asistentes personales digitales y tecnología inalámbrica, así como de una historia clínica electrónica única desde el consultorio del Médico de Familia al resto del sistema. Se concluye que los resultados obtenidos han comenzado lentamente a mejorar la eficiencia de este nivel de atención y nuestra especialidad. Se vislumbra que la continuación de estos proyectos y extensión de sus resultados originará un salto de calidad en los servicios de la Medicina General Integral en las áreas de salud, sobre todo, las rurales y más aisladas del país. Se recomienda formalizar un centro y red de investigación en salud-e desde la atención primaria, con presupuesto para proyectos de investigación, incluir técnicas de tele-cirugía y cirugía de mínimo acceso, sobre todo en áreas rurales, así como también facilitar la investigación en colaboración en salud-e y los entrenamientos e intercambios académicos en los países de avanzada(AU)