Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Viability of mesenchymal stem cells during electrospinning

Tissue engineering is a technique by which a live tissue can be re-constructed and one of its main goals is to associate cells with biomaterials. Electrospinning is a technique that facilitates the production of nanofibers and is commonly used to develop fibrous scaffolds to be used in tissue engineering. In the present study, a different approach for cell incorporation into fibrous scaffolds was tested. Mesenchymal stem cells were extracted from the wall of the umbilical cord and mononuclear cells from umbilical cord blood. Cells were re-suspended in a 10% polyvinyl alcohol solution and subjected to electrospinning for 30 min under a voltage of 21 kV. Cell viability was assessed before and after the procedure by exclusion of dead cells using trypan blue staining. Fiber diameter was observed by scanning electron microscopy and the presence of cells within the scaffolds was analyzed by confocal laser scanning microscopy. After electrospinning, the viability of mesenchymal stem cells was reduced from 88 to 19.6% and the viability of mononuclear cells from 99 to 8.38%. The loss of viability was possibly due to the high viscosity of the polymer solution, which reduced the access to nutrients associated with electric and mechanical stress during electrospinning. These results suggest that the incorporation of cells during fiber formation by electrospinning is a viable process that needs more investigation in order to find ways to protect cells from damage.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Laser assisted extraction of pacemaker and implantable defibrillator leads

OBJECTIVE: To analyze the results of laser-assisted extraction of permanent pacemaker and defibrillator leads. METHODS: We operated upon 36 patients, whose mean age was 54.2 years, and extracted 56 leads. The reasons for extracting the leads were as follows: infection in 19 patients, elective replacement in 13, and other causes in 4 patients. The mean time of catheter placement was 7.5±5.5 years. Forty-seven leads were from pacemakers, and 9 were from defibrillators. Thirty-eight leads were in use, 14 had been abandoned in the pacemaker pocket, and 4 had been abandoned inside the venous system. RESULTS: We successfully extracted 54 catheters, obtaining a 96.4% rate of success and an 82.1% rate for complete extraction. The 2 unsuccessful cases were due to the presence of calcium in the trajectory of the lead. The mean duration of laser light application was 123.0±104.5 s, using 5,215.2±4,924.0 pulses, in a total of 24.4±24.2 cycles of application. Thirty-four leads were extracted from the myocardium with countertraction after complete progression of the laser sheath, 12 leads came loose during the progression of the laser sheath, and the remaining 10 were extracted with other maneuvers. One patient experienced cardiac tamponade after extraction of the defibrillator lead, requiring open emergency surgery. CONCLUSION: The use of the excimer laser allowed extraction of the leads with a 96% rate of success; it was not effective in 2 patients who had calcification on the lead. One patient (2.8%) had a complication that required cardiac surgery on an emergency basis.

Morphological Study of the Larval Spiracular System in Eight Lutzomyia Species (Diptera: Psychodidae)

The morphology of the spiracles of fourth instar larva in eight sandfly species were examined by light and scanning electron microscopy. Species studied were: Lutzomyia longipalpis (Lutz & Neiva), L. ovallesi (Ortiz), L. youngi Feliciangeli & Murillo, L. evansi (Nuñez-Tovar), L. trinidadensis (Newstead), L. migonei (França), L. absonodonta Feliciangeli, and L. venezuelensis (Floch & Abonnenc). In larvae of all eight species both thoracic and abdominal spiracles are located at the top of a globular bulge. Their structure consists of a spiracular plate with a sclerotized central portion and a rose-like peripheral portion. The latter has circularly arranged papillae, separated from each other by elongated septa. Each papilla is longitudinally crossed by a fine cleft dividing it into two identical parts. The taxonomic and adaptative value of spiracular morphology is discussed

The postembryonic development of the ocellar system of Triatoma infestans Klug (Heteroptera: Reduviidae)

Simple eyes or ocelli coexist with compound eyes in many adult insects. The change in the morphology of the ocelli along the five larval instars of Triatoma infestans was studied by light and scanning electron microscopy. Our analysis showed that the development of the ocelli of these bugs occurs gradually along the larval life. The photoreceptor layer is present from the second-instar onwards. The cornea appears first at the imaginal stage and grows up to the 18-20th day after the last ecdysis, associated to an increase in the retinal mass. Findings are discussed in a comparative fashion and in relation to the functionality of the ocellar system in T. infestans.

Spinitectus osorioi (Nematoda: Cystidicolidae) in the Mexican endemic fish Atherinella alvarezi (Atherinopsidae) from the Atlantic River drainage system in Chiapas, Southern Mexico

Specimens of Spinitectus osorioi Choudhury and Pérez-Ponce de León, an intestinal nematode species previously considered to be specific to Chirostoma spp and endemic to some lakes in the Pacific drainage in Michoacán, were collected from the freshwater fish Atherinella alvarezi (Díaz-Pardo) (Atherinopsidae) of the Michol River near Palenque, Chiapas, Southern Mexico, which belongs to the Atlantic drainage system. Studies using light and scanning electron microscopy revealed some taxonomically important, previously unreported or erroneously reported features of S. osorioi, such as the location of the vulva, the actual number and distribution of postanal papillae and phasmids and the presence of a short median cuticular ridge anterior to the cloacal opening (in addition to two long subventral ridges). The recorded somewhat shorter spicules (420-465 and 105-111 μm) and mostly smaller eggs (33-36 × 18-20 μm) as compared to the original species description may be due to a different type of host, geographical region or generally smaller body measurements of these specimens. These biometrical differences are considered to be within the limits of the intraspecific variability of S. osorioi. A key to species of Spinitectus parasitizing freshwater fishes in Mexico is provided.

Whole-body MRI: comprehensive evaluation on a 48-channel 3T MRI system in less than 40 minutes. Preliminary results

OBJECTIVE: To evaluate a comprehensive MRI protocol that investigates for cancer, vascular disease, and degenerative/inflammatory disease from the head to the pelvis in less than 40 minutes on a new generation 48-channel 3T system. MATERIALS AND METHODS: All MR studies were performed on a 48-channel 3T MR scanner. A 20-channel head/neck coil, two 18-channel body arrays, and a 32-channel spine array were employed. A total of 4 healthy individuals were studied. The designed protocol included a combination of single-shot T2-weighted sequences, T1-weighted 3D gradient-echo pre- and post-gadolinium. All images were retrospectively evaluated by two radiologists independently for overall image quality. RESULTS: The image quality for cancer was rated as excellent in the liver, pancreas, kidneys, lungs, pelvic organs, and brain, and rated as fair in the colon and breast. For vascular diseases ratings were excellent in the aorta, major branch vessel origins, inferior vena cava, portal and hepatic veins, rated as good in pulmonary arteries, and as poor in the coronary arteries. For degenerative/inflammatory diseases ratings were excellent in the brain, liver and pancreas. The inter-observer agreement was excellent. CONCLUSION: A comprehensive and time efficient screening for important categories of disease processes may be achieved with high quality imaging in a new generation 48-channel 3T system.

Implementação de um sistema de eletroforese capilar com detecção de fluorescência induzida por laser

A capillary electrophoresis system using laser induced fluorescence detection is described. A Raman system equipped with a microscope has been used to focus the laser beam on the capillary giving a lateral resolution of 1.5 mm. The fluorescence signal of the analyte (ZnPcTS - tetrasulfonated zinc-phthalocyanine) was collected by the microscope objectives and analysed by a monochromator with confocal characteristics equipped with a CCD detector. Electropherograms obtained with this system were compared to those obtained on a commercial instrument, showing that the described system presents a lower detection limit and better resolution.

Estudos de QSAR 3D para um conjunto de inibidores de butirilcolinesterase humana

Alzheimer's disease (AD) is considered the main cause of cognitive decline in adults. The available therapies for AD treatment seek to maintain the activity of cholinergic system through the inhibition of the enzyme acetylcholinesterase. However, butyrylcholinesterase (BuChE) can be considered an alternative target for AD treatment. Aiming at developing new BuChE inhibitors, robust QSAR 3D models with high predictive power were developed. The best model presents a good fit (r²=0.82, q²=0.76, with two PCs) and high predictive power (r²predict=0.88). Analysis of regression vector shows that steric properties have considerable importance to the inhibition of the BuChE.

Kinetic study of the cobalt electrodeposition onto glassy carbon electrode from ammonium sulfate solutions

We carried out an electrochemical study of the cobalt electrodeposition onto glassy carbon electrode from an aqueous solution containing 10-2 M of CoSO4 + 1 M (NH4)2SO4 at natural pH 4.5. The potentiostatic study indicated a progressive 3D nucleation and growth during the deposition process. The average diffusion coefficient calculated for this system was 2.65 X 10-6 cm² s-1 while the ΔG for the formation of stable nucleus was 6.50 X 10-20 J/nuclei. The scanning electron microscopy images indicated the formation of small and homogeneous nucleus onto GCE of approximately 300 nm.

Encapsulation of naproxen in nanostructured system: structural characterization and in vitro release studies

Nanoparticles were produced by solvent emulsification evaporation method with the following characteristics: nanometric size (238 ± 3 nm), narrow polydispersity index (0.11), negative zeta potential (-15.1 mV), good yield of the process (73 ± 1.5%), excellent encapsulation efficiency (81.3 ± 4.2%) and spherical shape. X-rays diffraction demonstrated the loss of drug crystallinity after encapsulation; however, the profile of the diffractograms of the poly-ε-caprolactone (PCL) nanoparticles was kept. Differential scanning calorimetry thermograms, correspondingly, exhibited the loss of drug melting peak and the increasing of the melting point of the PCL nanoparticles, evidencing an interaction drug-polymer. Naproxen release was low and sustained obeying the Higuchi´s kinetic. The results show that nanoparticles are promising sustained release system to the naproxen.

Using an aerial system of remote sensing to detect different nutritional status in Brachiaria decumbens

The aim of this study was to use digital images acquired by cameras attached to a helium balloon to detect variation of the nutritional status in Brachiaria decumbens. The treatments consisted of five doses of nitrogen (0, 50, 100, 150 e 200kg ha-1) with six replications each, evaluated in a completely randomized statistical design. A remote sensing system composed of digital cameras and microcomputers was used for image acquisition, and a helium balloon lifted the cameras to the heights of 15, 20, 25 and 30m. A portable chlorophyll meter and analyses of leaf nitrogen content were used to make comparisons with data obtained by the remote sensing system. Data was acquired in two phases, in different climatic conditions. At the end of each phase, dry matter production was measured. Three vegetation indices were used to evaluate the detection of different nutritional status. The three indices were able to detect the effects of N doses. The indices constructed with the Green spectral band showed to be more efficient.

Portable flow board for storage of fruits and vegetables in mini-chambers with controlled atmosphere

ABSTRACT A portable flow board system was developed in the present study with the aim to facilitate lab-scale experiments of controlled atmosphere (CA) with fruits and vegetables. This sturdy flow board combines ease fabrication, low cost and gas economy. Its functionality is provided by manifolds and gas mixers. Each gaseous component is supplied by a gas cylinder through a differential valve of adjusted pressure control, generally at 6 kPa, and forced through 13 standardized restrictors coupled to each manifold output. Controlled atmospheres are then formed with one, two or three gases in 13 gas mixers affixed to the flow board base, which are further conducted through flexible tubes to storage mini-chambers that can also be used to study metabolic consumption and production of gaseous components. The restrictors used in the flow gaseous components were manufactured from microhematocrit test-type capillary glass tubes following the hot forming method under continuous air flow. The portable flow board showed to be low cost and simple post-harvest equipment that allows preparing controlled atmospheres in open systems with stable composition and flow, in a manner similar to traditional flow boards with control of gas escape by barostats.

Experimental validation of a 3-D vision-based measurement system applied to robot calibration

One of the problems that slows the development of off-line programming is the low static and dynamic positioning accuracy of robots. Robot calibration improves the positioning accuracy and can also be used as a diagnostic tool in robot production and maintenance. A large number of robot measurement systems are now available commercially. Yet, there is a dearth of systems that are portable, accurate and low cost. In this work a measurement system that can fill this gap in local calibration is presented. The measurement system consists of a single CCD camera mounted on the robot tool flange with a wide angle lens, and uses space resection models to measure the end-effector pose relative to a world coordinate system, considering radial distortions. Scale factors and image center are obtained with innovative techniques, making use of a multiview approach. The target plate consists of a grid of white dots impressed on a black photographic paper, and mounted on the sides of a 90-degree angle plate. Results show that the achieved average accuracy varies from 0.2mm to 0.4mm, at distances from the target from 600mm to 1000mm respectively, with different camera orientations.