A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

Effect of Low-Level Laser Therapy on Malondialdehyde Concentration in Random Cutaneous Flap Viability

Objective: The aim of this study was to assess the effects of 830 and 670 nm laser on malondialdehyde (MDA) concentration in random skin-flap survival. Background Data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and activating superoxide-dismutase delivery, thus helping the inhibition of free-radical action and consequently reducing necrosis. Materials and Methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each one. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group; group 2 received 830 nm laser radiation; and group 3 was submitted to 670 nm laser radiation. The animals underwent laser therapy with 36 J/cm(2) energy density immediately after surgery and on the 4 days subsequent to surgery. The application site of the laser radiation was 1 point, 2.5 cm from the flap's cranial base. The percentage of the skin-flap necrosis area was calculated 7 days postoperative using the paper-template method, and a skin sample was collected immediately after as a way of determining the MDA concentration. Results: Statistically significant differences were found between the necrosis percentages, with higher values seen in group 1 compared with groups 2 and 3. Groups 2 and 3 did not present statistically significant differences (p > 0.05). Group 3 had a lower concentration of MDA values compared to the control group (p < 0.05). Conclusion: LLLT was effective in increasing the random skin-flap viability in rats, and the 670 nm laser was efficient in reducing the MDA concentration.

Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

Prognostic Factors in Patients with Malignant Salivary Gland Neoplasms in a Brazilian Population

Due to the difficulty of follow-up for long periods, information about the survival rates of malignant salivary gland tumors is deficient in the global scientific literature. This study was aimed at investigating the epidemiological profile and prognostic factors that might affect survival in patients with primary malignant salivary gland tumors in Brazil. Patients were investigated regarding histopathological subtypes, age, gender, anatomic localization, smoking and alcohol intake, tumor size, clinical stage, histological grade, recurrence, metastasis, and treatment on clinicopathological outcomes. Survival curves were generated using the Kaplan-Meier method, and both univariate and multivariate analyses were performed using the log rank test and Cox regression, respectively. A total of 63 cases were analyzed, females beingslightly predominant (50.8%), with ages ranging from 13 to 87 years. The most common diagnosis was adenoid cystic carcinoma and the most affected anatomical location was the parotid. Tumors were predominantly classified as stage I and high-grade at the diagnosis. The 5- and 10-year overall survival rates were 84.6% and 74.7%, respectively. Disease-free survival (DFS) rates were 71.6% (5 years) and 56.6% (10 years). Univariate analysis showed significant effects of tumor size and clinical stage on the DFS (P < 0.0001 for both), and Cox regression analysis confirmed clinical stage as an independent prognostic factor (P = 0.035). Our results highlight the relevance of clinical stage as an independent prognostic parameter for malignant salivary gland tumors.

Tick-Borne Bacteria in Free-Living Jaguars (Panthera onca) in Pantanal, Brazil

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer >= 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.

Toxoplasma gondii Isolates From Free-Range Chickens From the Northeast Region of Brazil

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of 7: gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gonulii in 152 free-range chickens (Gallus domesticus) from 22 municipalities in 7 northeastern states (Pernambuco, Rio Grande do Norte, Maranh5o, Bahia, Ceara, Sergipe, and Alagoas) of Brazil was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT); 81 (53.3 %) chickens had titers of 1:5 in 26, 1:10 in 9, 1:20 in 4, 1:40 in 1, 1:80 in 6, 1:160 in 6, 1:320 in 13, 1:640 in 6, 1:1,280 in 3, 1:2,560 in 6, and 1:5,120 or higher in I. Hearts and brains of 81 seropositive chickens were bioassayed individually in mice. Toxoplasma gondii was isolated from 23 chickens with MAT titers of 1:5 or higher; the isolates were designated TgCKBr165-187. Five isolates killed all infected mice. Results indicate widespread contamination of rural environment in Brazil with T. gondii oocysts.

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Automated supervised classification of variable stars in the CoRoT programme Method and application to the first four exoplanet fields

Aims. In this work, we describe the pipeline for the fast supervised classification of light curves observed by the CoRoT exoplanet CCDs. We present the classification results obtained for the first four measured fields, which represent a one-year in-orbit operation. Methods. The basis of the adopted supervised classification methodology has been described in detail in a previous paper, as is its application to the OGLE database. Here, we present the modifications of the algorithms and of the training set to optimize the performance when applied to the CoRoT data. Results. Classification results are presented for the observed fields IRa01, SRc01, LRc01, and LRa01 of the CoRoT mission. Statistics on the number of variables and the number of objects per class are given and typical light curves of high-probability candidates are shown. We also report on new stellar variability types discovered in the CoRoT data. The full classification results are publicly available.

Novel method for measuring nanofriction by atomic force microscope

The authors describe a novel approach to the measurement of nanofriction, and demonstrate the application of the method by measurement of the coefficient of friction for diamondlike carbon (DLC) on DLC, Si on DLC, and Si on Si surfaces. The technique employs an atomic force microscope in a mode in which the tip moves only in the z (vertical) direction and the sample surface is sloped. As the tip moves vertically on the sloped surface, lateral tip slipping occurs, allowing the cantilever vertical deflection and the frictional (lateral) force to be monitored as a function of tip vertical deflection. The advantage of the approach is that cantilever calibration to obtain its spring constants is not necessary. Using this method, the authors have measured friction coefficients, for load range 0 < L M 6 mu N, of 0.047 +/- 0.002 for Si on Si, 0.0173 +/- 0.0009 for Si on DLC, and 0.0080 +/- 0.0005 for DLC on DLC. For load range 9 < L < 13 mu N, the DLC on DLC coefficient of friction increased to 0.051 +/- 0.003. (C) 2008 American Vacuum Society.

Free-standing urethane/urea elastomer films undoped and doped with ferro-nano-particles

We report on an experimental study of the structures presented by urethane/urea elastomeric films without and with ferromagnetic nanoparticles incorporated. The study is made by using the X-ray diffraction, nuclear magnetic resonance (NMR), optical, atomic and magnetic force (MFM) microscopy techniques, and mechanical assays. The structure of the elastomeric matrix is characterized by a distance of 0.46 nm between neighboring molecular segments, almost independent on the stretching applied. The shear casting performed in order to obtain the elastomeric films tends to orient the molecules parallel to the flow direction thus introducing anisotropy in the molecular network which is reflected on the values obtained for the orientational order parameter and its increase for the stretched films. In the case of nanoparticles-doped samples, the structure remains nearly unchanged although the local order parameter is clearly larger for the undoped films. NMR experiments evidence modifications in the molecular network local ordering. Micrometer size clusters were observed by MFM for even small concentration of magnetic particles.

Study of the influence of indium segregation on the optical properties of InGaAs/GaAs quantum wells via split-operator method

In the case of quantum wells, the indium segregation leads to complex potential profiles that are hardly considered in the majority of the theoretical models. The authors demonstrated that the split-operator method is useful tool for obtaining the electronic properties in these cases. Particularly, they studied the influence of the indium surface segregation in optical properties of InGaAs/GaAs quantum wells. Photoluminescence measurements were carried out for a set of InGaAs/GaAs quantum wells and compared to the results obtained theoretically via split-operator method, showing a good agreement.

Resummation of infrared divergences in the free energy of spin-two fields

We derive a closed form expression for the sum of all the infrared divergent contributions to the free energy of a gas of gravitons. An important ingredient of our calculation is the use of a gauge fixing procedure such that the graviton propagator becomes both traceless and transverse. This has been shown to be possible, in a previous work, using a general gauge fixing procedure, in the context of the lowest order expansion of the Einstein-Hilbert action, describing noninteracting spin-two fields. In order to encompass the problems involving thermal loops, such as the resummation of the free energy, in the present work, we have extended this procedure to the situations when the interactions are taken into account.

Pressure of massless hot scalar theory in the boundary effective theory framework

We use the boundary effective theory approach to thermal field theory in order to calculate the pressure of a system of massless scalar fields with quartic interaction. The method naturally separates the infrared physics, and is essentially nonperturbative. To lowest order, the main ingredient is the solution of the free Euler-Lagrange equation with nontrivial (time) boundary conditions. We derive a resummed pressure, which is in good agreement with recent calculations found in the literature, following a very direct and compact procedure.

Free energy of Lorentz-violating QED at high temperature

In this paper we study the one-and two-loop contribution to the free energy in QED with Lorentz symmetry breaking introduced via constant CPT-even Lorentz-breaking parameters at the high temperature limit. We find the impact of the Lorentz-violating term for the free energy and carry out a numerical estimation for the Lorentz-breaking parameter.

Accurate prediction of the Si/SiO(2) interface band offset using the self-consistent ab initio DFT/LDA-1/2 method

We use the density functional theory/local-density approximation (DFT/LDA)-1/2 method [L. G. Ferreira , Phys. Rev. B 78, 125116 (2008)], which attempts to fix the electron self-energy deficiency of DFT/LDA by half-ionizing the whole Bloch band of the crystal, to calculate the band offsets of two Si/SiO(2) interface models. Our results are similar to those obtained with a ""state-of-the-art"" GW approach [R. Shaltaf , Phys. Rev. Lett. 100, 186401 (2008)], with the advantage of being as computationally inexpensive as the usual DFT/LDA. Our band gap and band offset predictions are in excellent agreement with experiments.