Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappa B signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow ( BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells ( CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency ( than expected by chance) of NF-kappa B-binding sites (BS), including potentially novel NF-kappa B targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappa B, and other important TFs on more primitive HSC sets.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Low-Level Laser Intensity Application in Masseter Muscle for Treatment Purposes

Objective: This study evaluated with histochemical analysis how the number of laser applications can affect the masseter muscle. Background: In dentistry today, the laser is used in patients with temporomandibular disorders (TMDs), mainly for radiating pain in the masticatory muscles, whose origins may be associated with malocclusion, although the laser effects are not well understood on the cellular level. Materials and Methods: Thirty mice (HRS/J lineage) were randomly distributed into groups according to the number of laser applications (three, six, and 10). For each group of laser applications (experimental, n = 5), it was considered the control group (n = 5), which was not irradiated. All animals inhaled halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane, minimum 99%, Sigma Aldrich, India) before each laser irradiation performed on the left masseter muscle region, on alternate days with 20 J/cm(2), 40mW, for 20 sec. The muscle samples were collected for histochemical analysis with succinate dehydrogenase (SDH) enzyme 72 h after the last application. Results: (a) A decrease in area of light fibers type (35.91% +/- 6.9%; 32.08% +/- 6.3%, and 27.88% +/- 6.3%), according to the increase of laser applications (p < 0.05); (b) significant increase (p < 0.05) in the area of intermediate fibers, with an increase of laser application (11.08% +/- 3.9%; 16.52% +/- 5.7%, and 15.96% +/- 3.9%), although the increase with 10 applications was small; (c) area increase of dark fibers in the group with three laser applications (0.16% +/- 0.3%) (p < 0.05), and in groups with six and 10 laser applications, respectively (9.68% +/- 6.0% and 9.60% +/- 4.0%). Conclusions: The SDH enzyme activity revealed that the number of laser applications increases the metabolic pattern of the muscle fibers. A minimal difference in metabolic activity between six and 10 applications of a laser suggests that further analyses should be done to confirm that six applications are enough to produce the same clinical effects, thereby contributing data to professionals from different fields in regard to the cost-benefit ratio of this therapy.

Influence of Chinese breeds on pork quality of commercial pig lines

We compared carcass and meat quality of pigs from the same sire line and two different dam lines, one that included Chinese breeds and one that did not. Line A consisted of 1/4 Landrace, 1/2 Large White, 1/8 Chinese breeds (Meishan, Fengjing, Jiaxing), and 1/8 Large White, Duroc and Pietrain, and line B consisted of 1/2 Large White and 1/2 Pietrain. The animals (N = 144) were slaughtered at a live weight of 108 kg. Backfat thickness, percentage of lean meat, pH 24 h after slaughter, meat color, percentage of drip loss, and percentage of intramuscular fat were measured and compared using analysis of variance in a completely randomized design; the BioEstat 5.0 test was applied for the comparison of means at a significance level of 5% for all analyses. Backfat was significantly lower for line A (12.78 mm) than for line B (15.90 mm). The pH measured 24 h after slaughter was significantly lower in line A (5.68) compared to line B (5.84). Percent lean meat was significantly higher for line A (61.21%) compared to line B (59.72%). Percentage drip loss was significantly higher in line A (2.73%) than in line B (2.23%). Percentage intramuscular fat and meat color were not significantly different between the lines. The inclusion of Chinese breeds produced a higher percentage of lean meat and reduced fat thickness, along with increased heterosis, which are important characteristics for breeding programs.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Fast ray-tracing algorithm for circumstellar structures (FRACS) II. Disc parameters of the B[e] supergiant CPD-57 degrees 2874 from VLTI/MIDI data

Context. B[e] supergiants are luminous, massive post-main sequence stars exhibiting non-spherical winds, forbidden lines, and hot dust in a disc-like structure. The physical properties of their rich and complex circumstellar environment (CSE) are not well understood, partly because these CSE cannot be easily resolved at the large distances found for B[e] supergiants (typically greater than or similar to 1 kpc). Aims. From mid-IR spectro-interferometric observations obtained with VLTI/MIDI we seek to resolve and study the CSE of the Galactic B[e] supergiant CPD-57 degrees 2874. Methods. For a physical interpretation of the observables (visibilities and spectrum) we use our ray-tracing radiative transfer code (FRACS), which is optimised for thermal spectro-interferometric observations. Results. Thanks to the short computing time required by FRACS (<10 s per monochromatic model), best-fit parameters and uncertainties for several physical quantities of CPD-57 degrees 2874 were obtained, such as inner dust radius, relative flux contribution of the central source and of the dusty CSE, dust temperature profile, and disc inclination. Conclusions. The analysis of VLTI/MIDI data with FRACS allowed one of the first direct determinations of physical parameters of the dusty CSE of a B[e] supergiant based on interferometric data and using a full model-fitting approach. In a larger context, the study of B[e] supergiants is important for a deeper understanding of the complex structure and evolution of hot, massive stars.

Kinematics of galaxies in compact groups Studying the B-band Tully-Fischer relation

We obtained new Fabry-Perot data cubes and derived velocity fields, monochromatic, and velocity dispersion maps for 28 galaxies in the Hickson compact groups 37, 40, 47, 49, 54, 56, 68, 79, and 93. We also derived rotation curves for 9 of the studied galaxies, 6 of which are strongly asymmetric. Combining these new data with previously published 2D kinematic maps of compact group galaxies, we investigated the differences between the kinematic and morphological position angles for a sample of 46 galaxies. We find that one third of the unbarred compact group galaxies have position angle misalignments between the stellar and gaseous components. This and the asymmetric rotation curves are clear signatures of kinematic perturbations, probably because of interactions among compact group galaxies. A comparison between the B-band Tully-Fisher relation for compact group galaxies and for the GHASP field-galaxy sample shows that, despite the high fraction of compact group galaxies with asymmetric rotation curves, these lay on the TF relation defined by galaxies in less dense environments, although with more scatter. This agrees with previous results, but now confirmed for a larger sample of 41 galaxies. We confirm the tendency for compact group galaxies at the low-mass end of the Tully-Fisher relation (HCG 49b, 89d, 96c, 96d, and 100c) to have either a magnitude that is too bright for its mass (suggesting brightening by star formation) and/or a low maximum rotational velocity for its luminosity (suggesting tidal stripping). These galaxies are outside the Tully Fisher relation at the 1 sigma level, even when the minimum acceptable values of inclinations are used to compute their maximum velocities. Including such galaxies with nu < 100 km s(-1) in the determination of the zero point and slope of the compact group B-band Tully-Fisher relation would strongly change the fit, making it different from the relation for field galaxies, which has to be kept in mind when studying scaling relations of interacting galaxies, especially at high redshifts.

Transiting exoplanets from the CoRoT space mission XI. CoRoT-8b: a hot and dense sub-Saturn around a K1 dwarf

Aims. We report the discovery of CoRoT-8b, a dense small Saturn-class exoplanet that orbits a K1 dwarf in 6.2 days, and we derive its orbital parameters, mass, and radius. Methods. We analyzed two complementary data sets: the photometric transit curve of CoRoT-8b as measured by CoRoT and the radial velocity curve of CoRoT-8 as measured by the HARPS spectrometer**. Results. We find that CoRoT-8b is on a circular orbit with a semi-major axis of 0.063 +/- 0.001 AU. It has a radius of 0.57 +/- 0.02 R(J), a mass of 0.22 +/- 0.03 M(J), and therefore a mean density of 1.6 +/- 0.1 g cm(-3). Conclusions. With 67% of the size of Saturn and 72% of its mass, CoRoT-8b has a density comparable to that of Neptune (1.76 g cm(-3)). We estimate its content in heavy elements to be 47-63 M(circle plus), and the mass of its hydrogen-helium envelope to be 7-23 M(circle plus). At 0.063 AU, the thermal loss of hydrogen of CoRoT-8b should be no more than similar to 0.1% over an assumed integrated lifetime of 3 Ga.

H alpha spectropolarimetry of the B[e] supergiant GG Carinae

Aims. We study the geometry of the circumstellar environment of the B[e] supergiant star GG Car. Methods. We present observations acquired using the IAGPOL imaging polarimeter in combination with the Eucalyptus-IFU spectrograph to obtain spectropolarimetric measurements of GG Car across Ha at two epochs. Polarization effects along the emission line are analysed using the Q-U diagram. In particular, the polarization position angle (PA) obtained using the line effect is able to constrain the symmetry axis of the disk/envelope. Results. By analysing the fluxes, GG Car shows an increase in its double-peaked Ha line emission relative to the continuum within the interval of our measurements (similar to 43 days). The depolarization line effect around Ha is evident in the Q-U diagram for both epochs, confirming that light from the system is intrinsically polarized. A rotation of the PA along Ha is also observed, indicating a counter-clockwise rotating disk. The intrinsic PA calculated using the line effect (similar to 85 degrees.) is consistent between our two epochs, suggesting a clearly defined symmetry axis of the disk.

The effect of low-level laser irradiation (In-Ga-Al-AsP-660 nm) on melanoma in vitro and in vivo

Background: It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation. Methods: We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm(2), irradiance 2.5 W/cm(2) and irradiation times of 60s (dose 150 J/cm(2)) and 420s (dose 1050 J/cm(2)) respectively. Results: There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 +/- 1.40% and 4.26 +/- 0.60%) at 72 h postirradiation. This cancer-protective effect was not reproduced in the in vivo experiment where outcome measures for the 150 J/cm(2) dose group were not significantly different from controls. For the 1050 J/cm(2) dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups. Conclusion: LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm(2)) and high dose (1050 J/cm(2)) significantly increases melanoma tumor growth in vivo.

Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.

Evaluation of Sphingolipids in Wistar Rats Treated to Prolonged and Single Oral Doses of Fumonisin B(1)

The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B(1) (FB(1)). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB(1). Prolonged exposure to FB(1) caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB(1). Animals receiving a single dose of FB(1) presented variations in Sa and So levels and in the Sa/So ratio.

Effects of Aflatoxin B(1) and Fumonisin B(1) on the Viability and Induction of Apoptosis in Rat Primary Hepatocytes

The present study evaluated the effect of aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) either alone, or in association, on rat primary hepatocyte cultures. Cell viability was assessed by flow cytometry after propidium iodine intercalation. DNA fragmentation and apoptosis were assessed by agarose gel electrophoresis and acridine orange and ethidium bromide staining. At the concentrations of AFB(1) and FB(1) used, the toxins did not decrease cell viability, but did induce apoptosis in a concentration and time-dependent manner.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.