Heteroduplex formation and S1 digestion for mapping alternative splicing sites

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Melatonin treatment decreases c-fos expression in a headache model induced by capsaicin

The aim of the present work was to analyze c-fos response within the trigeminal nucleus caudalis (TNC) of pinealectomized rats and animals that received intraperitoneal melatonin, after intracisternal infusion of capsaicin, used to induce intracranial trigeminovascular stimulation. Experimental groups consisted of animals that received vehicle solution (saline-ethanol-Tween 80, 8:1:1, diluted 1:50) only (VEI, n = 5); animals that received capsaicin solution (200 nM) only (CAP, n = 6); animals submitted to pinealectomy (PX, n = 5); sham-operated animals (SH, n = 5); animals submitted to pinealectomy followed by capsaicin stimulation (200 nM) after 15 days (PX + CAP, n = 7); and animals that received capsaicin solution (200 nM) and intraperitoneal melatonin (10 mg/kg) (CAP + MEL, n = 5). Control rats, receiving vehicle in the cisterna magna, showed a small number of c-fos-positive cells in the TNC (layer I/II) as well as the sham-operated and pinealectomized rats, when compared to animals stimulated by capsaicin. On the other hand, pinealectomized rats, which received capsaicin, presented the highest number of c-fos-positive cells. Animals receiving capsaicin and melatonin treatment had similar expression of the vehicle group. Our data provide experimental evidence to support the role of melatonin and pineal gland in the pathophysiology of neurovascular headaches.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in Sao Paulo state, Brazil. We show that different viral genotypes entered Sao Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in Sao Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay

Background: Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. Methods: Glutathione S-transferase (GST) and GST-fusion proteins representing the N-terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation. Results: The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay ( ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea ( PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG. Conclusions: This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

(7,9,10)Be elastic scattering and total reaction cross sections on a (12)C target

Elastic scattering angular distributions for (7)Be, (9)Be, and (10)Be isotopes on (12)C target were measured at laboratory energies of 18.8, 26.0, and 23.2 MeV, respectively. The analysis was performed in terms of optical model potentials using Woods-Saxon and double-folding form factors. Also, continuum discretized coupled-channels calculations were performed for (7)Be and (9)Be + (12)C systems to infer the role of breakup in the elastic scattering. For the (10)Be + (12)C system, bound states coupled-channels calculations were considered. Moreover, total reaction cross sections were deduced from the elastic scattering analysis and compared with published data on other weakly and tightly bound projectiles elastically scattered on the (12)C target, as a function of energy.

Elastic scattering and total reaction cross sections for the (8)Li+(12)C system

The elastic-scattering angular distribution for (8)Li on (12)C has been measured at E(LAB) = 23.9 MeV with (8)Li radioactive nuclear beam produced by the Radioactive Ion Beams in Brazil facility. This angular distribution was analyzed in terms of optical-model with Woods-Saxon and double-folding Sao Paulo potential. The roles of the breakup and inelastic channels were also investigated with cluster folding and deformed potentials, respectively, through coupled-channels calculations. The angular distribution for the proton-transfer (12)C((8)Li, (9)Be)(11)B reaction was also measured at the same energy. The spectroscopic factor for the <(9)Be|(8)Li + p > bound system was obtained and compared with shell-model calculations and with other experimental values. Total reaction cross sections for the present system were also extracted from the elastic-scattering analysis. A systematic of the reduced reaction cross sections obtained from the present and published data on (6,7,8)Li isotopes on (12)C was performed as a function of energy.

Possible hyperdeformed band in (36)Ar observed in (12)C+(24)Mg elastic scattering

Fifteen strongly oscillating angular distributions of the elastic scattering of (12)C + (24)Mg at energies around the Coulomb barrier (E(c.m). = 10.67-16.00 MeV) are reproduced by adding five Breit-Wigner resonance terms to the l = 2, 4, 6, 7, and 8 elastic S matrix. The nonresonant, background elastic scattering S matrix S(l)(0) is calculated using the Sao Paulo potential. The J = 2, 4, 6, 7, and 8 (h) over bar molecular resonances fit well into a rotational molecular band, together with other higher lying resonances observed in the (16)O + (20)Ne elastic scattering. We propose that the presently observed, largely deformed molecular band corresponds to the hyperdeformed band, which has been found previously in alpha-cluster calculations, as well as in a new Nilsson model calculation. Systematic study of its possible clusterizations predicts the preference of the (12)C + (24)Mg and (16)O + (20)Ne molecular structure, in accordance with our present results.

Elastic scattering and total reaction cross sections for the (8)B, (7)Be, and (6)Li+(12)C systems

Angular distributions for the elastic scattering of (8)B, (7)Be, and (6)Li on a (12)C target have been measured at E(lab) = 25.8, 18.8, and 12.3 MeV, respectively. The analyses of these angular distributions have been performed in terms of the optical model using Woods-Saxon and double-folding type potentials. The effect of breakup in the elastic scattering of (8)B + (12)C is investigated by performing coupled-channels calculations with the continuum discretized coupled-channel method and cluster-model folding potentials. Total reaction cross sections were deduced from the elastic-scattering analysis and compared with published data on elastic scattering of other weakly and tightly bound projectiles on (12)C, as a function of energy. With the exception of (4)He and (16)O, the data can be described using a universal function for the reduced cross sections.

Transition-metal 13-atom clusters assessed with solid and surface-biased functionals

First-principles density-functional theory studies have reported open structures based on the formation of double simple-cubic (DSC) arrangements for Ru(13), Rh(13), Os(13), and Ir(13), which can be considered an unexpected result as those elements crystallize in compact bulk structures such as the face-centered cubic and hexagonal close-packed lattices. In this work, we investigated with the projected augmented wave method the dependence of the lowest-energy structure on the local and semilocal exchange-correlation (xc) energy functionals employed in density-functional theory. We found that the local-density approximation (LDA) and generalized-gradient formulations with different treatment of the electronic inhomogeneities (PBE, PBEsol, and AM05) confirm the DSC configuration as the lowest-energy structure for the studied TM(13) clusters. A good agreement in the relative total energies are obtained even for structures with small energy differences, e. g., 0.10 eV. The employed xc functionals yield the same total magnetic moment for a given structure, i.e., the differences in the bond lengths do not affect the moments, which can be attributed to the atomic character of those clusters. Thus, at least for those systems, the differences among the LDA, PBE, PBEsol, and AM05 functionals are not large enough to yield qualitatively different results. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3577999]

Pseudodifferential operators with C*-algebra-valued symbols: Abstract characterizations

Given a separable unital C*-algebra C with norm parallel to center dot parallel to, let E-n denote the Banach-space completion of the C-valued Schwartz space on R-n with norm parallel to f parallel to(2)=parallel to < f, f >parallel to(1/2), < f, g >=integral f(x)* g(x)dx. The assignment of the pseudodifferential operator A=a(x,D) with C-valued symbol a(x,xi) to each smooth function with bounded derivatives a is an element of B-C(R-2n) defines an injective mapping O, from B-C(R-2n) to the set H of all operators with smooth orbit under the canonical action of the Heisenberg group on the algebra of all adjointable operators on the Hilbert module E-n. In this paper, we construct a left-inverse S for O and prove that S is injective if C is commutative. This generalizes Cordes' description of H in the scalar case. Combined with previous results of the second author, our main theorem implies that, given a skew-symmetric n x n matrix J and if C is commutative, then any A is an element of H which commutes with every pseudodifferential operator with symbol F(x+J xi), F is an element of B-C(R-n), is a pseudodifferential operator with symbol G(x - J xi), for some G is an element of B-C(R-n). That was conjectured by Rieffel.

ON SPACES OF COMPACT OPERATORS ON C(K, X) SPACES

This paper concerns the spaces of compact operators kappa(E,F), where E and F are Banach spaces C([1, xi], X) of all continuous X-valued functions defined on the interval of ordinals [1, xi] and equipped with the supremun norm. We provide sufficient conditions on X, Y, alpha, beta, xi and eta, with omega <= alpha <= beta < omega 1 for the following equivalence: (a) kappa(C([1, xi], X), C([1, alpha], Y)) is isomorphic to kappa(C([1,eta], X), C([1, beta], Y)), (b) beta < alpha(omega). In this way, we unify and extend results due to Bessaga and Pelczynski (1960) and C. Samuel (2009). Our result covers the case of the classical spaces X = l(p) and Y = l(q) with 1 < p, q < infinity.

Eddy Formation in the Tropical Atlantic Induced by Abrupt Changes in the Meridional Overturning Circulation

The variability of the meridional overturning circulation (MOC) in the upper tropical Atlantic basin is investigated using a reduced-gravity model in a simplified domain. Four sets of idealized numerical experiments are performed: (i) switch-on of the MOC until a fixed value when a constant northward flow is applied along the western boundary; (ii) MOC with a variable flow; (iii) MOC in a quasi-steady flow; and (iv) shutdown of the MOC in the Northern Hemisphere. Results from experiments (i) show that eddies are generated at the equatorial region by shear instability and detached northward; eddies are responsible for an enhancement of the mean flow and the variability of the MOC. Results from experiments (ii) show a transitional behavior of the MOC related to the eddy generation in interannual-decadal time scales as the Reynolds number varies due to the variations in the MOC. In experiments (iii), a critical Reynolds number Re(c) around 30 is found, above which eddies are generated. Experiments (iv) demonstrate that even after the collapse of MOC in the Northern Hemisphere, eddies can still be generated and carry energy across the equator into the Northern Hemisphere; these eddies act to attenuate the impact of the MOC shutdown on short time scales. The results described here may be particularly pertinent to ocean general circulation models in which the Reynolds number lies close to the bifurcation point separating the laminar and turbulent regimes.

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naive cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.

Knoop Microhardness and FT-Raman Spectroscopic Evaluation of a Resin-Based Dental Material Light-Cured by an Argon Ion Laser and Halogen Lamp: An in Vitro Study

Objective: The purpose of this study was to evaluate in vitro the Knoop microhardness (Knoop hardness number [KHN]) and the degree of conversion using FT-Raman spectroscopy of a light-cured microhybrid resin composite (Z350-3M-ESPE) Vita shade A3 photopolymerized with a halogen lamp or an argon ion laser. Background Data: Optimal polymerization of resin-based dental materials is important for longevity of restorations in dentistry. Materials and Methods: Thirty specimens were prepared and inserted into a disc-shaped polytetrafluoroethylene mold that was 2.0 mm thick and 3 mm in diameter. The specimens were divided into three groups (n = 10 each). Group 1 (G1) was light-cured for 20 sec with an Optilux 501 halogen light with an intensity of 1000 mW/cm(2). Group 2 (G2) was photopolymerized with an argon laser with a power of 150 mW for 10 sec, and group 3 (G3) was photopolymerized with an argon laser at 200 mW of power for 10 sec. All specimens were stored in distilled water for 24 h at 37 degrees C and kept in lightproof containers. For the KHN test five indentations were made and a depth of 100 mu m was maintained in each specimen. One hundred and fifty readings were obtained using a 25-g load for 45 sec. The degree of conversion values were measured by Raman spectroscopy. KHN and degree of conversion values were obtained on opposite sides of the irradiated surface. KHN and degree of conversion data were analyzed by one-way ANOVA and Tukey tests with statistical significance set at p < 0.05. Results: The results of KHN testing were G1 = 37.428 +/- 4.765; G2 = 23.588 +/- 6.269; and G3 = 21.652 +/- 4.393. The calculated degrees of conversion (DC%) were G1 = 48.57 +/- 2.11; G2 = 43.71 +/- 3.93; and G3 = 44.19 +/- 2.71. Conclusions: Polymerization with the halogen lamp ( G1) attained higher microhardness values than polymerization with the argon laser at power levels of 150 and 200 mW; there was no difference in hardness between the two argon laser groups. The results showed no statistically significant different degrees of conversion for the polymerization of composite samples with the two light sources tested.

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome c

The alternative low-spin states of Fe3+ and Fe2+ cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met 80 by another strong field ligand at the sixth heme iron coordination position, Fe3+ ALSScytc exhibited 1-nm Soret band blue shift and e enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe3+ and Fe2+ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe3+ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential similar to 200 mV lower than the wild-type protein (1220 mV) and was more susceptible to the attack of free radicals.