Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Covariant Gauge on the Lattice: A New Implementation

We derive a new implementation of linear covariant gauges on the lattice, based on a minimizing functional that can be interpreted as the Hamiltonian of a spin-glass model in a random external magnetic field. We show that our method solves most problems encountered in earlier implementations, mostly related to the no-go condition formulated by Giusti [Nucl. Phys. B498, 331 (1997)]. We carry out tests in the SU(2) case in four space-time dimensions. We also present preliminary results for the transverse gluon propagator at different values of the gauge parameter xi.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.

Constraints on the infrared behavior of the ghost propagator in Yang-Mills theories

We present rigorous upper and lower bounds for the momentum-space ghost propagator G(p) of Yang-Mills theories in terms of the smallest nonzero eigenvalue (and of the corresponding eigenvector) of the Faddeev-Popov matrix. We apply our analysis to data from simulations of SU(2) lattice gauge theory in Landau gauge, using the largest lattice sizes to date. Our results suggest that, in three and in four space-time dimensions, the Landau gauge ghost propagator is not enhanced as compared to its tree-level behavior. This is also seen in plots and fits of the ghost dressing function. In the two-dimensional case, on the other hand, we find that G(p) diverges as p(-2-2 kappa) with kappa approximate to 0.15, in agreement with A. Maas, Phys. Rev. D 75, 116004 (2007). We note that our discussion is general, although we make an application only to pure gauge theory in Landau gauge. Our simulations have been performed on the IBM supercomputer at the University of Sao Paulo.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

Microstructuration induced differences in the thermo-optical and luminescence properties of Nd:YAG fine grain ceramics and crystals

The temperature and compositional dependences of thermo- optical properties of neodymium doped yttrium aluminum garnet (YAG) crystals and fine grain ceramics have been systematically investigated by means of time- resolved thermal lens spectrometry. We have found that Nd:YAG ceramics show a reduced thermal diffusivity compared to Nd:YAG single crystals in the complete temperature range investigated (80-300 K). The analysis of the time- resolved luminescent properties of Nd(3+) has revealed that the reduction in the phonon mean free path taking place in Nd:YAG ceramics cannot be associated with an increment in the density of lattice defects, indicating that phonon scattering at grain boundaries is the origin of the observed reduction in the thermal diffusivity of Nd: YAG ceramics. Finally, our results showed the ability of the time- resolved thermal lens to determine and optimize the thermo- optical properties of Nd: YAG ceramic based lasers. (C) 2008 American Institute of Physics. [DOI: 10.1063/1.2975335]

Constraints on the infrared behavior of the gluon propagator in Yang-Mills theories

We present rigorous upper and lower bounds for the zero-momentum gluon propagator D(0) of Yang-Mills theories in terms of the average value of the gluon field. This allows us to perform a controlled extrapolation of lattice data to infinite volume, showing that the infrared limit of the Landau-gauge gluon propagator in SU(2) gauge theory is finite and nonzero in three and in four space-time dimensions. In the two-dimensional case, we find D(0)=0, in agreement with Maas. We suggest an explanation for these results. We note that our discussion is general, although we apply our analysis only to pure gauge theory in the Landau gauge. Simulations have been performed on the IBM supercomputer at the University of Sao Paulo.

Regional scale analysis of landform configuration with base-level (isobase) maps

Base-level maps (or ""isobase maps"", as originally defined by Filosofov, 1960), express a relationship between valley order and topography. The base-level map can be seen as a ""simplified"" version of the original topographic surface, from which the ""noise"" of the low-order stream erosion was removed. This method is able to identify areas with possible tectonic influence even within lithologically uniform domains. Base-level maps have been recently applied in semi-detail scale (e.g., 1:50 000 or larger) morphotectonic analysis. In this paper, we present an evaluation of the method's applicability in regional-scale analysis (e.g., 1:250 000 or smaller). A test area was selected in northern Brazil, at the lower course of the Araguaia and Tocantins rivers. The drainage network extracted from SRTM30_PLUS DEMs with spatial resolution of approximately 900 m was visually compared with available topographic maps and considered to be compatible with a 1:1,000 000 scale. Regarding the interpretation of regional-scale morphostructures, the map constructed with 2nd and 3rd-order valleys was considered to present the best results. Some of the interpreted base-level anomalies correspond to important shear zones and geological contacts present in the 1:5 000 000 Geological Map of South America. Others have no correspondence with mapped Precambrian structures and are considered to represent younger, probably neotectonic, features. A strong E-W orientation of the base-level lines over the inflexion of the Araguaia and Tocantins rivers, suggest a major drainage capture. A N-S topographic swath profile over the Tocantins and Araguaia rivers reveals a topographic pattern which, allied with seismic data showing a roughly N-S direction of extension in the area, lead us to interpret this lineament as an E-W, southward-dipping normal fault. There is also a good visual correspondence between the base-level lineaments and geophysical anomalies. A NW-SE lineament in the southeast of the study area partially corresponds to the northern border of the Mosquito lava field, of Jurassic age, and a NW-SE lineament traced in the northeastern sector of the study area can be interpreted as the Picos-Santa Ines lineament, identifiable in geophysical maps but with little expression in hypsometric or topographic maps.

Gene Expression Complex Networks: Synthesis, Identification, and Analysis

Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdos-Renyi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabasi-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference method was sensitive to average degree k variation, decreasing its network recovery rate with the increase of k. The signal size was important for the inference method to get better accuracy in the network identification rate, presenting very good results with small expression profiles. However, the adopted inference method was not sensible to recognize distinct structures of interaction among genes, presenting a similar behavior when applied to different network topologies. In summary, the proposed framework, though simple, was adequate for the validation of the inferred networks by identifying some properties of the evaluated method, which can be extended to other inference methods.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Descriptions and phylogenetic significance of the fronto-lateral gland pores and dorsal lattice organs of cyprid larvae of seven species of barnacles (Cirripedia : Thoracica : Pedunculata)

The paired fronto-lateral gland pores and lattice organs (LO1, 2, 3, 4, and 5) of seven species of pedunculate barnacles belonging to two thoracican suborders, Heteralepadomorpha (family Heteralepadidae: Heteralepas sp. 1 and 2) and Lepadomorpha (families Poecilasmatidae: Poecilasma inaequilaterale and Octolasmis aymonini geryonophila and Lepadidae: Lepas pacifica, Dosima fascicularis, and Conchoderma virgatum), were investigated by scanning electron microscopy (SEM). While the fronto-lateral gland pores exhibit slight variation among species, with only L. pacifica showing a different morphology, the variations in the arrangement of LOs are phylogenetically instructive. The lattice organs in the foregoing species correspond in general to the inferred advanced type (Type C), but the distinct keel in the pore field in P. inaequilaterale and L. pacifica is reminiscent of, but not necessarily identical with the less advanced Type B. The arrangement of the anterior LOs (1-2) is rhomboidal in the two heteralepadomorph species, the two poecilasmatid species, and two of the three lepadid species, as it is in all previously and presently known lepadomorph cyprids except D. fascicularis. In this last species, they are deployed linearly along the hinge line. A linear arrangement of all the lattice organs is presumably the plesiomorphic condition for the Thoracica; an obvious exception being the pattern seen in Ibla cumingi. The arrangement of the first two pairs of posterior LOs (3-4) in O. a. geryonophila and C. virgatum differs from that of all previously described Lepadomorpha in being rhomboidal rather than aligned linearly along the hinge line. This same arrangement of LOs 3 and 4 in the two heteralepadomorph species is notable since it is not known in other thoracicans. Our results concerning variation in lattice organs of the lower Pedunculata are more or less consistent with current phylogenetic speculations and genetic information that ally Heteralepadomorpha with Lepadomorpha. Significance of this variation at lower taxonomic levels is also evident in the two similar forms of Heteralepas.

Placebo Response of Non-Pharmacological and Pharmacological Trials in Major Depression: A Systematic Review and Meta-Analysis

Background: Although meta-analyses have shown that placebo responses are large in Major Depressive Disorder (MDD) trials; the placebo response of devices such as repetitive transcranial magnetic stimulation (rTMS) has not been systematically assessed. We proposed to assess placebo responses in two categories of MDD trials: pharmacological (antidepressant drugs) and non-pharmacological (device-rTMS) trials. Methodology/Principal Findings: We performed a systematic review and meta-analysis of the literature from April 2002 to April 2008, searching MEDLINE, Cochrane, Scielo and CRISP electronic databases and reference lists from retrieved studies and conference abstracts. We used the keywords placebo and depression and escitalopram for pharmacological studies; and transcranial magnetic stimulation and depression and sham for non-pharmacological studies. All randomized, double-blinded, placebo-controlled, parallel articles on major depressive disorder were included. Forty-one studies met our inclusion criteria-29 in the rTMS arm and 12 in the escitalopram arm. We extracted the mean and standard values of depression scores in the placebo group of each study. Then, we calculated the pooled effect size for escitalopram and rTMS arm separately, using Cohen's d as the measure of effect size. We found that placebo response are large for both escitalopram (Cohen's d-random-effects model-1.48; 95% C.I. 1.26 to 1.6) and rTMS studies (0.82; 95% C.I. 0.63 to 1). Exploratory analyses show that sham response is associated with refractoriness and with the use of rTMS as an add-on therapy, but not with age, gender and sham method utilized. Conclusions/Significance: We confirmed that placebo response in MDD is large regardless of the intervention and is associated with depression refractoriness and treatment combination (add-on rTMS studies). The magnitude of the placebo response seems to be related with study population and study design rather than the intervention itself.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

LATENT RANK CHANGE DETECTION FOR ANALYSIS OF SPLICE-JUNCTION MICROARRAYS WITH NONLINEAR EFFECTS

Alternative splicing of gene transcripts greatly expands the functional capacity of the genome, and certain splice isoforms may indicate specific disease states such as cancer. Splice junction microarrays interrogate thousands of splice junctions, but data analysis is difficult and error prone because of the increased complexity compared to differential gene expression analysis. We present Rank Change Detection (RCD) as a method to identify differential splicing events based upon a straightforward probabilistic model comparing the over-or underrepresentation of two or more competing isoforms. RCD has advantages over commonly used methods because it is robust to false positive errors due to nonlinear trends in microarray measurements. Further, RCD does not depend on prior knowledge of splice isoforms, yet it takes advantage of the inherent structure of mutually exclusive junctions, and it is conceptually generalizable to other types of splicing arrays or RNA-Seq. RCD specifically identifies the biologically important cases when a splice junction becomes more or less prevalent compared to other mutually exclusive junctions. The example data is from different cell lines of glioblastoma tumors assayed with Agilent microarrays.