Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning Ache to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by Ache and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse–human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

Synthesis and characterization of Pd(II) and Pt(II) complexes with triazolopyrimidine derivatives: The crystal structure of [Pd2L2Cl4] where L=5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine

The Pd(II) and Pt(II) complexes with triazolopyrimidine C-nucleosides L-1 (5,7-dimethyl-3-(2',3',5'-tri-O-benzoyl-beta-D-ribofuranosyl-s-triazolo)[4,3-a]pyrimidine), L-2 (5,7-dimethyl-3-beta-D-ribofuranosyl-s-triazolo [4,3-a]pyrimidine) and L-3 (5,7-dimethyl[1,5-a]-s-triazolopyrimidine), [Pd(en)(L-1)](NO3)(2), (Pd(bpy)(L-1)](NO3)(2), cis-Pd(L-3)(2)Cl-2, [Pd-2(L-3)(2)Cl-4]center dot H2O, cis-Pd(L-2)(2)Cl-2 and [Pt-3(L-1)(2)Cl-6] were synthesized and characterized by elemental analysis and NMR spectroscopy. The structure of the [Pd-2(L-3)(2)Cl-4]center dot H2O complex was established by Xray crystallography. The two L-3 ligands are found in a head to tail orientation, with a (PdPd)-Pd-... distance of 3.1254(17) angstrom.L-1 coordinates to Pd(II) through N8 and N1 forming polymeric structures. L-2 coordinates to Pd(II) through N8 in acidic solutions (0.1 M HCl) forming complexes of cis-geometry. The Pd(II) coordination to L-2 does not affect the sugar conformation probably due to the high stability of the C-C glycoside bond. (c) 2006 Elsevier B.V. All rights reserved.

Feature analysis of spammers in social networks with active honeypots:a case study of Chinese microblogging networks

In this poster we presented our preliminary work on the study of spammer detection and analysis with 50 active honeypot profiles implemented on Weibo.com and QQ.com microblogging networks. We picked out spammers from legitimate users by manually checking every captured user's microblogs content. We built a spammer dataset for each social network community using these spammer accounts and a legitimate user dataset as well. We analyzed several features of the two user classes and made a comparison on these features, which were found to be useful to distinguish spammers from legitimate users. The followings are several initial observations from our analysis on the features of spammers captured on Weibo.com and QQ.com. ¦The following/follower ratio of spammers is usually higher than legitimate users. They tend to follow a large amount of users in order to gain popularity but always have relatively few followers. ¦There exists a big gap between the average numbers of microblogs posted per day from these two classes. On Weibo.com, spammers post quite a lot microblogs every day, which is much more than legitimate users do; while on QQ.com spammers post far less microblogs than legitimate users. This is mainly due to the different strategies taken by spammers on these two platforms. ¦More spammers choose a cautious spam posting pattern. They mix spam microblogs with ordinary ones so that they can avoid the anti-spam mechanisms taken by the service providers. ¦Aggressive spammers are more likely to be detected so they tend to have a shorter life while cautious spammers can live much longer and have a deeper influence on the network. The latter kind of spammers may become the trend of social network spammer. © 2012 IEEE.

Promoter effects in the polymerisation of a mixed hydrocarbon feed with silica-supported BF3

The activity of a silica-supported BF3–methanol solid acid catalyst in the cationic polymerisation of an industrial aromatic C9 feedstock has been investigated. Reuse has been achieved under continuous conditions. Titration of the catalyst acid sites with triethylphosphine oxide (TEPO) in conjunction with 31P MAS NMR shows the catalyst to have two types of acid sites. Further analysis with 2,6 di-tert-butyl-4-methylpyridine (DBMP) has revealed the majority of these acid sites to be Brønsted in nature. The role of α-methylstyrene in promoting resin polymerisation via chain transfer is proposed.

Quantitative analysis of solid-state processes studied with isothermal microcalorimetry

Quantitative analysis of solid-state processes from isothermal microcalorimetric data is straightforward if data for the total process have been recorded and problematic (in the more likely case) when they have not. Data are usually plotted as a function of fraction reacted (α); for calorimetric data, this requires knowledge of the total heat change (Q) upon completion of the process. Determination of Q is difficult in cases where the process is fast (initial data missing) or slow (final data missing). Here we introduce several mathematical methods that allow the direct calculation of Q by selection of data points when only partial data are present, based on analysis with the Pérez-Maqueda model. All methods in addition allow direct determination of the reaction mechanism descriptors m and n and from this the rate constant, k. The validity of the methods is tested with the use of simulated calorimetric data, and we introduce a graphical method for generating solid-state power-time data. The methods are then applied to the crystallization of indomethacin from a glass. All methods correctly recovered the total reaction enthalpy (16.6 J) and suggested that the crystallization followed an Avrami model. The rate constants for crystallization were determined to be 3.98 × 10-6, 4.13 × 10-6, and 3.98 × 10 -6 s-1 with methods 1, 2, and 3, respectively. © 2010 American Chemical Society.

Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.

Numerical analysis of wooden slabs with perforations under fire conditions

Wood is considered an ideal solution for floors and roofs building construction, due the mechanical and thermal properties, associated with acoustic conditions. These constructions have good sound absorption, heat insulation and relevant architectonic characteristics. They are used in many civil applications: concert and conference halls, auditoriums, ceilings, walls… However, the high vulnerability of wooden elements submitted to fire conditions requires the evaluation of its structural behaviour with accuracy. The main objective of this work is to present a numerical model to assess the fire resistance of wooden cellular slabs with different perforations. Also the thermal behaviour of the wooden slabs will be compared considering different material insulation, with different sizes, inside the cavities. A transient thermal analysis with nonlinear material behaviour will be solved using ANSYS© program. This study allows to verify the fire resistance, the temperature evolution and the char-layer, throughout a wooden cellular slab with perforations and considering the insulation effect inside the cavities.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in Sao Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Evaluation of the carbon content of aerosols from the burning of biomass in the Brazilian Amazon using thermal, optical and thermal-optical analysis methods

Aerosol samples were collected at a pasture site in the Amazon Basin as part of the project LBA-SMOCC-2002 (Large-Scale Biosphere-Atmosphere Experiment in Amazonia - Smoke Aerosols, Clouds, Rainfall and Climate: Aerosols from Biomass Burning Perturb Global and Regional Climate). Sampling was conducted during the late dry season, when the aerosol composition was dominated by biomass burning emissions, especially in the submicron fraction. A 13-stage Dekati low-pressure impactor (DLPI) was used to collect particles with nominal aerodynamic diameters (D(p)) ranging from 0.03 to 0.10 mu m. Gravimetric analyses of the DLPI substrates and filters were performed to obtain aerosol mass concentrations. The concentrations of total, apparent elemental, and organic carbon (TC, EC(a), and OC) were determined using thermal and thermal-optical analysis (TOA) methods. A light transmission method (LTM) was used to determine the concentration of equivalent black carbon (BC(e)) or the absorbing fraction at 880 nm for the size-resolved samples. During the dry period, due to the pervasive presence of fires in the region upwind of the sampling site, concentrations of fine aerosols (D(p) < 2.5 mu m: average 59.8 mu g m(-3)) were higher than coarse aerosols (D(p) > 2.5 mu m: 4.1 mu g m(-3)). Carbonaceous matter, estimated as the sum of the particulate organic matter (i.e., OC x 1.8) plus BC(e), comprised more than 90% to the total aerosol mass. Concentrations of EC(a) (estimated by thermal analysis with a correction for charring) and BC(e) (estimated by LTM) averaged 5.2 +/- 1.3 and 3.1 +/- 0.8 mu g m(-3), respectively. The determination of EC was improved by extracting water-soluble organic material from the samples, which reduced the average light absorption Angstrom exponent of particles in the size range of 0.1 to 1.0 mu m from >2.0 to approximately 1.2. The size-resolved BC(e) measured by the LTM showed a clear maximum between 0.4 and 0.6 mu m in diameter. The concentrations of OC and BC(e) varied diurnally during the dry period, and this variation is related to diurnal changes in boundary layer thickness and in fire frequency.

Non-linear boundary element formulation applied to contact analysis using tangent operator

This work presents a non-linear boundary element formulation applied to analysis of contact problems. The boundary element method (BEM) is known as a robust and accurate numerical technique to handle this type of problem, because the contact among the solids occurs along their boundaries. The proposed non-linear formulation is based on the use of singular or hyper-singular integral equations by BEM, for multi-region contact. When the contact occurs between crack surfaces, the formulation adopted is the dual version of BEM, in which singular and hyper-singular integral equations are defined along the opposite sides of the contact boundaries. The structural non-linear behaviour on the contact is considered using Coulomb`s friction law. The non-linear formulation is based on the tangent operator in which one uses the derivate of the set of algebraic equations to construct the corrections for the non-linear process. This implicit formulation has shown accurate as the classical approach, however, it is faster to compute the solution. Examples of simple and multi-region contact problems are shown to illustrate the applicability of the proposed scheme. (C) 2011 Elsevier Ltd. All rights reserved.

Dual boundary element formulation applied to analysis of multi-fractured domains

This paper deals with analysis of multiple random crack propagation in two-dimensional domains using the boundary element method (BEM). BEM is known to be a robust and accurate numerical technique for analysing this type of problem. The formulation adopted in this work is based on the dual BEM, for which singular and hyper-singular integral equations are used. We propose an iterative scheme to predict the crack growth path and the crack length increment at each time step. The proposed scheme able us to simulate localisation and coalescence phenomena, which is the main contribution of this paper. Considering the fracture mechanics analysis, the displacement correlation technique is applied to evaluate the stress intensity factors. The propagation angle and the equivalent stress intensity factor are calculated using the theory of maximum circumferential stress. Examples of simple and multi-fractured domains, loaded up to the rupture, are considered to illustrate the applicability of the proposed scheme. (C) 2010 Elsevier Ltd. All rights reserved.

Analyzing static three-dimensional elastic domains with a new infinite boundary element formulation

A new two-dimensionally mapped infinite boundary element (IBE) is presented. The formulation is based on a triangular boundary element (BE) with linear shape functions instead of the quadrilateral IBEs usually found in the literature. The infinite solids analyzed are assumed to be three-dimensional, linear-elastic and isotropic, and Kelvin fundamental solutions are employed. One advantage of the proposed formulation over quadratic or higher order elements is that no additional degrees of freedom are added to the original BE mesh by the presence of the IBEs. Thus, the IBEs allow the mesh to be reduced without compromising the accuracy of the result. Two examples are presented, in which the numerical results show good agreement with authors using quadrilateral IBEs and analytical solutions. (C) 2010 Elsevier Ltd. All rights reserved.