A fragment-based approach for ligand binding affinity and selectivity for the liver X receptor beta

Selective modulation of liver X receptor beta (LXR beta) has been recognized as an important approach to prevent or reverse the atherosclerotic process. In the present work, we have developed robust conformation-independent fragment-based quantitative structure-activity and structure-selectivity relationship models for a series of quinolines and cinnolines as potent modulators of the two LXR sub-types. The generated models were then used to predict the potency of an external test set and the predicted values were in good agreement with the experimental results, indicating the potential of the models for untested compounds. The final 2D molecular recognition patterns obtained were integrated to 3D structure-based molecular modeling studies to provide useful insights into the chemical and structural determinants for increased LXR beta binding affinity and selectivity. (C) 2011 Elsevier Inc. All rights reserved.

The nuclear electric quadrupole moment of hafnium from the molecular method

The molecular method is used to obtain nuclear electric quadrupole moment (NQM) values for hafnium through electric field gradients (EFGs) at this nucleus in HfO and HfS. Dirac-Coulomb calculations with the Coupled Cluster approach, DC-CCSD (T) and DC-CCSD-T, were carried out to achieve the most accurate estimates of these EFGs. Higher order corrections are also added. Hence, the most reliable values for 177Hf and 179Hf determined here are 3319(33) and 3750(37) mbarn, respectively, in nice accordance with the best currently accepted NQMs for this element. (C) 2012 Elsevier B.V. All rights reserved.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

A rigorous approach to facilitate and guarantee the correctness of the genetic testing management in human genome information systems

Abstract Background Recent medical and biological technology advances have stimulated the development of new testing systems that have been providing huge, varied amounts of molecular and clinical data. Growing data volumes pose significant challenges for information processing systems in research centers. Additionally, the routines of genomics laboratory are typically characterized by high parallelism in testing and constant procedure changes. Results This paper describes a formal approach to address this challenge through the implementation of a genetic testing management system applied to human genome laboratory. We introduced the Human Genome Research Center Information System (CEGH) in Brazil, a system that is able to support constant changes in human genome testing and can provide patients updated results based on the most recent and validated genetic knowledge. Our approach uses a common repository for process planning to ensure reusability, specification, instantiation, monitoring, and execution of processes, which are defined using a relational database and rigorous control flow specifications based on process algebra (ACP). The main difference between our approach and related works is that we were able to join two important aspects: 1) process scalability achieved through relational database implementation, and 2) correctness of processes using process algebra. Furthermore, the software allows end users to define genetic testing without requiring any knowledge about business process notation or process algebra. Conclusions This paper presents the CEGH information system that is a Laboratory Information Management System (LIMS) based on a formal framework to support genetic testing management for Mendelian disorder studies. We have proved the feasibility and showed usability benefits of a rigorous approach that is able to specify, validate, and perform genetic testing using easy end user interfaces.

A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

Abstract Background The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup. Methods Larvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches. Results Bayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach. Conclusions Our study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.

Molecular phylogeny, morphology and their implications for the taxonomy of Eriocaulaceae

The pantropical family Eriocaulaceae includes ten genera and c. 1,400 species, with diversity concentrated in the New World. The last complete revision of the family was published more than 100 years ago, and until recently the generic and infrageneric relationships were poorly resolved. However, a multi-disciplinary approach over the last 30 years, using morphological and anatomical characters, has been supplemented with additional data from palynology, chemistry, embryology, population genetics, cytology and, more recently, molecular phylogenetic studies. This led to a reassessment of phylogenetic relationships within the family. In this paper we present new data for the ITS and trnL-F regions, analysed separately and in combination, using maximum parsimony and Bayesian inference. The data confirm previous results, and show that many characters traditionally used for differentiating and circumscribing the genera within the family are homoplasious. A new generic key with characters from various sources and reflecting the current taxonomic changes is presented.

Applying many-body expansion aiming to make ab in initio molecular dynamics more efficient

In this present work we present a methodology that aims to apply the many-body expansion to decrease the computational cost of ab initio molecular dynamics, keeping acceptable accuracy on the results. We implemented this methodology in a program which we called ManBo. In the many-body expansion approach, we partitioned the total energy E of the system in contributions of one body, two bodies, three bodies, etc., until the contribution of the Nth body [1-3]: E = E1 + E2 + E3 + …EN. The E1 term is the sum of the internal energy of the molecules; the term E2 is the energy due to interaction between all pairs of molecules; E3 is the energy due to interaction between all trios of molecules; and so on. In Manbo we chose to truncate the expansion in the contribution of two or three bodies, both for the calculation of the energy and for the calculation of the atomic forces. In order to partially include the many-body interactions neglected when we truncate the expansion, we can include an electrostatic embedding in the electronic structure calculations, instead of considering the monomers, pairs and trios as isolated molecules in space. In simulations we made we chose to simulate water molecules, and use the Gaussian 09 as external program to calculate the atomic forces and energy of the system, as well as reference program for analyzing the accuracy of the results obtained with the ManBo. The results show that the use of the many-body expansion seems to be an interesting approach for reducing the still prohibitive computational cost of ab initio molecular dynamics. The errors introduced on atomic forces in applying such methodology are very small. The inclusion of an embedding electrostatic seems to be a good solution for improving the results with only a small increase in simulation time. As we increase the level of calculation, the simulation time of ManBo tends to largely decrease in relation to a conventional BOMD simulation of Gaussian, due to better scalability of the methodology presented. References [1] E. E. Dahlke and D. G. Truhlar; J. Chem. Theory Comput., 3, 46 (2007). [2] E. E. Dahlke and D. G. Truhlar; J. Chem. Theory Comput., 4, 1 (2008). [3] R. Rivelino, P. Chaudhuri and S. Canuto; J. Chem. Phys., 118, 10593 (2003).

Synthetic biology: a novel approach for the construction of industrial microorganisms

The recent achievement of synthesising a functioning bacterial chromosome marks a coming of age for engineering living organisms. In the future this should allow the construction of novel organisms to help solve the problems facing the human race, including health care, food, energy and environmental protection. In this minireview, the current state of the field is described and the role of synthetic biology in biotechnology in the short and medium term is discussed. It is particularly aimed at the needs of food technologists, nutritionists and other biotechnologists, who might not be aware of the potential significance of synthetic biology to the research and development in their fields. The potential of synthetic biology to produce interesting new polyketide compounds is discussed in detail.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

Towards the generation of photoactive nanomaterials: a supramolecular approach

Objective of these four first chapters is to have a complete understanding of the supramolecular organisation of several complementary modules able to form 2-D networks first in solution using optical spectroscopy measurements as function of solvent polarity , concentration and temperature, and then on solid surface using microscopy techniques such as STM, AFM and TEM. The last chapter presents another type of supramolecular material for application in solar cells technology involving fullerenes and OPV systems. We describes the photoinduced energy and electron process using transient absorption experiments. All these systems provide an exceptional example for the potential of the supramolecular approach as an alternative to the restricted lithographic method for the fabrication of adressable molecular devices.

Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

Researches on berry composition in red grape: agronomical, biochemical and molecular approaches

It is well known that the best grape quality can occur only through the achievement of optimal source/sink ratio. Vine balance is in fact a key parameter in controlling berry sugar, acidity and secondary metabolites content (Howell, 2001; Vanden Heuvel et al., 2004). Despite yield reduction and quality improvement are not always strictly related, cluster thinning is considered a technique which could lead to improvement in grape sugar and anthocyanin composition (Dokoozlian and Hirschfelt, 1995; Guidoni et al., 2002). Among several microclimatic variables which may impact grape composition, the effect of cluster light exposure and temperature, which probably act in synergistic and complex way, has been widely explored showing positive even sometimes contradictory results (Spayd et al., 2001; Tarara et al., 2008). Pre-bloom and véraison defoliation are very efficient techniques in inducing cluster microclimatic modification. Furthermore pre-bloom defoliation inducing a lower berry set percentage On these basis the aim of the first experiment of the thesis was to verify in cv Sangiovese the effects on ripening and berry composition of management techniques which may increase source/sink ratio and /or promote light incidence on berries throughout grape ripening. An integrated agronomic, biochemical and microarray approach, aims to understand which mechanisms are involved in berry composition and may be conditioned in the berries during ripening in vines submitted to three treatments. In particular the treatments compared were: a) cluster thinning (increasing in source/sink ratio) b) leaf removal at véraison (increasing cluster light exposure) c) pre-bloom defoliation (increasing source sink ratio and cluster light exposure). Vine response to leaf removal at véraison was further evaluated in the second experiment on three different varieties (Cabernet Sauvignon, Nero d’Avola, Raboso Piave) chosen for their different genetic traits in terms of anthocyanin amount and composition. The integrated agronomic, biochemical and microarray approach, employed in order to understand those mechanisms involved in berry composition of Sangiovese vines submitted to management techniques which may increase source/sink ratio and induce microclimatic changes, bring to interesting results. This research confirmed the main role of source/sink ratio in conditioning sugars metabolism and revealed also that carbohydrates availability is a crucial issue in triggering anthocyanin biosynthesis. More complex is the situation of pre-bloom defoliation, where source/sink and cluster light increase effects are associated to determine final berry composition. It results that the application of pre-bloom defoliation may be risky, as too much dependent on seasonal conditions (rain and temperature) and physiological vine response (leaf area recovery, photosynthetic compensation, laterals regrowth). Early induced stress conditions could bring cluster at véraison in disadvantage to trigger optimal berry ripening processes compared to untreated vines. This conditions could be maintained until harvest, if no previously described physiological recovery occurs. Certainly, light exposure increase linked to defoliation treatments, showed a positive and solid effect on flavonol biosynthesis, as in our conditions temperature was not so different among treatments. Except the last aspects, that could be confirmed also for véraison defoliation, microclimatic changes by themselves seemed not able to induce any modification in berry composition. Further studies are necessary to understand if the peculiar anthocyanic and flavonols composition detected in véraison defoliation could play important role in both color intensity and stability of wines.

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

Bioremediation implies the use of living organisms, primarily microorganisms, to convert environmental contaminants into less toxic forms. The impact of the consequences of hydrocarbon release in the environment maintain a high research interest in the study of microbial metabolisms associated with the biodegradation of aromatic and aliphatic hydrocarbons but also in the analysis of microbial enzymes that can convert petroleum substrates to value-added products. The studies described in this Thesis fall within the research field that directs the efforts into identifying gene/proteins involved in the catabolism of n-alkanes and into studying the regulatory mechanisms leading to their oxidation. In particular the studies were aimed at investigating the molecular aspects of the ability of Rhodococcus sp. BCP1 to grow on aliphatic hydrocarbons as sole carbon and energy sources. We studied the ability of Rhodococcus sp. BCP1 to grow on gaseous (C2-C4), liquid (C5-C16) and solid (C17-C28) n-alkanes that resulted to be biochemically correlated with the activity of one or more monooxygenases. In order to identify the alkane monooxygenase that is involved in the n-alkanes degradation pathway in Rhodococcus sp. BCP1, PCR-based methodology was applied by using degenerate primers targeting AlkB monooxygenase family members. As result, a chromosomal region, including the alkB gene cluster, was cloned from Rhodococcus sp. BCP1 genome. We characterized the products of this alkB gene cluster and the products of the orfs included in the flanking regions by comparative analysis with the homologues in the database. alkB gene expression studies were carried out by RT-PCR and by the construction of a promoter probe vector containing the lacZ gene downstream of the alkB promoter. B-galactosidase assays revealed the alkB promoter activity induced by n-alkanes and by n-alkanes metabolic products. Furthermore, the transcriptional start of alkB gene was determined by primer extension procedure. A proteomic approach was subsequently applied to compare the protein patterns expressed by BCP1 growing on n-butane, n-hexane, n-hexadecane or n-eicosane with the protein pattern expressed by BCP1 growing on succinate. The accumulation of enzymes specifically induced on n-alkanes was determined. These enzymes were identified by tandem mass spectrometry (LC/MS/MS). Finally, a prm gene, homologue to the gene family coding for soluble di-iron monooxygenases (SDIMOs), has been isolated from Rhodococcus sp. BCP1 genome. This gene product could be involved in the degradation of gaseous n-alkanes in this Rhodococcus strain. The versatility in utilizing hydrocarbons and the discovery of new remarkable metabolic activities outline the potential applications of this microorganism in environmental and industrial biotechnologies.

Development of synthetic molecular circuits for the control of cell systems

Synthetic biology is a young field of applicative research aiming to design and build up artificial biological devices, useful for human applications. How synthetic biology emerged in past years and how the development of the Registry of Standard Biological Parts aimed to introduce one practical starting solution to apply the basics of engineering to molecular biology is presented in chapter 1 in the thesis The same chapter recalls how biological parts can make up a genetic program, the molecular cloning tecnique useful for this purpose, and an overview of the mathematical modeling adopted to describe gene circuit behavior. Although the design of gene circuits has become feasible the increasing complexity of gene networks asks for a rational approach to design gene circuits. A bottom-up approach was proposed, suggesting that the behavior of a complicated system can be predicted from the features of its parts. The option to use modular parts in large-scale networks will be facilitated by a detailed and shared characterization of their functional properties. Such a prediction, requires well-characterized mathematical models of the parts and of how they behave when assembled together. In chapter 2, the feasibility of the bottom-up approach in the design of a synthetic program in Escherichia coli bacterial cells is described. The rational design of gene networks is however far from being established. The synthetic biology approach can used the mathematical formalism to identify biological information not assessable with experimental measurements. In this context, chapter 3 describes the design of a synthetic sensor for identifying molecules of interest inside eukaryotic cells. The Registry of Standard parts collects standard and modular biological parts. To spread the use of BioBricks the iGEM competition was started. The ICM Laboratory, where Francesca Ceroni completed her Ph.D, partecipated with teams of students and Chapter 4 summarizes the projects developed.

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes -12s and 16s -, and two protein coding ones - cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogeny. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses.