MediPlEx - a tool to combine in silico & experimental gene expression profiles of the model legume Medicago truncatula

Background: Expressed Sequence Tags (ESTs) are in general used to gain a first insight into gene activities from a species of interest. Subsequently, and typically based on a combination of EST and genome sequences, microarray-based expression analyses are performed for a variety of conditions. In some cases, a multitude of EST and microarray experiments are conducted for one species, covering different tissues, cell states, and cell types. Under these circumstances, the challenge arises to combine results derived from the different expression profiling strategies, with the goal to uncover novel information on the basis of the integrated datasets. Findings: Using our new analysis tool, MediPlEx (MEDIcago truncatula multiPLe EXpression analysis), expression data from EST experiments, oligonucleotide microarrays and Affymetrix GeneChips® can be combined and analyzed, leading to a novel approach to integrated transcriptome analysis. We have validated our tool via the identification of a set of well-characterized AM-specific and AM-induced marker genes, identified by MediPlEx on the basis of in silico and experimental gene expression profiles from roots colonized with AM fungi. Conclusions: MediPlEx offers an integrated analysis pipeline for different sets of expression data generated for the model legume Medicago truncatula. As expected, in silico and experimental gene expression data that cover the same biological condition correlate well. The collection of differentially expressed genes identified via MediPlEx provides a starting point for functional studies in plant mutants.

Construção de modelos de interação in silico e in vitro do inibidor do tipo Kunitz de Adenanthera pavonina L. para as enzimas cisteínicas e serínicas

Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases

Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Porównawcza analiza strukturalno-funkcjonalna in silico białek STAT i IRF, w celu identyfikacji związków chemicznych, działających jako specyficzne inhibitory funkcjonalne

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Protein expression of Myt272-3 recombinant clone and in silico prediction of a possible vaccine candidate against Mycobacterium tuberculosis

Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis . Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation & Pulldown. Protein sequence was analysed in silico using bioinformatics software for the prediction of allergenicity, antigenicity, MHC-I and MHC-II binding, and B-cell epitope binding. Results: The candidate protein was a non-allergen with 15.19 % positive predictive value. It was also predicted to be antigenic, with binding affinity to MHC-I and MHC-II, as well as B-cell epitope binding. Conclusion: The predicted results obtained in this study provide a guide for practical design of a new tuberculosis vaccine.

Análise da interação entre moléculas desenhadas a partir do ácido anacárdico e a proteína PPAR usando ferramentas in silico

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.

In silico adjuvant design and validation

Adjuvants are substances that boost the protective immune response to vaccine antigens. The majority of known adjuvants have been identified through the use of empirical approaches. Our aim was to identify novel adjuvants with well-defined cellular and molecular mechanisms by combining a knowledge of immunoregulatory mechanisms with an in silico approach. CD4 + CD25 + FoxP3 + regulatory T cells (Tregs) inhibit the protective immune responses to vaccines by suppressing the activation of antigen presenting cells such as dendritic cells (DCs). In this chapter, we describe the identification and functional validation of small molecule antagonists to CCR4, a chemokine receptor expressed on Tregs. The CCR4 binds the chemokines CCL22 and CCL17 that are produced in large amounts by activated innate cells including DCs. In silico identified small molecule CCR4 antagonists inhibited the migration of Tregs both in vitro and in vivo and when combined with vaccine antigens, significantly enhanced protective immune responses in experimental models.

Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

Estudo in silico de sítios de restrição enzimática de genes da proteína capsidial e análise de diversidade genética de Apple chlorotic leaf spot virus em fruteiras de semente e caroço.

Cultivares comerciais de macieiras são infectadas por 3 espécies principais de vírus: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) e Apple stem pitting virus (ASPV), geralmente em infecções complexas. O objetivo do estudo foi caracterizar a diversidade genética de genes da proteína capsidial (CP) de isolados de ACLSV.

Validazione di un modello In Silico di progressione dell'osteoporosi per la predizione del rischio di frattura dell'anca

Questo lavoro di tesi contribuisce allo sviluppo di una metodologia in silico, che può essere utilizzata sia come strumento clinico per la predizione del rischio di frattura, sia come strumento per la valutazione dell’efficacia di trattamenti farmacologici. Lo scopo di questo lavoro è di validare un modello di progressione dell’osteoporosi utilizzando dati clinici prospettici. Per fare ciò, è stata utilizzata una procedura già precedentemente validata da Bhattacharya et al. su una coorte retrospettica di Sheffield, con validazione effettuata unicamente per l’ARF0. In questo lavoro di tesi è stata intergata una legge di invecchiamento dell’osso e predetto il carico di rottura del femore ed il rischio di frattura fino a cinque anni successivi all’esecuzione della CT (ARF5). I risultati ottenuti sono poi stati confrontati con i dati reali, ovvero con le reali fratture che sono avvenute o meno nei pazienti reali oggetto dello studio. La validazione su una coorte prospettica ha evidenziato una discriminazione tra fratturati e non fratturati migliore rispetto a quella trovata per la coorte retrospettica. Tuttavia, l’ARF5 e l’ARF0, sui trentaquattro soggetti considerati, hanno delle prestazioni confrontabili.

Homozygous Inactivating Mutation In Nanos3 In Two Sisters With Primary Ovarian Insufficiency.

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

Analysis of anti-Müllerian hormone (AMH) and its receptor (AMHR2) genes in patients with persistent Müllerian duct syndrome

OBJECTIVE: To screen for mutations in AMH and AMHR2 genes in patients with persistent Müllerian duct syndrome (PMDS). PATIENTS AND METHOD: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. RESULTS: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A>G, and p.Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A>G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. CONCLUSION: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A>G, and p.Arg502Leu in AMH; and p.Gly323Ser in AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.