Live neighbor-joining.

BACKGROUND: In phylogenetic reconstruction the result is a tree where all taxa are leaves and internal nodes are hypothetical ancestors. In a live phylogeny, both ancestral and living taxa may coexist, leading to a tree where internal nodes may be living taxa. The well-known Neighbor-Joining heuristic is largely used for phylogenetic reconstruction. RESULTS: We present Live Neighbor-Joining, a heuristic for building a live phylogeny. We have investigated Live Neighbor-Joining on datasets of viral genomes, a plausible scenario for its application, which allowed the construction of alternative hypothesis for the relationships among virus that embrace both ancestral and descending taxa. We also applied Live Neighbor-Joining on a set of bacterial genomes and to sets of images and texts. Non-biological data may be better explored visually when their relationship in terms of content similarity is represented by means of a phylogeny. CONCLUSION: Our experiments have shown interesting alternative phylogenetic hypothesis for RNA virus genomes, bacterial genomes and alternative relationships among images and texts, illustrating a wide range of scenarios where Live Neighbor-Joining may be used.

A Support Vector Machine based method to distinguish long non-coding RNAs from protein coding transcripts.

BACKGROUND: In recent years, a rapidly increasing number of RNA transcripts has been generated by thousands of sequencing projects around the world, creating enormous volumes of transcript data to be analyzed. An important problem to be addressed when analyzing this data is distinguishing between long non-coding RNAs (lncRNAs) and protein coding transcripts (PCTs). Thus, we present a Support Vector Machine (SVM) based method to distinguish lncRNAs from PCTs, using features based on frequencies of nucleotide patterns and ORF lengths, in transcripts. METHODS: The proposed method is based on SVM and uses the first ORF relative length and frequencies of nucleotide patterns selected by PCA as features. FASTA files were used as input to calculate all possible features. These features were divided in two sets: (i) 336 frequencies of nucleotide patterns; and (ii) 4 features derived from ORFs. PCA were applied to the first set to identify 6 groups of frequencies that could most contribute to the distinction. Twenty-four experiments using the 6 groups from the first set and the features from the second set where built to create the best model to distinguish lncRNAs from PCTs. RESULTS: This method was trained and tested with human (Homo sapiens), mouse (Mus musculus) and zebrafish (Danio rerio) data, achieving 98.21%, 98.03% and 96.09%, accuracy, respectively. Our method was compared to other tools available in the literature (CPAT, CPC, iSeeRNA, lncRNApred, lncRScan-SVM and FEELnc), and showed an improvement in accuracy by ≈3.00%. In addition, to validate our model, the mouse data was classified with the human model, and vice-versa, achieving ≈97.80% accuracy in both cases, showing that the model is not overfit. The SVM models were validated with data from rat (Rattus norvegicus), pig (Sus scrofa) and fruit fly (Drosophila melanogaster), and obtained more than 84.00% accuracy in all these organisms. Our results also showed that 81.2% of human pseudogenes and 91.7% of mouse pseudogenes were classified as non-coding. Moreover, our method was capable of re-annotating two uncharacterized sequences of Swiss-Prot database with high probability of being lncRNAs. Finally, in order to use the method to annotate transcripts derived from RNA-seq, previously identified lncRNAs of human, gorilla (Gorilla gorilla) and rhesus macaque (Macaca mulatta) were analyzed, having successfully classified 98.62%, 80.8% and 91.9%, respectively. CONCLUSIONS: The SVM method proposed in this work presents high performance to distinguish lncRNAs from PCTs, as shown in the results. To build the model, besides using features known in the literature regarding ORFs, we used PCA to identify features among nucleotide pattern frequencies that contribute the most in distinguishing lncRNAs from PCTs, in reference data sets. Interestingly, models created with two evolutionary distant species could distinguish lncRNAs of even more distant species.

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a ß-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

The First Contact of Human Dendritic Cells With Trypanosoma cruzi Reveals Response to Virus as an Unexplored Central Pathway.

Chagas disease is a debilitating and neglected disease caused by the protozoan Trypanosoma cruzi. Soon after infection, interactions among T. cruzi and host innate immunity cells can drive/contribute to disease outcome. Dendritic cells (DCs), present in all tissues, are one of the first immune cells to interact with Trypanosoma cruzi metacyclic trypomastigotes. Elucidating the immunological events triggered immediately after parasite-human DCs encounter may aid in understanding the role of DCs in the establishment of infection and in the course of the disease. Therefore, we performed a transcriptomic analysis of a 12 h interaction between T. cruzi and MoDCs (monocyte-derived DCs) from three human donors. Enrichment analyses of the 468 differentially expressed genes (DEGs) revealed viral infection response as the most regulated pathway. Additionally, exogenous antigen processing and presentation through MHC-I, chemokine signaling, lymphocyte co-stimulation, metallothioneins, and inflammasome activation were found up-regulated. Notable, we were able to identify the increased gene expression of alternative inflammasome sensors such as AIM2, IFI16, and RIG-I for the first time in a T. cruzi infection. Both transcript and protein expression levels suggest proinflammatory cytokine production during early T. cruzi-DCs contact. Our transcriptome data unveil antiviral pathways as an unexplored process during T. cruzi-DC initial interaction, disclosing a new panorama for the study of Chagas disease outcomes.

Selection of Antibody Fragments for CAR-T Cell Therapy from Phage Display Libraries.

CAR-T cell therapy emerged in the last years as a great promise to cancer treatment. Nowadays, there is a run to improve the breadth of its use, and thus, new chimeric antigen receptors (CAR) are being proposed. The antigen-binding counterpart of CAR is an antibody fragment, scFv (single chain variable fragment), that recognizes a membrane protein associated to a cancer cell. In this chapter, the use of human scFv phage display libraries as a source of new mAbs against surface antigen is discussed. Protocols focusing in the use of extracellular domains of surface protein in biotinylated format are proposed as selection antigen. Elution with unlabeled peptide and selection in solution is described. The analysis of enriched scFvs throughout the selection using NGS is also outlined. Taken together these protocols allow for the isolation of new scFvs able to be useful in the construction of new chimeric antigen receptors for application in cancer therapy.

Isolation of osteosarcoma-associated human antibodies from a combinatorial Fab phage display library.

Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain) and Vkappa (kappa chain variable domain) regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

Expression of functional recombinant human factor IX in milk of mice.

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk beta-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.

Early transcriptional response of Paracoccidioides brasiliensis upon internalization by murine macrophages.

Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.

HIV type 1 genetic variability in central Brazil.

This study analyzed the genes pol and env to determine the genetic variability of HIV-1 in Central Brazil. Forty-one isolates of HIV-1-infected individuals had protease, reverse transcriptase, and C2C3/ env amplified by nested PCR and sequenced. The subtype was determined by the program REGA and phylogenetic analyses. The samples identified as putative recombinant forms were analyzed by SimPlot. A high prevalence of subtype B (95.1%) was observed, followed by mosaic viruses B/F (4.9%). The amino acid sequences from 30 HIV-1 isolates were analyzed for the antigenic intrasubtype diversity. The most prevalent gp120 V3 loop motif was the GPGR (United States/Europe) (43.3%), described in B and F subtypes, followed by the GPGK tetrapeptide (10%). The Brazilian variant B" (GWGR), GFGR, and GLGR tetrapeptides were found in 6.7%. Other V3 variants were found in eight isolates (26.7%). Phylogenetic tree analysis was also performed in order to verify the relationship of the HIV-1 samples from Central Brazil with other HIV-1 sequences that circulate in Brazil. The subtype B sequences from Central Brazil formed a polyphyletic cluster in the tree, indicating that these strains are similar to those from other geographic regions. These results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells.

BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

Overview and perspectives the transcriptome of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.

Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients.

Osteosarcoma is the commonest type of primary malignant bone tumor, frequently found in adolescents at sites of rapid bone growth. Despite current management protocols, up to half of the patients succumb to this disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. Since phage display methodology allows the selection of human antibody fragments with potential use in clinical applications, we applied this procedure to construct a recombinant Fab (antigen binding fragment) library from patients with osteosarcoma. We used peripheral blood lymphocyte total RNA from 11 osteosarcoma patients and cloned recombinant Fab representing the micro, gamma and kappa chain antibody repertoires of these individuals. The resulting library was cloned in the pComb3X vector and attained 1.45 x 10(8) different functional forms. BstO I fingerprinting and DNA sequencing analysis of randomly selected clones revealed the diversity of the library, demonstrating that Fab harbors Vkappa chains from subgroups I to V, biased towards the A27 fragment, as normally reported for the human repertoire. Analysis of the VH repertoire revealed that our library has a slight bias towards the VH4 family, instead of the usually reported VH3. This is the first description of a phage display library from osteosarcoma patients. We believe these human Fab fragments will provide a valuable tool for the study of this neoplasia and could also contribute to improvements in the diagnosis of this disease.

Bioinformatics of the Paracoccidioides brasiliensis EST Project.

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project--PbGenome Project was developed to study the infection process by analyzing expressed sequence tags--ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible.

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

Knowledge-based reasoning to annotate noncoding RNA using multi-agent system.

Noncoding RNAs (ncRNAs) have been focus of intense research over the last few years. Since characteristics and signals of ncRNAs are not entirely known, researchers use different computational tools together with their biological knowledge to predict putative ncRNAs. In this context, this work presents ncRNA-Agents, a multi-agent system to annotate ncRNAs based on the output of different tools, using inference rules to simulate biologists' reasoning. Experiments with data from the fungus Saccharomyces cerevisiae allowed to measure the performance of ncRNA-Agents, with better sensibility, when compared to Infernal, a widely used tool for annotating ncRNA. Besides, data of the Schizosaccharomyces pombe and Paracoccidioides brasiliensis fungi identified novel putative ncRNAs, which demonstrated the usefulness of our approach. NcRNA-Agents can be be found at: http://www.biomol.unb.br/ncrna-agents.

Draft Genome Sequence of FT9, a Novel Bacillus cereus Strain Isolated from a Brazilian Thermal Spring.

A Bacillus cereus strain, FT9, isolated from a hot spring in the midwest region of Brazil, had its entire genome sequenced.

Clustering rfam 10.1: clans, families, and classes.

The Rfam database contains information about non-coding RNAs emphasizing their secondary structures and organizing them into families of homologous RNA genes or functional RNA elements. Recently, a higher order organization of Rfam in terms of the so-called clans was proposed along with its "decimal release". In this proposition, some of the families have been assigned to clans based on experimental and computational data in order to find related families. In the present work we investigate an alternative classification for the RNA families based on tree edit distance. The resulting clustering recovers some of the Rfam clans. The majority of clans, however, are not recovered by the structural clustering. Instead, they get dispersed into larger clusters, which correspond roughly to well-described RNA classes such as snoRNAs, miRNAs, and CRISPRs. In conclusion, a structure-based clustering can contribute to the elucidation of the relationships among the Rfam families beyond the realm of clans and classes.

Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.