Integrated proteomics, phosphoproteomics and metabolomics analyses reveal similarities among giant cell granulomas of the jaws with different genetic mutations.

BACKGROUND: Giant cell granuloma of the jaws are benign osteolytic lesions of the jaws. These lesions are genetically characterized by mutually exclusive somatic mutations at TRPV4, KRAS, and FGFR1, and a fourth molecular subgroup which is wild-type for the three mutations. Irrespective of the molecular background, giant cell granulomas show MAPK/ERK activation. However, it remains unclear if these mutations lead to differences in their molecular signaling in giant cell granulomas. METHODS: Metabolomics, proteomics, and phosphoproteomics analyses were carried out in formalin-fixed paraffin-embedded samples of giant cell granuloma of the jaws. The study cohort consisted of five lesions harboring mutations in FGFR1, six in KRAS, five in TRPV4, and five that were wild-type for these mutations. RESULTS: Lesions harboring KRAS or FGFR1 mutations showed overall similar proteomics and metabolomics profiles. In all four groups, metabolic pathways showed similarity in apoptosis, cell signaling, gene expression, cell differentiation, and erythrocyte activity. Lesions harboring TRPV4 mutations showed a greater number of enriched pathways related to tissue architecture. On the other hand, the wild-type group presented increased number of enriched pathways related to protein metabolism compared to the other groups. CONCLUSION: Despite some minor differences, our results revealed an overall similar molecular profile among the groups with different mutational profile at the metabolic, proteic, and phosphopeptidic levels.

Quantitative proteomic study reveals differential expression of matricellular proteins between fibrous dysplasia and cemento-ossifying fibroma pathogenesis.

BACKGROUND: Fibrous dysplasia (FD) and cemento-ossifying fibroma (COF) are the most common gnathic fibro-osseous lesions. These diseases exhibit remarkable overlap of several clinicopathological aspects, and differential diagnosis depends on the combination of histopathological, radiographic, and clinical aspects. Their molecular landscape remains poorly characterized, and herein, we assessed their proteomic and phosphoproteomic profiles. METHODS: The quantitative differences in protein profile of FD and COF were assessed by proteomic and phosphoproteomic analyses of formalin-fixed paraffin-embedded tissue samples. Pathway enrichment analyses with differentially regulated proteins were performed. RESULTS: FD and COF exhibited differential regulation of pathways related to extracellular matrix organization, cell adhesion, and platelet and erythrocytes activities. Additionally, these lesions demonstrated distinct abundance of proteins involved in osteoblastic differentiation and tumorigenesis and differential abundance of phosphorylation of Ser61 of Yes-associated protein 1 (YAP1). CONCLUSIONS: In summary, despite the morphological similarity between these diseases, our results demonstrated that COF and DF present numerous quantitative differences in their proteomic profiles. These findings suggest that these fibro-osseous lesions trigger distinct molecular mechanisms during their pathogenesis. Moreover, some proteins identified in our analysis could serve as potential biomarkers for differential diagnosis of these diseases after further validation.

Yvis: antibody high-density alignment visualization and analysis platform with an integrated database.

As antibodies are a very important tool for diagnosis, therapy, and experimental biology, a large number of antibody structures and sequences have become available in recent years. Therefore, tools that allow the analysis, comparison, and visualization of this large amount of antibody data are crucially needed. We developed the antibody high-density alignment visualization and analysis (Yvis) platform to provide an innovative, robust and high-density data visualization of antibody sequence alignments, called Collier de Diamants. The Yvis platform also provides an integrated structural database, which is updated weekly, and many different search and filter options. This platform can help to formulate hypotheses concerning the key residues in antibody structures or interactions to improve the understanding of antibody properties. The Yvis platform is available at http://bioinfo.icb.ufmg.br/yvis/.

PEPOP 2.0: new approaches to mimic non-continuous epitopes.

BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.

Searching whole genome sequences for biochemical identification features of emerging and reemerging pathogenic Corynebacterium species.

Biochemical tests are traditionally used for bacterial identification at the species level in clinical microbiology laboratories. While biochemical profiles are generally efficient for the identification of the most important corynebacterial pathogen Corynebacterium diphtheriae, their ability to differentiate between biovars of this bacterium is still controversial. Besides, the unambiguous identification of emerging human pathogenic species of the genus Corynebacterium may be hampered by highly variable biochemical profiles commonly reported for these species, including Corynebacterium striatum, Corynebacterium amycolatum, Corynebacterium minutissimum, and Corynebacterium xerosis. In order to identify the genomic basis contributing for the biochemical variabilities observed in phenotypic identification methods of these bacteria, we combined a comprehensive literature review with a bioinformatics approach based on reconstruction of six specific biochemical reactions/pathways in 33 recently released whole genome sequences. We used data retrieved from curated databases (MetaCyc, PathoSystems Resource Integration Center (PATRIC), The SEED, TransportDB, UniProtKB) associated with homology searches by BLAST and profile Hidden Markov Models (HMMs) to detect enzymes participating in the various pathways and performed ab initio protein structure modeling and molecular docking to confirm specific results. We found a differential distribution among the various strains of genes that code for some important enzymes, such as beta-phosphoglucomutase and fructokinase, and also for individual components of carbohydrate transport systems, including the fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase (PTS) and the ribose-specific ATP-binging cassette (ABC) transporter. Horizontal gene transfer plays a role in the biochemical variability of the isolates, as some genes needed for sucrose fermentation were seen to be present in genomic islands. Noteworthy, using profile HMMs, we identified an enzyme with putative alpha-1,6-glycosidase activity only in some specific strains of C. diphtheriae and this may aid to understanding of the differential abilities to utilize glycogen and starch between the biovars.

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners.

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

Classification epitopes in groups based on their protein family.

BACKGROUND: The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. RESULTS: We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. CONCLUSIONS: We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen's protein family. We expect that these findings will help to improve current computational mapping methods based on physico-chemical due it's potential application during epitope discovery.

PnPP-19, a Synthetic and Nontoxic Peptide Designed from a Phoneutria nigriventer Toxin, Potentiates Erectile Function via NO/cGMP.

PURPOSE: We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity. MATERIALS AND METHODS: Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19. RESULTS: PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase. CONCLUSIONS: PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.

Unveiling the multifaceted landscape of N-glycosylation in antibody variable domains: Insights and implications.

N-glycosylation at the antibody variable domain has emerged as an important modification influencing antibody function. Despite its significance, information regarding its role and regulation remains limited. To address this gap, we comprehensively explored antibody structures housing N-glycosylation within the Protein Data Bank, yielding fresh insights into this intricate landscape. Our findings revealed that among 208 structures, N-glycosylation was more prevalent in human and mouse antibodies containing IGHV1-8 and IGHV2-2 germline genes, respectively. Moreover, our research highlights the potential for somatic hypermutation to introduce N-glycosylation sites by substituting polar residues (Ser or Thr) in germline variable genes with asparagine. Notably, our study underscores the prevalence of N-glycosylation in antiviral antibodies, especially anti-HIV. Besides antigen-antibody interaction, our findings suggest that N-glycosylation may impact antibody specificity, affinity, and avidity by influencing Fab dimer formation and complementary-determining region orientation. We also identified different glycan structures in HIV and SARS-CoV-2 antibody proteomic datasets, highlighting disparities from the N-glycan structures between PDB antibodies and biological repertoires further highlighting the complexity of N-glycosylation patterns. Our findings significantly enrich our understanding of the N-glycosylation's multifaceted characteristics within the antibody variable domain. Additionally, they underscore the pressing imperative for a more comprehensive characterization of its impact on antibody function.

Dynamic changes in B cell subpopulations in response to triple-negative breast cancer development.

Despite presenting a worse prognosis and being associated with highly aggressive tumors, triple-negative breast cancer (TNBC) is characterized by the higher frequency of tumor-infiltrating lymphocytes, which have been implicated in better overall survival and response to therapy. Though recent studies have reported the capacity of B lymphocytes to recognize overly-expressed normal proteins, and tumor-associated antigens, how tumor development potentially modifies B cell response is yet to be elucidated. Our findings reveal distinct effects of 4T1 and E0771 murine tumor development on B cells in secondary lymphoid organs. Notably, we observe a significant expansion of total B cells and plasma cells in the tumor-draining lymph nodes (tDLNs) as early as 7 days after tumor challenge in both murine models, whereas changes in the spleen are less pronounced. Surprisingly, within the tumor microenvironment (TME) of both models, we detect distinct B cell subpopulations, but tumor development does not appear to cause major alterations in their frequency over time. Furthermore, our investigation into B cell regulatory phenotypes highlights that the B10 Breg phenotype remains unaffected in the evaluated tissues. Most importantly, we identified an increase in CD19 + LAG-3 + cells in tDLNs of both murine models. Interestingly, although CD19 + LAG-3 + cells represent a minor subset of total B cells (< 3%) in all evaluated tissues, most of these cells exhibit elevated expression of IgD, suggesting that LAG-3 may serve as an activation marker for B cells. Corroborating with these findings, we detected distinct cell cycle and proliferation genes alongside LAG-3 analyzing scRNA-Seq data from a cohort of TNBC patients. More importantly, our study suggests that the presence of LAG-3 B cells in breast tumors could be associated with a good prognosis, as patients with higher levels of LAG-3 B cell transcripts had a longer progression-free interval (PFI). This novel insight could pave the way for targeted therapies that harness the unique properties of LAG-3 + B cells, potentially offering new avenues for improving patient outcomes in TNBC. Further research is warranted to unravel the mechanistic pathways of these cells and to validate their prognostic value in larger, diverse patient cohorts.

Yclon: Ultrafast clustering of B cell clones from high-throughput immunoglobulin repertoire sequencing data.

MOTIVATION: The next-generation sequencing technologies have transformed our understanding of immunoglobulin (Ig) profiles in various immune states. Clonotyping, which groups Ig sequences into B cell clones, is crucial in investigating the diversity of repertoires and changes in antigen exposure. Despite its importance, there is no widely accepted method for clonotyping, and existing methods are computationally intensive for large sequencing datasets. RESULTS: To address this challenge, we introduce YClon, a fast and efficient approach for clonotyping Ig repertoire data. YClon uses a hierarchical clustering approach, similar to other methods, to group Ig sequences into B cell clones in a highly sensitive and specific manner. Notably, our approach outperforms other methods by being more than 30 to 5000 times faster in processing the repertoires analyzed. Astonishingly, YClon can effortlessly handle up to 2 million Ig sequences on a standard laptop computer. This enables in-depth analysis of large and numerous antibody repertoires. AVAILABILITY AND IMPLEMENTATION: YClon was implemented in Python3 and is freely available on GitHub.

The C-terminal mutation beyond the catalytic site of brown spider phospholipase D significantly impacts its biological activities.

Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with ß-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.

BioSin UFMG Club: Engaging a multilevel and multidisciplinary community in synthetic biology.

Learning synthetic biology is often seen as a far distant possibility, restricted to those who have the privilege of an academic career. We propose a student-centered discussion group around synthetic biology, aimed at people from high school onwards with different backgrounds to interact and learn about synthetic biology. We developed a 14-week long program with three modules: "Leveling," "Introducing," and "Discussion." By completing the first two modules, the members should be more comfortable with biological names, structures, concepts, and techniques. The modules developed are available in Portuguese, Spanish, and English via the Open Lab Idea Real website (https://ideareal.org/clube-de-biologia-sintetica/) and can be used to implement the Club either in place or virtually around the world. We put it to practice at Universidade Federal de Minas Gerais (UFMG) creating the Club named BioSin. There are programs such as the International Genetically Engineered Machine (iGEM) competition focused on disseminating synthetic biology. Although iGEM is one fantastic way of learning about synthetic biology, there is a high cost. Because of that, a study and discussion Club is a tool to spread knowledge and engage with the study area.

Overview of protein posttranslational modifications in Arthropoda venoms.

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation.

Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.

The major role of junctional diversity in the horse antibody repertoire.

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.

Thromboelastometry demonstrates endogenous coagulation activation in nonsevere and severe COVID-19 patients and has applicability as a decision algorithm for intervention.

In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.

Computing biological functions using BioPsi, a formal description of biological processes based on elementary bricks of actions.

MOTIVATION: In the available databases, biological processes are described from molecular and cellular points of view, but these descriptions are represented with text annotations that make it difficult to handle them for computation. Consequently, there is an obvious need for formal descriptions of biological processes. RESULTS: We present a formalism that uses the BioPsi concepts to model biological processes from molecular details to networks. This computational approach, based on elementary bricks of actions, allows us to calculate on biological functions (e.g. process comparison, mapping structure-function relationships, etc.). We illustrate its application with two examples: the functional comparison of proteases and the functional description of the glycolysis network. This computational approach is compatible with detailed biological knowledge and can be applied to different kinds of systems of simulation. AVAILABILITY: www.sysdiag.cnrs.fr/publications/supplementary-materials/BioPsi_Manager/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BioNetCAD: design, simulation and experimental validation of synthetic biochemical networks.

MOTIVATION: Synthetic biology studies how to design and construct biological systems with functions that do not exist in nature. Biochemical networks, although easier to control, have been used less frequently than genetic networks as a base to build a synthetic system. To date, no clear engineering principles exist to design such cell-free biochemical networks. RESULTS: We describe a methodology for the construction of synthetic biochemical networks based on three main steps: design, simulation and experimental validation. We developed BioNetCAD to help users to go through these steps. BioNetCAD allows designing abstract networks that can be implemented thanks to CompuBioTicDB, a database of parts for synthetic biology. BioNetCAD enables also simulations with the HSim software and the classical Ordinary Differential Equations (ODE). We demonstrate with a case study that BioNetCAD can rationalize and reduce further experimental validation during the construction of a biochemical network. AVAILABILITY AND IMPLEMENTATION: BioNetCAD is freely available at http://www.sysdiag.cnrs.fr/BioNetCAD. It is implemented in Java and supported on MS Windows. CompuBioTicDB is freely accessible at http://compubiotic.sysdiag.cnrs.fr/.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.