Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson's disease. Currently, Parkinson's disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography-IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and -80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

Biomechanical Assessment of Bicuspid Aortic Valve Phenotypes: A Fluid-Structure Interaction Modelling Approach.

PURPOSE: Bicuspid aortic valve (BAV) is a congenital heart malformation with phenotypic heterogeneity. There is no prior computational study that assesses the haemodynamic and valve mechanics associated with BAV type 2 against a healthy tricuspid aortic valve (TAV) and other BAV categories. METHODS: A proof-of-concept study incorporating three-dimensional fluid-structure interaction (FSI) models with idealised geometries (one TAV and six BAVs, namely type 0 with lateral and anterior-posterior orientations, type 1 with R-L, N-R and N-L leaflet fusion and type 2) has been developed. Transient physiological boundary conditions have been applied and simulations were run using an Arbitrary Lagrangian-Eulerian formulation. RESULTS: Our results showed the presence of abnormal haemodynamics in the aorta and abnormal valve mechanics: type 0 BAVs yielded the best haemodynamical and mechanical outcomes, but cusp stress distribution varied with valve orifice orientation, which can be linked to different cusp calcification location onset; type 1 BAVs gave rise to similar haemodynamics and valve mechanics, regardless of raphe position, but this position altered the location of abnormal haemodynamic features; finally, type 2 BAV constricted the majority of blood flow, exhibiting the most damaging haemodynamic and mechanical repercussions when compared to other BAV phenotypes. CONCLUSION: The findings of this proof-of-concept work suggest that there are specific differences across haemodynamics and valve mechanics associated with BAV phenotypes, which may be critical to subsequent processes associated with their pathophysiology processes.

The eukaryotic flagellum makes the day: novel and unforeseen roles uncovered after post-genomics and proteomics data.

This review will summarize and discuss the current biological understanding of the motile eukaryotic flagellum, as posed out by recent advances enabled by post-genomics and proteomics approaches. The organelle, which is crucial for motility, survival, differentiation, reproduction, division and feeding, among other activities, of many eukaryotes, is a great example of a natural nanomachine assembled mostly by proteins (around 350-650 of them) that have been conserved throughout eukaryotic evolution. Flagellar proteins are discussed in terms of their arrangement on to the axoneme, the canonical "9+2" microtubule pattern, and also motor and sensorial elements that have been detected by recent proteomic analyses in organisms such as Chlamydomonas reinhardtii, sea urchin, and trypanosomatids. Such findings can be remarkably matched up to important discoveries in vertebrate and mammalian types as diverse as sperm cells, ciliated kidney epithelia, respiratory and oviductal cilia, and neuro-epithelia, among others. Here we will focus on some exciting work regarding eukaryotic flagellar proteins, particularly using the flagellar proteome of C. reinhardtii as a reference map for exploring motility in function, dysfunction and pathogenic flagellates. The reference map for the eukaryotic flagellar proteome consists of 652 proteins that include known structural and intraflagellar transport (IFT) proteins, less wellcharacterized signal transduction proteins and flagellar associated proteins (FAPs), besides almost two hundred unannotated conserved proteins, which lately have been the subject of intense investigation and of our present examination.

Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice.

BACKGROUND: Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants. FINDINGS: To construct the DNA vaccine, the hsp60 gene of C. pseudotuberculosis was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/hsp60). CONCLUSION: This vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

Pathogenomics analysis of Leishmania spp.: flagellar gene families of putative virulence factors.

The trypanosomatid flagellar apparatus contains conventional and unique features, whose roles in infectivity are still enigmatic. Although the flagellum and the flagellar pocket are critical organelles responsible for all vesicular trafficking between the cytoplasm and cell surface, still very little is known about their roles in pathogenesis and how molecules get to and from the flagellar pocket. The ongoing analysis of the genome sequences and proteome profiles of Leishmania major and L infantum, Trypanosoma cruzi, T. brucei, and T. gambiensi ( www.genedb.org ), coupled with our own work on L. chagasi (as part of the Brazilian Northeast Genome Program- www.progene.ufpe.br ), prompted us to scrutinize flagellar genes and proteins of Leishmania spp. promastigotes that could be virulence factors in leishmaniasis. We have identified some overlooked parasite factors such as the MNUDC-1 (a protein involved in nuclear development and genomic fusion) and SQS (an enzyme of sterol biosynthesis), among the described flagellar gene families. A database concerning the results of this work, as well as of other studies of Leishmania and its organelles, is available at http://nugen.lcc.uece.br/LPGate . It will serve as a convenient bioinformatics resource on genomics and pathology of the etiological agents of leishmaniasis.