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Lenalidomide-Induced Myocarditis, Rare Nevertheless Perhaps Fatal Toxic body of a Popular Immunotherapy.
Septins are dynamic filament-forming proteins that are recognized as important components of the cytoskeleton and are involved in numerous functions inside the cells, such as cytokinesis, exocytosis, and ciliogenesis and even in defense against pathogenic bacteria. Despite being highly conserved in eukaryotes, there is scarce literature on the role of septins in organisms other than humans and yeast. Therefore, septins from Schistosoma mansoni represent an interesting model to study an unexplored branch of this protein family. Here we described standard protocols for recombinant production and initial characterization of septins from S. mansoni. Septins are notably difficult to purify, mostly due to their tendency to assemble into filaments. Therefore, specific protocols to stabilize these proteins have been developed. In this chapter, we systematically describe protocols to clone, express, and purify schistosome septins. We also describe the use of circular dichroism to assess the folding and stability of septins and use of chromatography to characterize their oligomeric state, bound guanine nucleotide, and GTP hydrolysis. We expect that these protocols may help researchers involved in the study of schistosome septins as well as assist to establish protocols for septins from other organisms.An important aspect of host-pathogen interactions is the interference of secreted proteins with the fibrinolytic system. Herein, we describe a modified ELISA method used to evaluate the interaction of a recombinant Schistosoma mansoni protein with plasminogen (PLG). Using this protocol, we demonstrated that a secreted protein, recombinant venom allergen-like protein 18 (rSmVAL18) acts as a plasminogen receptor increasing its activation into plasmin in the presence of the urokinase-type plasminogen activator (uPA). PLG binding was determined by immobilizing human PLG in the plate and incubating with the recombinant protein; competitive binding with a lysine analog demonstrated the interaction of the protein lysine residues with PLG Kringle domains. To assess the activation of S. mansoni recombinant protein-bound PLG, the amidolytic activity of generated plasmin was measured using the D-Val-Leu-Lys 4-nitroanilide dihydrochloride substrate.Electrophysiology is the standard method for characterizing ion channel function. Two-electrode voltage clamp is a robust and relatively simple version which can be applied to the characterization of glutamate-gated chloride channels from Schistosoma mansoni, a potential schistomicidal target. Here, the method is described in detail, with an emphasis on the investigation of S. mansoni. GluCls.Dihydrofolate reductase (DHFR) is an essential enzyme for nucleotide metabolism used to obtain energy and structural nucleic acids. Schistosoma mansoni has all the pathways for pyrimidine biosynthesis, which include the thymidylate cycle and, consequentially, the DHFR enzyme. Here, we describe the characterization of Schistosoma mansoni DHFR (SmDHFR) using isothermal titration calorimetry for the enzymatic activity and thermodynamic determination, also the folate analogs inhibition. Moreover, X-ray crystallography was used to determine the enzyme atomic model at 1.95 Å.Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.Transposable elements (TEs) represent a significant portion of eukaryotic genomes and are important players in their dynamics and evolution. Therefore, the description of TEs and the analysis of their distribution in the genomes are important steps to understand their influence in the architecture of genomes. Here we describe the protocol used by us to identify and curate consensus TEs sequences from S. mansoni, as well the protocol to map these elements in the S. mansoni genome. We expect that these protocols may help researchers interested in studying TEs content in S. mansoni or other organisms.In the last few years, long non-coding RNAs (lncRNAs) have been widely studied in humans, and their relevance for physiological and pathological conditions has been demonstrated. In parasites, there are only a few works, such as in Plasmodium falciparum, where it was shown that an lncRNA regulates the expression of a gene associated with immune system evasion, also indicating the relevance of understanding the role of this class of RNAs in parasites. In Schistosoma mansoni, in the last 2 years, there were four published articles related to the annotation of lncRNAs in different life cycle stages using RNA-Seq libraries. In order to make this process of lncRNA identification and annotation more accessible to biologists with no bioinformatics training, considering the growing number of S. mansoni RNA-Seq libraries publicly available from different sources, such as ovary tissues from bi-sex and single-sex infections, and the potential of lncRNAs as therapeutic targets, we provide this step-by-step protocol of lncRNA identification and quantification. This guide includes the download of RNA-Seq libraries from a public database and reads processing and mapping against the genome, transcript reconstruction, novel lncRNA identification, transcripts expression level determination, and the identification of differentially expressed lncRNAs.DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.The tegument (outer surface) of Schistosoma mansoni and other trematodes is in intimate contact with the host and plays an important role in host-parasite interactions. It is a complex structure that contains hundreds of proteins implicated in a variety of functions, although, so far, only a few proteins have been well characterized. Indeed, a few of these proteins have been shown to be effective vaccine and diagnostic candidates against S. mansoni and other schistosomes, and so the proteomic characterization of tegumental molecules could open new avenues for the development of novel control and surveillance strategies to combat schistosomiasis. Here, we describe the step by step isolation of tegumental proteins from the different tegument compartments using a biotinylation approach, as well as the materials and reagents needed.Schistosomiasis is one of the most important helminthic parasitic infections in the world, with over 700 million people at risk of infection. Species of Schistosoma have a complex life cycle involving the infection of freshwater snails before infecting their mammalian definitive host. Taking about 130,000 lives per annum, S. mansoni is the major cause of intestinal schistosomiasis worldwide. Within Biomphalaria glabrata snails, asexual replication of the parasite gives rise to cercariae larvae. Cercariae actively penetrate the host's skin to complete their life cycle and eventually transform into adult worms. If left untreated, intestinal schistosomiasis can lead to peripheral destruction of the portal vein system, gastric hemorrhage from esophageal varices, as well as hepatic failure. Mass spectrometry (MS) is the method of choice for proteomics analysis. The bottom-up proteomics approach-also known as "shotgun proteomics"-typically includes a protein extraction and solubilization step followed by proteolytic digestion and tandem MS (MS/MS) analysis. Proteins are later identified by peptide de novo sequencing upon MS and MS/MS spectra of digest peptides. In this chapter, we introduce an analytical workflow for proteome profiling of S. mansoni cercariae using bottom-up proteomics. The cercariae were isolated and lysed. Proteins were then extracted, enzymatically digested, and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins were identified using MaxQuant software. Cercariae are the first life stage of the parasite S. LJI308 cell line mansoni which humans encounter, and conducting proteomic analysis on this life cycle stage can shed light on possible drug or vaccine candidates to help disable the parasite's ability to infect or arm the immune system for parasite clearance.Schistosomes are deadly pathogens responsible for the neglected tropical disease schistosomiasis. The parasite's virulence is aided by a skin-like tissue called the tegument. The study of the tegument is hampered by a lack of tools suitable for visualizing the tissue. Here we describe a novel methodology employing fluorophore-conjugated dextrans that allows specific fluorescent labeling of the tegument and that is compatible with downstream fluorescence-labeling techniques including phalloidin labeling, RNA FISH, and immunofluorescence.Individual developmental stages of blood fluke Schistosoma mansoni excrete or secrete a different set of molecules. Here we describe optimized protocols for collection of excretory/secretory products (E/S products) from cercariae, schistosomula, adult worms, and eggs. These E/S products are essential for successful parasitism functioning at the host-parasite interface, enabling invasion into the host and contributing to the survival of the parasite by modulation of host physiology and immune responses. Collection of sufficient amounts of E/S products is required for detailed research of these processes.In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.
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