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There are over a 100 driver gene mutations in patients with diffuse large B‐cell lymphoma (DLBCL), but their clinical significance remains unclear. Here, we first analyzed the DLBCL dataset from the UK‐based Haematological Malignancy Research Network. Patients were divided into high‐ and low‐risk groups based on whether lymphoma progressed within 24 months. Genes showing significantly different frequencies between groups were selected. Survival data for patients with the selected mutant genes were analyzed. The results were validated using two other large databases to evaluate the relationship between the selected mutant genes and prognosis. The mutation frequencies of 11 genes (MYD88[L265P], SGK1, MPEG1, TP53, SPEN, NOTCH1, ETV6, TNFRSF14, MGA, CIITA, and PIM1) significantly differed between the high‐ and low‐risk groups. The relationships between these mutant genes and patient survival were analyzed. Patients who harbored SGK1 (serum and glucocorticoid‐inducible kinase 1) mutations exhibited the best prognosis. Most patients with SGK1 mutation are germinal center B‐cell (GCB) subtype. Among patients with GCB DLBCL, those harboring SGK1 mutations exhibited better prognosis than those without SGK1 mutations. Most SGK1 mutations were single‐base substitutions, primarily scattered throughout the catalytic domain‐encoding region. Multiple SGK1 mutations were identified in a single patient. Thus, SGK1 mutations are a marker of good prognosis for DLBCL and occur predominantly in the GCB subtype of DLBCL. SGK1 mutation status can further stratify patients with GCB DLBCL into different prognostic subgroups. [ABSTRACT FROM AUTHOR]

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes. [ABSTRACT FROM AUTHOR]

Tissue morphogenesis critically depends on the coordination of cellular growth patterns. In plants, many organs consist of clonally distinct cell layers, such as the epidermis, whose cells undergo divisions that are oriented along the plane of the layer. The developmental control of such planar growth is poorly understood. We have previously identified the Arabidopsis AGCVIII-class protein kinase UNICORN (UCN) as a central regulator of this process. Plants lacking UCN activity show spontaneous formation of ectopic multicellular protrusions in integuments and malformed petals indicating that UCN suppresses uncontrolled growth in those tissues. In the current model UCN regulates planar growth of integuments in part by directly repressing the putative transcription factor ABERRANT TESTA SHAPE (ATS). Here we report on the identification of 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE 1 (PDK1) as a novel factor involved in UCN-mediated growth control. PDK1 constitutes a basic component of signaling mediated by AGC protein kinases throughout eukaryotes. Arabidopsis PDK1 is implied in stress responses and growth promotion. Here we show that loss-of-function mutations in PDK1 suppress aberrant growth in integuments and petals of ucn mutants. Additional genetic, in vitro, and cell biological data support the view that UCN functions by repressing PDK1. Furthermore, our data indicate that PDK1 is indirectly required for deregulated growth caused by ATS overexpression. Our findings support a model proposing that UCN suppresses ectopic growth in integuments through two independent processes: the attenuation of the protein kinase PDK1 in the cytoplasm and the repression of the transcription factor ATS in the nucleus. [ABSTRACT FROM AUTHOR]

Background: The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. Methodology/Principal Finding: A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z’ and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. Conclusions/Significance: Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors. [ABSTRACT FROM AUTHOR]

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts. [ABSTRACT FROM AUTHOR]

Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich Strombidinopsis sp., and their respective algal food using Illumina RNAseq. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We detected approximately 10,000 and 7,600 amino acid sequences for S. rassoulzadegani and Strombidinopsis sp., respectively. About half of these transcripts had significant BLASTP hits (E-value <10−6) against previously-characterized sequences, mostly from the model ciliate Oxytricha trifallax. Transcriptomes from both the mixotroph and the heterotroph species provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance and functioning in the transcriptome of this ciliate. While chloroplasts are known sources of reactive oxygen species (ROS), we found the same complement of antioxidant pathways in both ciliates, except for one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy. [ABSTRACT FROM AUTHOR]