Diverse Effects on Mitochondrial and Nuclear Functions Elicited by Drugs and Genetic Knockdowns in Bloodstream Stage Trypanosoma brucei

The parasite Trypanosoma brucei causes human African trypanosomiasis, which is fatal unless treated. Currently used drugs are toxic, difficult to administer, and often are no longer effective due to drug resistance. The search for new drugs is long and expensive, and determining which compounds are worth pursuing is a key challenge in that process. In this study we sought to determine whether different compounds elicited different responses in the mammalian-infective stage of the parasite. We also examined whether genetic knockdown of parasite molecules led to similar responses. Our results show that, depending on the treatment, the replication of the parasite genomes, proper division of the cell, and mitochondrial function can be affected. Surprisingly, these different responses were not able to predict which compounds affected the long term proliferative potential of T. brucei. We found that some of the compounds had irreversible effects on the parasites within one day, so that even cells that appeared healthy could not proliferate. We suggest that determining which compounds set the parasites on a one-way journey to death may provide a means of identifying those that could lead to drugs with high efficacy

Late Stage Infection in Sleeping Sickness

At the turn of the 19th century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles

Emergence and maintenance of actionable genetic drivers at medulloblastoma relapse

BACKGROUND: 90% of tumors) and established genetic drivers (e.g. SHH/WNT/P53 mutations; 60% of rMB events) were maintained from diagnosis. Critically, acquired and maintained rMB events converged on targetable pathways which were significantly enriched at relapse (e.g. DNA damage-signaling) and specific events (e.g. 3p loss) predicted survival post-relapse. CONCLUSIONS: rMB is defined by the emergence of novel events and pathways, in concert with selective maintenance of established genetic drivers. Together, these define the actionable genetic landscape of rMB and provide a basis for improved clinical management and development of stratified therapeutics, across disease-course

Absence of polysialylated NCAM is an unfavorable prognostic phenotype for advanced stage neuroblastoma

<p>Abstract</p> <p>Background</p> <p>The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.</p> <p>Methods</p> <p>A tumor tissue microarray (TMA) of 36 paraffin-embedded neuroblastoma samples was utilized to detect polySia-NCAM expression with a polySia-binding fluorescent fusion protein, and polySia-NCAM expression was compared with clinical stage, age, <it>MYCN </it>amplification status, histology (INPC), and proliferation index (PI).</p> <p>Results</p> <p>PolySia-NCAM-positive neuroblastoma patients had more often metastases at diagnosis, and polySia-NCAM expression associated with advanced disease (<it>P </it>= 0.047). Most interestingly, absence of polySia-NCAM-expressing tumor cells in TMA samples, however, was a strong unfavorable prognostic factor for overall survival in advanced disease (<it>P </it>= 0.0004), especially when <it>MYCN </it>was not amplified. PolySia-NCAM-expressing bone marrow metastases were easily detected in smears, aspirates and biopsies.</p> <p>Conclusion</p> <p>PolySia-NCAM appears to be a new clinically significant molecular marker in neuroblastoma, hopefully with additional value in neuroblastoma risk stratification.</p

Diffuse Glioneuronal tumour with Oligodendroglioma‐like features and Nuclear Clusters (DGONC) – a molecularly‐defined glioneuronal CNS tumour class displaying recurrent monosomy 14

Aims: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. Patients and methods: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. Results: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. Conclusions: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters

Quantitative real-time RT-PCR of CD24 mRNA in the detection of prostate cancer

BACKGROUND: Gene expression profiling has recently shown that the mRNA for CD24 is overexpressed in prostate carcinomas (Pca) compared to benign or normal prostate epithelial tissues. Immunohistochemical studies have reported the usefulness of anti-CD24 for detecting prostate cancer over the full range of prostate specimens encountered in surgical pathology, e.g. needle biopsies, transurethral resection of prostate chips, or prostatectomies. It is a small mucin-like cell surface protein and thus promises to become at least a standard adjunctive stain for atypical prostate biopsies. We tested the usefulness of real-time RT-PCR for specific and sensitive detection of CD24 transcripts as a supplementary measure for discriminating between malignant and benign lesions in prostatic tissues. METHODS: Total RNA was isolated from snap-frozen chips in 55 cases of benign prostatic hyperplasia (BPH) and from frozen sections in 59 prostatectomy cases. The latter contain at least 50% malignant epithelia. Relative quantification of CD24 transcripts was performed on the LightCycler instrument using hybridization probes for detection and porphobilinogen deaminase transcripts (PBGD) for normalization. RESULTS: Normalized CD24 transcript levels showed an average 2.69-fold increase in 59 Pca-cases (mean 0.21) when compared to 55 cases of BPH (mean 0.08). This difference was highly significant (p < 0.0001). The method has a moderate specificity (47.3%) but a high sensitivity (86.4%) if the cutoff is set at 0.0498. CD24 expression levels among Pca cases were not statistically associated with the tumor and lymph-node stage, the grading (WHO), the surgical margins, or the Gleason score. CONCLUSION: The present study demonstrates the feasibility of quantitative CD24 RNA transcript detection in prostatic tissues even without previous laser microdissection

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

OPA1 mutations induce mitochondrial DNA instability and optic atrophy ‘plus’ phenotypes

Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA ‘plus’ phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani