The effects of Trypanosoma brucei and mammalian-derived extracellular cathepsin-L on myocardial function

African trypanosomiasis is a neglected tropical disease affecting both animals and humans in sub-Saharan Africa. The disease is caused bythe protozoan parasite Trypanosoma brucei, which is transmitted by the tsetse fly (Glossina sp.) vector. In animals, infection leads to severe muscle atrophy and anaemia resulting in significant production and economic losses. In humans, infection leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, and indeed is responsible for the colloquial name “Sleeping Sickness”, the cardiac pathogenesis remains unknown. Previous studies interpreted cardiac dysfunction as being due to immune/inflammatory responses. However, recent work examining the parasite’s interaction with the blood brain barrier, the traversal of which is important for development of neurological signs, has identified direct immune/inflammatory independent mechanisms involving calcium (Ca2+) signalling. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter Ca2+ signalling and cardiac function independent of a systemic immune/inflammatory response. Using a high-throughput method of observing spontaneous contractile activity in isolated cardiomyocytes, we were able to determine that the presence of T. b. brucei parasites resulted in more cardiomyocytes exhibiting spontaneous contractile events. Moreover, when the parasites were removed by careful centrifugation, the culture supernatant had the same effect. Confocal Ca2+ imaging identified an increase in the frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves). Studies utilising specific inhibitors, recombinant protein and RNA interference all demonstrated that this altered SR function was due to cathepsin-L; a cysteine protease produced by T. brucei (TbCatL). Experiments utilising a Langendorff perfusion method revealed that trypanosome culture supernatant could induce ventricular premature contractions in 50% of a cohort of ex vivo whole rat hearts. Mechanistic experiments were performed on single isolated cardiomyocytes stimulated at 1.0 Hz and perfused first with control media followed by trypanosome culture supernatant. The protocol utilised triple caffeine applications: (i) prior to stimulation to empty the SR of Ca2+, (ii) after perfusion with control media and after supernatant to determine the SR Ca2+ content and sarcolemmal extrusion of Ca2+ following each solution. Results were normalised to a parallel set of cardiomyocytes perfused with control media only as time controls. These experiments revealed a 10-15% increase in SR Ca2+ reuptake by the SR Ca2+ ATPase (SERCA) but a reduced SR Ca2+ content suggesting a concomitant increase in SR-mediated Ca2+ leak. This conclusion was supported by the data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide (AIP), highlighting a role for Ca2+/calmodulin kinase II (CaMKII) in the TbCatL action on SR function. When cytosolic diastolic Ca2+ was measured in cardiomyocytes with SR function inhibited by ryanodine and thapsigargin, trypanosome supernatant prevented a decline in cytosolic diastolic Ca2+ that was observed in control media. AIP did not abolish this effect suggesting that TbCatL may raise diastolic Ca2+ that could activate CaMKII leading to the observed effects. These data demonstrated for the first time that African trypanosomes alter cardiac function independent of a systemic immune response via a mechanism involving extracellular cathepsin-L-mediated changes in SR function. Utilising the same (culture adapted and monomorphic) strain of T. brucei as the in vitro experiments, Lister 427, in a rat model of infection we found no significant increase in the arrhythmia frequency as measured by a 15 min electrocardiogram (ECG). However, when hearts were removed and Langendorff perfused with the addition of isoproterenol the arrhythmia frequency was increased. When the pleomorphic strain T. b. brucei TREU 927 was used in rats with continuous ECG recording from biopotential telemetry there was a significant increase in arrhythmia frequency in the infected rats. When hearts were removed and Langendorff perfused with isoproterenol there was a similar increase in arrhythmia frequency as observed with the 427 infected hearts. This suggests that a cardiac dysfunction phenotype is present during trypanosome infections in an animal model providing the basis for future therapeutic work. The relationship between arrhythmogenic SR-mediated Ca2+ release and TbCatL has parallels with endogenous extracellular cathepsin-L (CatL). It has been demonstrated that a basal level of CatL is necessary for normal cardiac function. However, in coronary heart disease (CHD) CatL levels are increased in the serum of patients correlating with the severity of disease. The effects of raised CatL on cardiac function remain unknown. Work in our lab has identified that ex vivo Langendorff perfused hearts that have undergone a 30 min period of ischaemia followed by 90 min reperfusion show greater CatL activity in coronary effluent than hearts perfused without ischaemia. In addition, preliminary data collected in this thesis suggest that human patients that have suffered a myocardial infarction and have undergone reperfusion via percutaneous coronary intervention (PCI) showed higher CatL levels in post-reperfusion serum samples compared to pre-reperfusion serum. When severity of heart function in patients (measured as left ventricular volume at systole and diastole, ejection fraction, infarct size and area at risk) was assessed by magnetic resonance imaging (MRI) in a preliminary study, there was a positive correlation with serum CatL levels. Using recombinant CatL on isolated rat ventricular cardiomyocytes it was found that the SR Ca2+ content and the stimulated Ca2+ transient were significantly reduced in a concentration dependent manner. This suggests a CatL dependent SR dysfunction. This conclusion was supported by an increase in Ca2+ wave frequency measured by confocal Ca2+ imaging in isolated cardiomyocytes. The work in this thesis demonstrates a role for both mammalian-derived and exogenous extracellular cathepsin-L proteases in arrhythmogenic SR-mediated Ca2+ release

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish

T. brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims: African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results: Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion: These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

Peer reviewedPublisher PD

No evidence for rare recessive and compound heterozygous disruptive variants in schizophrenia

Recessive inheritance of gene disrupting alleles, either through homozygosity at a specific site or compound heterozygosity, have been demonstrated to underlie many Mendelian diseases and some complex psychiatric disorders. On the basis of exome sequencing data, an increased burden of complete knockout (homozygous or compound heterozygous) variants has been identified in autism. In addition, using single-nucleotide polymorphism microarray data, an increased rate of homozygosity by descent, or autozygosity, has been linked to the risk of schizophrenia (SCZ). Here, in a large Swedish case-control SCZ sample (11 244 individuals, 5079 of whom have exome sequence data available), we survey the contribution of both autozygosity and complete knockouts to disease risk. We do not find evidence for association with SCZ, either genome wide or at specific loci. However, we note the possible impact of sample size and population genetic factors on the power to detect and quantify any burden that may exist

Discovery and Statistical Genotyping of Copy-Number Variation from Whole-Exome Sequencing Depth

Sequencing of gene-coding regions (the exome) is increasingly used for studying human disease, for which copy-number variants (CNVs) are a critical genetic component. However, detecting copy number from exome sequencing is challenging because of the noncontiguous nature of the captured exons. This is compounded by the complex relationship between read depth and copy number; this results from biases in targeted genomic hybridization, sequence factors such as GC content, and batching of samples during collection and sequencing. We present a statistical tool (exome hidden Markov model [XHMM]) that uses principal-component analysis (PCA) to normalize exome read depth and a hidden Markov model (HMM) to discover exon-resolution CNV and genotype variation across samples. We evaluate performance on 90 schizophrenia trios and 1,017 case-control samples. XHMM detects a median of two rare

A Rare Functional Noncoding Variant at the GWAS-Implicated MIR137/MIR2682 Locus Might Confer Risk to Schizophrenia and Bipolar Disorder

Schizophrenia (SZ) genome-wide association studies (GWASs) have identified common risk variants in >100 susceptibility loci; however, the contribution of rare variants at these loci remains largely unexplored. One of the strongly associated loci spans MIR137 (miR137) and MIR2682 (miR2682), two microRNA genes important for neuronal function. We sequenced ∼6.9 kb MIR137/MIR2682 and upstream regulatory sequences in 2,610 SZ cases and 2,611 controls of European ancestry. We identified 133 rare variants with minor allele frequency (MAF) <0.5%. The rare variant burden in promoters and enhancers, but not insulators, was associated with SZ (p = 0.021 for MAF < 0.5%, p = 0.003 for MAF < 0.1%). A rare enhancer SNP, 1:g.98515539A>T, presented exclusively in 11 SZ cases (nominal p = 4.8 × 10−4). We further identified its risk allele T in 2 of 2,434 additional SZ cases, 11 of 4,339 bipolar (BP) cases, and 3 of 3,572 SZ/BP study controls and 1,688 population controls; yielding combined p values of 0.0007, 0.0013, and 0.0001 for SZ, BP, and SZ/BP, respectively. The risk allele T of 1:g.98515539A>T reduced enhancer activity of its flanking sequence by >50% in human neuroblastoma cells, predicting lower expression of MIR137/MIR2682. Both empirical and computational analyses showed weaker transcription factor (YY1) binding by the risk allele. Chromatin conformation capture (3C) assay further indicated that 1:g.98515539A>T influenced MIR137/MIR2682, but not the nearby DPYD or LOC729987. Our results suggest that rare noncoding risk variants are associated with SZ and BP at MIR137/MIR2682 locus, with risk alleles decreasing MIR137/MIR2682 expression

Detection of recurrent copy number alterations in the genome: taking among-subject heterogeneity seriously

Se adjunta un fichero pdf con los datos de investigación titulado "Supplementary Material for \Detection of Recurrent Copy Number Alterations in the Genome: taking among-subject heterogeneity seriously"Background: Alterations in the number of copies of genomic DNA that are common or recurrent among diseased individuals are likely to contain disease-critical genes. Unfortunately, defining common or recurrent copy number alteration (CNA) regions remains a challenge. Moreover, the heterogeneous nature of many diseases requires that we search for common or recurrent CNA regions that affect only some subsets of the samples (without knowledge of the regions and subsets affected), but this is neglected by most methods. Results: We have developed two methods to define recurrent CNA regions from aCGH data. Our methods are unique and qualitatively different from existing approaches: they detect regions over both the complete set of arrays and alterations that are common only to some subsets of the samples (i.e., alterations that might characterize previously unknown groups); they use probabilities of alteration as input and return probabilities of being a common region, thus allowing researchers to modify thresholds as needed; the two parameters of the methods have an immediate, straightforward, biological interpretation. Using data from previous studies, we show that we can detect patterns that other methods miss and that researchers can modify, as needed, thresholds of immediate interpretability and develop custom statistics to answer specific research questions. Conclusion: These methods represent a qualitative advance in the location of recurrent CNA regions, highlight the relevance of population heterogeneity for definitions of recurrence, and can facilitate the clustering of samples with respect to patterns of CNA. Ultimately, the methods developed can become important tools in the search for genomic regions harboring disease-critical genesFunding provided by Fundación de Investigación Médica Mutua Madrileña. Publication charges covered by projects CONSOLIDER: CSD2007-00050 of the Spanish Ministry of Science and Innovation and by RTIC COMBIOMED RD07/0067/0014 of the Spanish Health Ministr

Small rock-slope failures conditioned by Holocene permafrost degradation:a new approach and conceptual model based on Schmidt-hammer exposure-age dating, Jotunheimen, southern Norway

Rock-slope failures (RSFs) constitute significant natural hazards but the geophysical processes which control their timing are poorly understood. However, robust chronologies can provide valuable information on the environmental controls on RSF occurrence: information which can inform models of RSF activity in response to climatic forcing. This paper uses Schmidt-hammer exposure-age dating (SHD) of boulder deposits to construct a detailed regional Holocene chronology of the frequency and magnitude of small rock-slope failures (SRSFs) in Jotunheimen, Norway. By focusing on the depositional fans of SRSFs (≤ 103 m3), rather than on the corresponding features of massive RSFs (~108 m3), 92 single-event RSFs are targeted for chronology building. A weighted SHD age-frequency distribution and probability density function analysis indicate four centennial- to millennial-scale periods of enhanced SRSF frequency, with a dominant mode at ~4.5 ka. Using change detection and discreet Meyer wavelet analysis, in combination with existing permafrost depth models, we propose that enhanced SRSF activity was primarily controlled by permafrost degradation. Long-term relative change in permafrost depth provides a compelling explanation for the high-magnitude departures from the SRSF background rate and accounts for (i1) the timing of peak SRSF frequency, (2ii) the significant lag (~2.2 ka) between the Holocene Thermal Maximum and the SRSF frequency peak, and (3iii) the marked decline in frequency in the late-Holocene. This interpretation is supported by geomorphological evidence, as the spatial distribution of SRSFs is strongly correlated with the aspect-dependent lower altitudinal limit of mountain permafrost in cliff faces. Results are indicative of a causal relationship between episodes of relatively warm climate, permafrost degradation and the transition to a seasonal-freezing climatic regime. This study highlights permafrost degradation as a conditioning factor for cliff collapse, and hence the importance of paraperiglacial processes; a result with implications for slope instability in glacial and periglacial environments under global warming scenarios

Mutational heterogeneity in cancer and the search for new cancer genes

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention