Molecular Analysis of Trypanosome Infections in Algerian Camels.

PURPOSE: Surra is an economically important livestock disease in many low- and middle-income countries, including those of Northern Africa. The disease is caused by the biting fly-transmitted subspecies Trypanosoma brucei evansi, which is very closely related to the tsetse-transmitted subspecies T. b. brucei and the sexually transmitted subspecies T. b. equiperdum. At least two phylogenetically distinct groups of T. b. evansi can be distinguished, called type A and type B. These evolved from T. b. brucei independently. The close relationships between the T. brucei subspecies and the multiple evolutionary origins of T. b. evansi pose diagnostic challenges. METHODS: Here we use previously established and newly developed PCR assays based on nuclear and mitochondrial genetic markers to type the causative agent of recent trypanosome infections of camels in Southern Algeria. RESULTS/CONCLUSION: We confirm that these infections have been caused by T. b. evansi type A. We also report a newly designed PCR assay specific for T. b. evansi type A that we expect will be of diagnostic use for the community.

Domain adaptation and self-supervised learning for surgical margin detection.

PURPOSE: One in five women who undergo breast conserving surgery will need a second revision surgery due to remaining tumor. The iKnife is a mass spectrometry modality that produces real-time margin information based on the metabolite signatures in surgical smoke. Using this modality and real-time tissue classification, surgeons could remove all cancerous tissue during the initial surgery, improving many facets of patient outcomes. An obstacle in developing a iKnife breast cancer recognition model is the destructive, time-consuming and sensitive nature of the data collection that limits the size of the datasets. METHODS: We address these challenges by first, building a self-supervised learning model from limited, weakly labeled data. By doing so, the model can learn to contextualize the general features of iKnife data from a more accessible cancer type. Second, the trained model can then be applied to a cancer classification task on breast data. This domain adaptation allows for the transfer of learnt weights from models of one tissue type to another. RESULTS: Our datasets contained 320 skin burns (129 tumor burns, 191 normal burns) from 51 patients and 144 breast tissue burns (41 tumor and 103 normal) from 11 patients. We investigate the effect of different hyper-parameters on the performance of the final classifier. The proposed two-step method performed statistically significantly better than a baseline model (p-value < 0.0001), by achieving an accuracy, sensitivity and specificity of 92%, 88% and 92%, respectively. CONCLUSION: This is the first application of domain transfer for iKnife REIMS data. We showed that having a limited number of breast data samples for training a classifier can be compensated by self-supervised learning and domain adaption on a set of unlabeled skin data. We plan to confirm this performance by collecting new breast samples and extending it to incorporate other cancer tissues.

Age-Dependent Decline in Cardiac Function in Guanidinoacetate-N-Methyltransferase Knockout Mice.

AIM: Guanidinoacetate N-methyltransferase (GAMT) is the second essential enzyme in creatine (Cr) biosynthesis. Short-term Cr deficiency is metabolically well tolerated as GAMT-/- mice exhibit normal exercise capacity and response to ischemic heart failure. However, we hypothesized long-term consequences of Cr deficiency and/or accumulation of the Cr precursor guanidinoacetate (GA). METHODS: Cardiac function and metabolic profile were studied in GAMT-/- mice >1 year. RESULTS: In vivo LV catheterization revealed lower heart rate and developed pressure in aging GAMT-/- but normal lung weight and survival versus age-matched controls. Electron microscopy indicated reduced mitochondrial volume density in GAMT-/- hearts (P < 0.001), corroborated by lower mtDNA copy number (P < 0.004), and citrate synthase activity (P < 0.05), however, without impaired mitochondrial respiration. Furthermore, myocardial energy stores and key ATP homeostatic enzymes were barely altered, while pathology was unrelated to oxidative stress since superoxide production and protein carbonylation were unaffected. Gene expression of PGC-1α was 2.5-fold higher in GAMT-/- hearts while downstream genes were not activated, implicating a dysfunction in mitochondrial biogenesis signaling. This was normalized by 10 days of dietary Cr supplementation, as were all in vivo functional parameters, however, it was not possible to differentiate whether relief from Cr deficiency or GA toxicity was causative. CONCLUSION: Long-term Cr deficiency in GAMT-/- mice reduces mitochondrial volume without affecting respiratory function, most likely due to impaired biogenesis. This is associated with hemodynamic changes without evidence of heart failure, which may represent an acceptable functional compromise in return for reduced energy demand in aging mice.

Moderate elevation of intracellular creatine by targeting the creatine transporter protects mice from acute myocardial infarction.

AIMS: Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: CrT-OE mice were selected for left ventricular (LV) creatine 20-100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury. CONCLUSION: Elevation of myocardial creatine by 20-100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia.

Mice over-expressing the myocardial creatine transporter develop progressive heart failure and show decreased glycolytic capacity.

The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32- week-old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel (1)H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the heart's metabolic enzymes and identified seven proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: alpha- and beta-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59+/-0.02 micromol ATP produced/microg protein/min; CrT-OE, 0.31+/-0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102+/-3 micromol/g wet myocardium; CrT-OE, 78+/-13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the beta-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study demonstrates that chronically increased myocardial [Cr] in the CrT-OE model leads to the development of progressive hypertrophy and heart failure, which may be mediated by a compromise in glycolytic capacity at the level of enolase.

Cardiac phenotype of mitochondrial creatine kinase knockout mice is modified on a pure C57BL/6 genetic background.

UNLABELLED: Discrepant results for the phenotype of mitochondrial creatine kinase knockout mice (Mt-CK(-/-)) could be due to mixed genetic background and use of non-littermate controls. We therefore backcrossed with C57BL/6J for >8 generations, followed by extensive in vivo cardiac phenotyping. Echocardiography and in vivo LV haemodynamics were performed in independent cohorts at 20-40 weeks and 1 year. No significant differences were observed for ECG, LV volumes, pressures, and systolic or diastolic function compared to littermate controls. Furthermore, responses to dobutamine were not different, indicating preserved contractile reserve. Contrary to published reports using Mt-CK(-/-) on a mixed background, we observed normal LV weights even in year old mice, and gene expression of common hypertrophic markers were not elevated. However, previously undetected adaptations were observed: an increase in activity of the cytosolic MM-CK isoenzyme (+20% vs WT, P=0.0009), and of citrate synthase (+18% vs WT, P=0.0007), a marker for mitochondrial volume. In a 3-week voluntary wheel running protocol, Mt-CK(-/-) ran significantly less per day (P=0.009) and attained lower maximum speed compared to controls (P=0.0003), suggesting impaired skeletal muscle function. MM-CK isoenzyme activity was significantly elevated in soleus but not gastrocnemius muscle of KO mice, and citrate synthase activities were normal in both, suggesting compensatory mechanisms are incomplete in skeletal muscle. CONCLUSIONS: in contrast to previous reports using a mixed genetic background, Mt-CK(-/-) on a C57BL/6 background do not develop LV hypertrophy or dysfunction even up to 1 year, and this may be explained by a compensatory increase in MM-CK activity and mitochondrial volume.

Creatine uptake in mouse hearts with genetically altered creatine levels.

Creatine plays an important role in energy metabolism in the heart. Cardiomyocytes accumulate creatine via a specific creatine transporter (CrT), the capacity of which is reduced in the failing heart, resulting in lower myocardial creatine concentration. Therefore, to gain insight into how the CrT is regulated, we studied two mouse models of severely altered myocardial creatine levels. Cardiac creatine uptake levels were measured in isolated hearts from creatine-free guanidinoacetate-N-methyl transferase knock out (GAMT(-/-)) mice and from mice overexpressing the myocardial CrT (CrT-OE) using (14)C-radiolabeled creatine. CrT mRNA levels were measured using real time RT-PCR and creatine levels with HPLC. Hearts from GAMT(-/-) mice showed a 7-fold increase in V(max) of creatine uptake and a 1.4-fold increase in CrT mRNA levels. The increase in Cr uptake and in CrT mRNA levels, however, was almost completely prevented when mice were fed a creatine supplemented diet, indicating that creatine uptake is subject to negative feedback regulation. Cardiac creatine uptake levels in CrT-OE mice were increased on average 2.7-fold, showing a considerable variation, in line with a similar variation in creatine content. Total CrT mRNA levels correlated well with myocardial creatine content (r=0.67; p<0.0001) but endogenous CrT mRNA levels did not correlate at all with myocardial creatine content (r=0.01; p=0.96). This study shows that creatine uptake can be massively upregulated in the heart, by almost an order of magnitude and that this upregulation is subject to feedback inhibition. In addition, our results strongly suggest that CrT activity is predominantly regulated by mechanisms other than alterations in gene expression.

Aminopeptidase O contains a functional nucleolar localization signal and is implicated in vascular biology.

We have identified a gene trap integration into Aminopeptidase O, the gene encoding a member of the M1 family of metalloproteases. Using the betagal reporter of the gene trap vector, we have revealed that at least some ApO isoforms are expressed predominantly in embryonic and adult blood vessels leading us to propose that ApO plays a role in vascular cell biology. The protein produced from an engineered Gfp-ApO fusion cDNA localises to the nucleolus in transfected COS7 cells. We confirm that indeed the APO protein contains a functional nucleolar localisation domain by demonstrating that GFP-APO fusion proteins that lack the predicted nucleolar localisation signal are retained in the cytoplasm. We report the existence of multiple alternatively spliced Apo isoforms that differ with respect to the presence of exons encoding important functional domains. Alternative splicing predictably produces protein products with or without the catalytic domain and/or a nucleolar localisation signal and therefore likely represents an important mechanism in regulating the biological activity of APO that has been reported to cleave one of the peptides of the renin angiotensin pathway.

The creatine kinase energy transport system in the failing mouse heart.

Characteristic alterations of the creatine kinase (CK) system occur in heart failure and may contribute to contractile dysfunction. We examined two mouse models of chronic cardiac stress, transverse aortic constriction (TAC) and coronary artery ligation (CAL), and examined the relationship of CK system changes with hypertrophy and heart failure development. C57Bl/6 mice were subjected to TAC or sham surgery and sacrificed after 2-10 weeks according to echocardiographic criteria of myocardial hypertrophy and function to create four groups representing progressive dysfunction from normal, through compensated hypertrophy, to heart failure. Only mice with congestive heart failure had LV total creatine concentration and total CK activity significantly lower than sham values (11% and 30% lower, respectively). However for all aortic banded mice, a linear relationship was observed between ejection fraction and estimated maximal CK reaction velocity. Mice with heart failure also had corresponding decreases in the activities of the Mito-, MM-, and MB-CK isoenzymes, while the BB isoform remained unchanged. To determine whether these changes were model specific, mice were subjected to CAL or sham operation and followed for 7 weeks. Quantitative changes in total creatine, total CK activity, Mito-CK and MM-CK activities were similar for CAL and TAC mice. We conclude that alterations in the creatine kinase system occur during heart failure in mice qualitatively similar to those occurring in larger animals and humans, suggesting that mice are a suitable model for studying the role of such changes in the pathogenesis of heart failure.

Supranormal myocardial creatine and phosphocreatine concentrations lead to cardiac hypertrophy and heart failure: insights from creatine transporter-overexpressing transgenic mice.

BACKGROUND: Heart failure is associated with deranged cardiac energy metabolism, including reductions of creatine and phosphocreatine. Interventions that increase myocardial high-energy phosphate stores have been proposed as a strategy for treatment of heart failure. Previously, it has not been possible to increase myocardial creatine and phosphocreatine concentrations to supranormal levels because they are subject to tight regulation by the sarcolemmal creatine transporter (CrT). METHODS AND RESULTS: We therefore created 2 transgenic mouse lines overexpressing the myocardial creatine transporter (CrT-OE). Compared with wild-type (WT) littermate controls, total creatine (by high-performance liquid chromatography) was increased in CrT-OE hearts (66+/-6 nmol/mg protein in WT versus 133+/-52 nmol/mg protein in CrT-OE). Phosphocreatine levels (by 31P magnetic resonance spectroscopy) were also increased but to a lesser extent. Surprisingly, CrT-OE mice developed left ventricular (LV) dilatation (LV end-diastolic volume: 21.5+/-4.3 microL in WT versus 33.1+/-9.6 microL in CrT-OE; P=0.002), substantial LV dysfunction (ejection fraction: 64+/-9% in WT versus 49+/-13% in CrT-OE; range, 22% to 70%; P=0.003), and LV hypertrophy (by 3-dimensional echocardiography and magnetic resonance imaging). Myocardial creatine content correlated closely with LV end-diastolic volume (r=0.51, P=0.02), ejection fraction (r=-0.74, P=0.0002), LV weight (r=0.59, P=0.006), LV end-diastolic pressure (r=0.52, P=0.02), and dP/dt(max) (r=-0.69, P=0.0008). Despite increased creatine and phosphocreatine levels, CrT-OE hearts showed energetic impairment, with increased free ADP concentrations and reduced free-energy change levels. CONCLUSIONS: Overexpression of the CrT leads to supranormal levels of myocardial creatine and phosphocreatine, but the heart is incapable of keeping the augmented creatine pool adequately phosphorylated, resulting in increased free ADP levels, LV hypertrophy, and dysfunction. Our data demonstrate that a disturbance of the CrT-mediated tight regulation of cardiac energy metabolism has deleterious functional consequences. These findings caution against the uncritical use of creatine as a therapeutic agent in heart disease.

Reduced inotropic reserve and increased susceptibility to cardiac ischemia/reperfusion injury in phosphocreatine-deficient guanidinoacetate-N-methyltransferase-knockout mice.

BACKGROUND: The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase-knockout (GAMT-/-) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT-/- mice. METHODS AND RESULTS: GAMT-/- mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and beta-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT-/- mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT-/- hearts (101+/-8 mm Hg in WT versus 59+/-7 mm Hg in GAMT-/-; P<0.05). (31)P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT-/- hearts utilized P-GA. During ischemia/reperfusion, GAMT-/- hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23+/-9 in WT and 51+/-5 mm Hg in GAMT-/- during reperfusion; P<0.05) and incomplete resynthesis of P-GA. CONCLUSIONS: GAMT-/- mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT-/- mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.

Creatine kinase knockout mice show left ventricular hypertrophy and dilatation, but unaltered remodeling post-myocardial infarction.

OBJECTIVE: Creatine kinase (CK) is responsible for the transport of high-energy phosphates in excitable tissue and is of central importance in myocardial energy homeostasis. Significant changes in myocardial energetics have been reported in mice lacking the various CK isoenzymes. Our hypothesis was that ablation of CK isoenzymes leads to cardiac hypertrophy, impaired function, and aggravation of left ventricular remodeling post-myocardial infarction. METHODS: CK-deficient mice (CK KO) were examined by cardiac magnetic resonance imaging (MRI) to determine left ventricular volumes, ejection fraction, and mass: ten wild-type (WT), 6 mitochondrial CK KO (Mito-CK-/-), 10 cytosolic CK KO (M-CK-/-), and 10 mice with combined KO (M/Mito-CK-/-). RESULTS: While ejection fraction was similar in all groups, there was significant LV dilatation with a approximately 30% increase in LV end-diastolic volumes in Mito-CK-/- and in M/Mito-CK-/-. Compared to WT, there was a striking 73% and 64% increase of LV mass in Mito-CK-/- and in M/Mito-CK-/- mice, respectively, but no significant increase of LV mass (+33%; p=n.s.) in M-CK-/-. Furthermore, significant re-expression of beta-MHC, a marker of myocardial hypertrophy, was found in all CK-deficient hearts. LV remodeling was investigated by MRI in hearts of 7 WT and 10 M/Mito-CK-/- mice 4 weeks postmyocardial infarction (MI). Four weeks post-LAD ligation (MI size approximately 32%), WT and M/Mito-CK-/- showed a similar degree of cardiac dysfunction, dilatation, and hypertrophy. CONCLUSION: Mito-CK-/- and M/Mito-CK-/- mice show significant LV dilatation and marked LV hypertrophy, but LV remodeling post-MI is not aggravated. CK ablation leads to substantial adaptational changes in heart.

A novel transmembrane MSP-containing protein that plays a role in right ventricle development.

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.

In vivo cardiac 1H-MRS in the mouse.

The mouse is the predominant animal model to study the effect of gene manipulations. Imaging techniques to define functional effects on the heart caused by genomic alterations are becoming increasingly routine in mice, yet methods for in vivo investigation of metabolic phenotypes in the mouse heart are lacking. In this work, cardiac 1H-MRS was developed and applied in mouse hearts in vivo using a single-voxel technique (PRESS). In normal C57Bl/6J mice, stability and reproducibility achieved by dedicated cardiac and respiratory gating was demonstrated by measuring amplitude and zero-order phase changes of the unsuppressed water signal. Various cardiac metabolites, such as creatine, taurine, carnitine, or intramyocardial lipids were successfully detected and quantified relative to the total water content in voxels as small as 2 microl, positioned in the interventricular septum. The method was applied to a murine model of guanidinoacetate N-methyltransferase (GAMT) deficiency, which is characterized by substantially decreased myocardial creatine levels. Creatine deficiency was confirmed noninvasively in myocardium of anesthetized GAMT-/- mice. This is the first study to report the application of cardiac 1H-MRS in mice in vivo.

Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.

We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.