Mitochondrial Bioenergetic Alterations in Mouse Neuroblastoma Cells Infected with Sindbis Virus: Implications to Viral Replication and Neuronal Death

The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis virus-induced encephalitis

Alzheimer disease models and human neuropathology: similarities and differences

Animal models aim to replicate the symptoms, the lesions or the cause(s) of Alzheimer disease. Numerous mouse transgenic lines have now succeeded in partially reproducing its lesions: the extracellular deposits of Aβ peptide and the intracellular accumulation of tau protein. Mutated human APP transgenes result in the deposition of Aβ peptide, similar but not identical to the Aβ peptide of human senile plaque. Amyloid angiopathy is common. Besides the deposition of Aβ, axon dystrophy and alteration of dendrites have been observed. All of the mutations cause an increase in Aβ 42 levels, except for the Arctic mutation, which alters the Aβ sequence itself. Overexpressing wild-type APP alone (as in the murine models of human trisomy 21) causes no Aβ deposition in most mouse lines. Doubly (APP × mutated PS1) transgenic mice develop the lesions earlier. Transgenic mice in which BACE1 has been knocked out or overexpressed have been produced, as well as lines with altered expression of neprilysin, the main degrading enzyme of Aβ. The APP transgenic mice have raised new questions concerning the mechanisms of neuronal loss, the accumulation of Aβ in the cell body of the neurons, inflammation and gliosis, and the dendritic alterations. They have allowed some insight to be gained into the kinetics of the changes. The connection between the symptoms, the lesions and the increase in Aβ oligomers has been found to be difficult to unravel. Neurofibrillary tangles are only found in mouse lines that overexpress mutated tau or human tau on a murine tau −/− background. A triply transgenic model (mutated APP, PS1 and tau) recapitulates the alterations seen in AD but its physiological relevance may be discussed. A number of modulators of Aβ or of tau accumulation have been tested. A transgenic model may be analyzed at three levels at least (symptoms, lesions, cause of the disease), and a reading key is proposed to summarize this analysis

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Solid stress and elastic energy as measures of tumour mechanopathology

olid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that 2D spatial maps of the solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and a wide range of sizes can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that (i) solid stress depends on both cancer cells and their microenvironments, (ii) solid stress increases with tumour size and (iii) mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to new discoveries and therapies. Increased tissue stiffness is a widely accepted and actively studied biomechanical property of fibrotic tumours and has been linked to several hallmarks of cancer, including growth, metabolism, invasion and metastasis 1,2,3,4,5,6,7 . However, the abnormal mechanics of tumours are not limited to tissue stiffening. We recently demonstrated that solid stress represents a new mechanopathology that is consistently elevated in mouse and human tumours 8,9 . Solid stress is distinct from interstitial fluid pressure, as the former is contained in—and transmitted by—solid and elastic elements of the extracellular matrix and cells rather than fluids 10 . Therefore, tumours are not only more rigid than many normal tissues, but cancer cells also produce and are exposed to these physical forces. Composed of a combination of tension and compression, these forces are significant in tumours, but negligible in most normal tissues. Early evidence for the existence of solid stress in tumours came from the discovery that blood and lymphatic vessels are mechanically compressed 11,12,13 . This can contribute to hypoxia 9,14 ,promote tumour progression and decrease the efficacy of chemo-, radio- and immunotherapies 15 . In addition to these physiological consequences, forces can directly affect tumour biology: the exogenous application of solid stress in vivo can mechanically stimulate tumorigenic pathways, increasing β-catenin signalling in colon epithelia 16 , and the controlled application of compressive forces in vitro affects the growth8 of cancer cells and promotes their collective migration 17 . Strategies designed to reduce solid stress and decompress blood vessels by reducing levels of collagen and hyaluronic acid 14,18,19 have led to therapeutic approaches for enhancing the efficiency of conventional anticancer treatments and are currently being tested in clinical trials 20,21 . Despite the important implications of solid stress and the immense potential for finding new mechanically activated pathways and targets, there are currently no high-resolution methods for quantifying solid stress in experimental or human tumours. Unlike stiffness, which can be measured using widely available multiscale techniques, measuring solid stress in biological tissues has proved challenging. Previous studies in our laboratory 8,9 , based on previous observations22,23 that arterial wall tissue relaxes if the contained forces are surgically released, demonstrated the presence of residual tissue stresses in tumours. However, this approach is based on a partial cut through a spherical model of the tumour, which makes the precise release of solid stress and the measurement of the ensuing deformation challenging. This method is also limited to bulk estimation of solid stress and is not applicable in situ. The optical-depth limitations of alternative imaging-based methods, such as fluorescent oil microdroplet injection 24 and single-molecule fluorescent force sensors 25 , restrict their use to cellular- and subcellular-scale force detection. We have developed experimental and mathematical frameworks to provide 2D mapping of solid stress in tumours (planar-cut method), sensitive estimations of the solid stress in small tumours with small magnitudes of solid stress, as is the case for metastatic lesions (slicing method), and in situ quantification of solid stress in tumours, which retains the effects of the normal surrounding tissues (needle-biopsy method). All three methods are based on the concept of releasing the solid stress in a controlled way with a defined geometry and then quantifying the stress-induced deformation via high-resolution ultrasonography or optical microscopy. Given the specific topography of the stress relaxation and the geometric and material properties of the tumour, solid stress and discharged elastic energy are estimated using mathematical modelling. Applying these methods to multiple mouse cancer models in primary and metastatic settings has led to the following findings: (i) solid stress and elastic energy may differ between primary and metastatic settings, as they depend on both cancer-cell type and their microenvironment; (ii) tumours with higher elastic energy are not necessarily stiffer, and the stiffer tumours do not necessarily have higher elastic energy; (iii) solid stress increases with tumour size; and (iv) the normal tissue surrounding a tumour substantially contributes to intratumoral solid stress

Measurement of the D+->pi(+) pi(0) and D+-> K+ pi(0) branching fractions

Contains fulltext : 128229.pdf (publisher's version ) (Open Access

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