Redox Regulation of Heart Regeneration: An Evolutionary Tradeoff

Heart failure is a costly and deadly disease, affecting over 23 million patients worldwide, half of which die within 5 years of diagnosis. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate lost or damaged myocardium. Although limited myocyte turnover does occur in the adult heart, it is insufficient for restoration of contractile function1-6. In contrast to lower vertebrates which can regenerate their myocardium through cardiomyocyte proliferation,7-13, adult mammalian heart cardiomyogenesis very limited1-5. Studies in the late 90s elegantly demonstrated that mammalian cardiomyocytes continue to divide for a few days after birth 14-16, only to undergo permanent cell cycle arrest shortly thereafter. Recently, we demonstrated that resection of up to 15% of the apex of the left ventricle of postnatal day 1 (P1) mice results in complete regeneration within 21 days following injury, without significant fibrosis and cardiac dysfunction17. Moreover, we described a similar regenerative response following ischemic myocardial infarction 18. This response was well characterized by robust cardiomyocyte proliferation, with gradual restoration of normal cardiac morphology and function. In addition to the histological evidence of proliferating myocytes, genetic fate-mapping studies confirmed that the majority of newly formed cardiomyocytes are derived from proliferation of preexisting cardiomyocytes17. Intriguingly, this regenerative capacity is lost by P7, after which injury results in the cardiomyocyte hypertrophy and scar-formation, which coincides with binucleation and cell cycle exit of cardiomyocytes 14, 19. An important approach to understanding the loss of regenerative ability of the mammalian heart is to first consider why, and not only how, this happens. The regenerative ability of the early postnatal heart following resection or ischemic infarction involves regeneration of the lost myocardium and vasculature with restoration of normal myocardial thickness and architecture, and long-term functional recovery. Why would the heart permanently forego such a remarkable regenerative program shortly after birth? The answer may lie in within the fundamental principal of evolutionary tradeoff

Phase field modelling voids nucleation and growth in binary systems

We present a comprehensive study of voids formation, nucleation and growth in a prototype model of binary alloys subjected to irradiation by using a combined approach based on phase field and rate theories. It is shown that voids formation is caused by interaction of irradiation-produced vacancies through elastic deformation of a lattice and vacancy coupling with composition field of the alloy. Phase diagrams illustrating the formation of states related to solid solution, phase decomposition, and patterning are obtained. Formation of voids from supersaturated ensemble of vacancies is accompanied by composition rearrangement of alloy components. It was found that elastic inhomogeneity leading to the formation of anisotropic precipitates in an initially prepared binary alloy results in the formation of a void super-lattice under irradiation. It was shown that voids nucleate and grow with dose according to diffusion controlled precipitation processes, where universal dynamics of voids growth is revealed. Estimations of main quantitative and statistical characteristics of voids by using material parameters relevant to most of alloys and steels give good agreement with experimental observations.Comment: 21 pages, 10 figure

Merkel cell polyomavirus small t antigen induces cancer and embryonic merkel cell proliferation in a transgenic mouse model

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT: Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting

Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication

Response of Merkel cell polyomavirus-positive Merkel cell carcinoma xenografts to a survivin inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ∼80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs. © 2013 Dresang et al

Quantitative Analysis of Viral Load per Haploid Genome Revealed the Different Biological Features of Merkel Cell Polyomavirus Infection in Skin Tumor

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n = 9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n = 45], actinic keratosis [AK, n = 52], Bowen’s disease [n = 34], seborrheic keratosis [n = 5], primary cutaneous anaplastic large-cell lymphoma [n = 5], malignant melanoma [n = 5], and melanocytic nevus [n = 6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119–42.8) and AK (0.02–0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection

Driving the atom by atomic fluorescence: analytic results for the power and noise spectra

We study how the spectral properties of resonance fluorescence propagate through a two-atom system. Within the weak-driving-field approximation we find that, as we go from one atom to the next, the power spectrum exhibits both sub-natural linewidth narrowing and large asymmetries while the spectrum of squeezing narrows but remains otherwise unchanged. Analytical results for the observed spectral features of the fluorescence are provided and their origin is thoroughly discussed.Comment: 13 pages, 5 figures; to be published in Phys. Rev. A Changed title and conten

Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients

The HSP70 modulator MAL3-101 inhibits Merkel cell carcinoma

Merkel Cell Carcinoma (MCC) is a rare and highly aggressive neuroendocrine skin cancer for which no effective treatment is available. MCC represents a human cancer with the best experimental evidence for a causal role of a polyoma virus. Large T antigens (LTA) encoded by polyoma viruses are oncoproteins, which are thought to require support of cellular heat shock protein 70 (HSP70) to exert their transforming activity. Here we evaluated the capability of MAL3-101, a synthetic HSP70 inhibitor, to limit proliferation and survival of various MCC cell lines. Remarkably, MAL3-101 treatment resulted in considerable apoptosis in 5 out of 7 MCC cell lines. While this effect was not associated with the viral status of the MCC cells, quantitative mRNA expression analysis of the known HSP70 isoforms revealed a significant correlation between MAL3-101 sensitivity and HSC70 expression, the most prominent isoform in all cell lines. Moreover, MAL3-101 also exhibited in vivo antitumor activity in an MCC xenograft model suggesting that this substance or related compounds are potential therapeutics for the treatment of MCC in the future. © 2014 Adam et al

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection