Virus-Induced Cancers of the Skin and Mucosa:Are We Dealing with "Smoking Guns" or "Smoke and Mirrors" in the Operating Theatre?

Introduction: Human papillomavirus (HPV) alone is thought to cause ~610,000 cases of cancer per year, and is the dominant aetiological agent for ano-genital (esp. cervical) and head and neck cancers (esp. oropharyngeal). Merkel cell polyomavirus (MCV) is a more recently discovered virus which causes Merkel cell carcinoma, a rare but highly aggressive skin malignancy. Methods: We explored the available published evidence to see if transmission of live HPV or MCV virus in smoke generated by laser or diathermy was feasible, and would pose an infection risk. Long-term infection with such carcinogenic viruses would then pose an increased risk for the development of virus-induced cancers in medical personnel. Results: The morphological structures of both HPV and MCV are very similar, and the size, external capsids and genomic structures show striking similarity. Both viruses have a non-enveloped external protein capsid consisting of 72 capsomeres, and a double-stranded DNA core. Sizes of both viruses range from 50 to 60 nm. There are now recent data demonstrating live and infectious HPV in smoke, and that these viruses can be used to infect cells in vitro. Further, anecdotal reports of virus transmission leading to disease causation in the production of respiratory airway viral warts (benign disease), and, finally, reports of HPV-induced oropharyngeal carcinoma (malignant disease) in two gynaecological surgeons as an occupational health hazard have been published recently. Conclusion: There is now sufficient evidence to support the hypotheses that live infectious carcinogenic viruses can be transmitted via smoke generated from surgical procedures, and, in rare instances, actually cause significant disease. Protective measures such as smoke extraction and airway protection should be instituted for all healthcare personnel, particularly those with multiple repeated exposures such as gynaecological surgeons

Predicting live birth, preterm and low birth weight infant after in-vitro fertilisation: a prospective study of 144018 treatment cycles

Background The extent to which baseline couple characteristics affect the probability of live birth and adverse perinatal outcomes after assisted conception is unknown. Methods and Findings We utilised the Human Fertilisation and Embryology Authority database to examine the predictors of live birth in all in vitro fertilisation (IVF) cycles undertaken in the UK between 2003 and 2007 (n = 144,018). We examined the potential clinical utility of a validated model that pre-dated the introduction of intracytoplasmic sperm injection (ICSI) as compared to a novel model. For those treatment cycles that resulted in a live singleton birth (n = 24,226), we determined the associates of potential risk factors with preterm birth, low birth weight, and macrosomia. The overall rate of at least one live birth was 23.4 per 100 cycles (95% confidence interval [CI] 23.2–23.7). In multivariable models the odds of at least one live birth decreased with increasing maternal age, increasing duration of infertility, a greater number of previously unsuccessful IVF treatments, use of own oocytes, necessity for a second or third treatment cycle, or if it was not unexplained infertility. The association of own versus donor oocyte with reduced odds of live birth strengthened with increasing age of the mother. A previous IVF live birth increased the odds of future success (OR 1.58, 95% CI 1.46–1.71) more than that of a previous spontaneous live birth (OR 1.19, 95% CI 0.99–1.24); p-value for difference in estimate <0.001. Use of ICSI increased the odds of live birth, and male causes of infertility were associated with reduced odds of live birth only in couples who had not received ICSI. Prediction of live birth was feasible with moderate discrimination and excellent calibration; calibration was markedly improved in the novel compared to the established model. Preterm birth and low birth weight were increased if oocyte donation was required and ICSI was not used. Risk of macrosomia increased with advancing maternal age and a history of previous live births. Infertility due to cervical problems was associated with increased odds of all three outcomes—preterm birth, low birth weight, and macrosomia. Conclusions Pending external validation, our results show that couple- and treatment-specific factors can be used to provide infertile couples with an accurate assessment of whether they have low or high risk of a successful outcome following IVF

Axonal inclusions in spinocerebellar ataxia type 3

Protein aggregation is a major pathological hallmark of many neurodegenerative disorders including polyglutamine diseases. Aggregation of the mutated form of the disease protein ataxin-3 into neuronal nuclear inclusions is well described in the polyglutamine disorder spinocerebellar ataxia type 3 (SCA3 or Machado–Joseph disease), although these inclusions are not thought to be directly pathogenic. Neuropil aggregates have not yet been described in SCA3. We performed a systematic immunohistochemical study of serial thick sections through brains of seven clinically diagnosed and genetically confirmed SCA3 patients. Using antibodies against ataxin-3, p62, ubiquitin, the polyglutamine marker 1C2 as well as TDP-43, we analyzed neuronal localization, composition and distribution of aggregates within SCA3 brains. The analysis revealed widespread axonal aggregates in fiber tracts known to undergo neurodegeneration in SCA3. Similar to neuronal nuclear inclusions, the axonal aggregates were ubiquitinated and immunopositive for the proteasome and autophagy associated shuttle protein p62, indicating involvement of neuronal protein quality control mechanisms. Rare TDP-43 positive axonal inclusions were also observed. Based on the correlation between affected fiber tracts and degenerating neuronal nuclei, we hypothesize that these novel axonal inclusions may be detrimental to axonal transport mechanisms and thereby contribute to degeneration of nerve cells in SCA3

High-throughput sequencing of Astrammina rara: Sampling the giant genome of a giant foraminiferan protist

<p>Abstract</p> <p>Background</p> <p>Foraminiferan protists, which are significant players in most marine ecosystems, are also genetic innovators, harboring unique modifications to proteins that make up the basic eukaryotic cell machinery. Despite their ecological and evolutionary importance, foraminiferan genomes are poorly understood due to the extreme sequence divergence of many genes and the difficulty of obtaining pure samples: exogenous DNA from ingested food or ecto/endo symbionts often vastly exceed the amount of "native" DNA, and foraminiferans cannot be cultured axenically. Few foraminiferal genes have been sequenced from genomic material, although partial sequences of coding regions have been determined by EST studies and mass spectroscopy. The lack of genomic data has impeded evolutionary and cell-biology studies and has also hindered our ability to test ecological hypotheses using genetic tools.</p> <p>Results</p> <p>454 sequence analysis was performed on a library derived from whole genome amplification of microdissected nuclei of the Antarctic foraminiferan <it>Astrammina rara</it>. Xenogenomic sequence, which was shown not to be of eukaryotic origin, represented only 12% of the sample. The first foraminiferal examples of important classes of genes, such as tRNA genes, are reported, and we present evidence that sequences of mitochondrial origin have been translocated to the nucleus. The recovery of a 3' UTR and downstream sequence from an actin gene suggests that foraminiferal mRNA processing may have some unusual features. Finally, the presence of a co-purified bacterial genome in the library also permitted the first calculation of the size of a foraminiferal genome by molecular methods, and statistical analysis of sequence from different genomic sources indicates that low-complexity tracts of the genome may be endoreplicated in some stages of the foraminiferal life cycle.</p> <p>Conclusions</p> <p>These data provide the first window into genomic organization and genetic control in these organisms, and also complement and expands upon information about foraminiferal genes based on EST projects. The genomic data obtained are informative for environmental and cell-biological studies, and will also be useful for efforts to understand relationships between foraminiferans and other protists.</p

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

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

RNA-Seq Identifies SNP Markers for Growth Traits in Rainbow Trout

Fast growth is an important and highly desired trait, which affects the profitability of food animal production, with feed costs accounting for the largest proportion of production costs. Traditional phenotype-based selection is typically used to select for growth traits; however, genetic improvement is slow over generations. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals; therefore, they are most suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. SNPs found within or near a coding sequence are of particular interest because they are more likely to alter the biological function of a protein. We aimed to use SNPs to identify markers and genes associated with genetic variation in growth. RNA-Seq whole-transcriptome analysis of pooled cDNA samples from a population of rainbow trout selected for improved growth versus unselected genetic cohorts (10 fish from 1 full-sib family each) identified SNP markers associated with growth-rate. The allelic imbalances (the ratio between the allele frequencies of the fast growing sample and that of the slow growing sample) were considered at scores >5.0 as an amplification and <0.2 as loss of heterozygosity. A subset of SNPs (n = 54) were validated and evaluated for association with growth traits in 778 individuals of a three-generation parent/offspring panel representing 40 families. Twenty-two SNP markers and one mitochondrial haplotype were significantly associated with growth traits. Polymorphism of 48 of the markers was confirmed in other commercially important aquaculture stocks. Many markers were clustered into genes of metabolic energy production pathways and are suitable candidates for genetic selection. The study demonstrates that RNA-Seq at low sequence coverage of divergent populations is a fast and effective means of identifying SNPs, with allelic imbalances between phenotypes. This technique is suitable for marker development in non-model species lacking complete and well-annotated genome reference sequences

Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease