siRNA-induced immunostimulation through TLR7 promotes antitumoral activity against HPV-driven tumors in vivo

Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2′O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy

Optimal Conservation of Migratory Species

Background. Migratory animals comprise a significant portion of biodiversity worldwide with annual investment for their conservation exceeding several billion dollars. Designing effective conservation plans presents enormous challenges. Migratory species are influenced by multiple events across land and sea-regions that are often separated by thousands of kilometres and span international borders. To date, conservation strategies for migratory species fail to take into account how migratory animals are spatially connected between different periods of the annual cycle (i.e. migratory connectivity) bringing into question the utility and efficiency of current conservation efforts. Methodology/Principal Findings. Here, we report the first framework for determining an optimal conservation strategy for a migratory species. Employing a decision theoretic approach using dynamic optimization, we address the problem of how to allocate resources for habitat conservation for a Neotropical-Nearctic migratory bird, the American redstart Setophaga ruticilla, whose winter habitat is under threat. Our first conservation strategy used the acquisition of winter habitat based on land cost, relative bird density, and the rate of habitat loss to maximize the abundance of birds on the wintering grounds. Our second strategy maximized bird abundance across the entire range of the species by adding the constraint of maintaining a minimum percentage of birds within each breeding region in North America using information on migratory connectivity as estimated from stable-hydrogen isotopes in feathers. We show that failure to take into account migratory connectivity may doom some regional populations to extinction, whereas including information on migratory connectivity results in the protection of the species across its entire range. Conclusions/Significance. We demonstrate that conservation strategies for migratory animals depend critically upon two factors: knowledge of migratory connectivity and the correct statement of the conservation problem. Our framework can be used to identify efficient conservation strategies for migratory taxa worldwide, including insects, birds, mammals, and marine organisms

Improving the prediction of disease-related variants using protein three-dimensional structure

Background: Single Nucleotide Polymorphisms (SNPs) are an important source of human genome variability. Non-synonymous SNPs occurring in coding regions result in single amino acid polymorphisms (SAPs) that may affect protein function and lead to pathology. Several methods attempt to estimate the impact of SAPs using different sources of information. Although sequence-based predictors have shown good performance, the quality of these predictions can be further improved by introducing new features derived from three-dimensional protein structures.Results: In this paper, we present a structure-based machine learning approach for predicting disease-related SAPs. We have trained a Support Vector Machine (SVM) on a set of 3,342 disease-related mutations and 1,644 neutral polymorphisms from 784 protein chains. We use SVM input features derived from the protein's sequence, structure, and function. After dataset balancing, the structure-based method (SVM-3D) reaches an overall accuracy of 85%, a correlation coefficient of 0.70, and an area under the receiving operating characteristic curve (AUC) of 0.92. When compared with a similar sequence-based predictor, SVM-3D results in an increase of the overall accuracy and AUC by 3%, and correlation coefficient by 0.06. The robustness of this improvement has been tested on different datasets and in all the cases SVM-3D performs better than previously developed methods even when compared with PolyPhen2, which explicitly considers in input protein structure information.Conclusion: This work demonstrates that structural information can increase the accuracy of disease-related SAPs identification. Our results also quantify the magnitude of improvement on a large dataset. This improvement is in agreement with previously observed results, where structure information enhanced the prediction of protein stability changes upon mutation. Although the structural information contained in the Protein Data Bank is limiting the application and the performance of our structure-based method, we expect that SVM-3D will result in higher accuracy when more structural date become available. \ua9 2011 Capriotti; licensee BioMed Central Ltd

The mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies

Urinary C-Peptide Measurement as a Marker of Nutritional Status in Macaques

Studies of the nutritional status of wild animals are important in a wide range of research areas such as ecology, behavioural ecology and reproductive biology. However, they have so far been strongly limited by the indirect nature of the available non-invasive tools for the measurement of individual energetic status. The measurement of urinary C-peptide (UCP), which in humans and great apes shows a close link to individual nutritional status, may be a more direct, non-invasive tool for such studies in other primates as well and possibly even in non-primate mammals. Here, we test the suitability of UCPs as markers of nutritional status in non-hominid primates, investigating relationships between UCPs and body-mass-index (BMI), skinfold fatness, and plasma C-peptide levels in captive and free-ranging macaques. We also conducted a food reduction experiment, with daily monitoring of body weight and UCP levels. UCP levels showed significant positive correlations with BMI and skinfold fatness in both captive and free-ranging animals and with plasma C-peptide levels in captive ones. In the feeding experiment, UCP levels were positively correlated with changes in body mass and were significantly lower during food reduction than during re-feeding and the pre-experimental control condition. We conclude that UCPs may be used as reliable biomarkers of body condition and nutritional status in studies of free-ranging catarrhines. Our results open exciting opportunities for energetic studies on free-ranging primates and possibly also other mammals

Junín Virus Infection of Human Hematopoietic Progenitors Impairs In Vitro Proplatelet Formation and Platelet Release via a Bystander Effect Involving Type I IFN Signaling

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN α/β) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2−/− mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels

Identification of Amino Acids in HA and PB2 Critical for the Transmission of H5N1 Avian Influenza Viruses in a Mammalian Host

Since 2003, H5N1 influenza viruses have caused over 400 known cases of human infection with a mortality rate greater than 60%. Most of these cases resulted from direct contact with virus-contaminated poultry or poultry products. Although only limited human-to-human transmission has been reported to date, it is feared that efficient human-to-human transmission of H5N1 viruses has the potential to cause a pandemic of disastrous proportions. The genetic basis for H5N1 viral transmission among humans is largely unknown. In this study, we used guinea pigs as a mammalian model to study the transmission of six different H5N1 avian influenza viruses. We found that two viruses, A/duck/Guangxi/35/2001 (DKGX/35) and A/bar-headed goose/Qinghai/3/2005(BHGQH/05), were transmitted from inoculated animals to naïve contact animals. Our mutagenesis analysis revealed that the amino acid asparagine (Asn) at position 701 in the PB2 protein was a prerequisite for DKGX/35 transmission in guinea pigs. In addition, an amino acid change in the hemagglutinin (HA) protein (Thr160Ala), resulting in the loss of glycosylation at 158–160, was responsible for HA binding to sialylated glycans and was critical for H5N1 virus transmission in guinea pigs. These amino acids changes in PB2 and HA could serve as important molecular markers for assessing the pandemic potential of H5N1 field isolates