Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data

The Photo-Activatable Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation (PAR-CLIP) method was recently developed for global identification of RNAs interacting with proteins. The strength of this versatile method results from induction of specific T to C transitions at sites of interaction. However, current analytical tools do not distinguish between non-experimentally and experimentally induced transitions. Furthermore, geometric properties at potential binding sites are not taken into account. To surmount these shortcomings, we developed a two-step algorithm consisting of a non-parametric two-component mixture model and a wavelet-based peak calling procedure. Our algorithm can reduce the number of false positives up to 24% thereby identifying high confidence interaction sites. We successfully employed this approach in conjunction with a modified PAR-CLIP protocol to study the functional role of nuclear Moloney leukemia virus 10, a putative RNA helicase interacting with Argonaute2 and Polycomb. Our method, available as the R package wavClusteR, is generally applicable to any substitution-based inference problem in genomics.     

Decision-making during NHEJ: a network of interactions in human Polμ implicated in substrate recognition and end-bridging

Human Polμ is a DNA polymerase belonging to the X family that has been implicated in the non-homologous end-joining (NHEJ) pathway during repair of double-strand breaks in DNA. Loop1 is a flexible piece of Polμ which has a critical role during terminal transferase and end-joining activities: it acts as a pseudo-template when the template strand is discontinuous or unavailable, whilst diffusing away if present to avoid steric clashes. Mutational analysis and inspection of the 3D structures available allowed us to identify a network of residues in charge of sensing the presence or absence of discontinuities in the template strand, which will in turn determine the final position adopted by Loop1. This network is formed by the previously uncharacterized thumb mini-loop (NSH motif) and the positively charged helix N, which contribute to the correct positioning of Loop1 and to juxtapose the discontinuous template strand during NHEJ of incompatible ends. Accordingly, single mutation of specific conserved residues in these motifs, whilst irrelevant in most of the cases for gap filling, largely affected terminal transferase and end-joining activities. Other point mutations in the ‘hinges’ of Loop1, such as residues Phe³⁸⁵ or Phe³⁸⁹, corroborated the flexibility requirements of this motif.     

New perspectives in radiation oncology: Young radiation oncologist point of view and challenges

AimTo assess the role of the young radiation oncologist in the context of important recent advancements in the field of radiation oncology, and to explore new perspectives and competencies of the young radiation oncologist.BackgroundRadiation oncology is a field that has rapidly advanced over the last century. It holds a rich tradition of clinical care and evidence-based practice, and more recently has advanced with revolutionary innovations in technology and computer science, as well as pharmacology and molecular biology.Materials and methodsSeveral young radiation oncologists from different countries evaluated the current status and future directions of radiation oncology.ResultsFor young radiation oncologists, it is important to reflect on the current practice and future directions of the specialty as it relates to the role of the radiation oncologist in the comprehensive management of cancer patients. Radiation oncologists are responsible for the radiation treatment provided to patients and its subsequent impact on patients’ quality of life. Young radiation oncologists must proactively master new clinical, biological and technical information, as well as lead radiation oncology teams consisting of physicists, dosimetrists, nurses and technicians.ConclusionsThe role of the young radiation oncologist in the field of oncology should be proactive in developing new competencies. Above all, it is important to remember that we are dealing with the family members and loved ones of many individuals during the most difficult part of their lives.     

Halobacterial megaplasmids are negatively supercoiled

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal-field alternating gel electro-phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.     

Deconstructing the long-standing a priori assumption that serial homology generally involves ancestral similarity followed by anatomical divergence

It has long been assumed that serial homologues are ancestrally similar—polysomerism resulting from a “duplication” or “repetition” of forms—and then often diverge—anisomerism, for example, as they become adapted to perform different tasks as is the case with the forelimb and hind limbs of humans. However, such an assumption, with crucial implications for comparative, evolutionary, and developmental biology, and for evolutionary developmental biology, has in general not really been tested by a broad analysis of the available empirical data. Perhaps not surprisingly, more recent anatomical comparisons, as well as molecular knowledge of how, for example, serial appendicular structures are patterned along with different anteroposterior regions of the body axis of bilateral animals, and how “homologous” patterning domains do not necessarily mark “homologous” morphological domains, are putting in question this paradigm. In fact, apart from showing that many so-called “serial homologues” might not be similar at all, recent works have shown that in at least some cases some “serial” structures are indeed more similar to each other in derived taxa than in phylogenetically more ancestral ones, as pointed out by authors such as Owen. In this article, we are taking a step back to question whether such assumptions are actually correct at all, in the first place. In particular, we review other cases of so-called “serial homologues” such as insect wings, arthropod walking appendages, Dipteran thoracic bristles, and the vertebrae, ribs, teeth, myomeres, feathers, and hairs of chordate animals. We show that: (a) there are almost never cases of true ancestral similarity; (b) in evolution, such structures—for example, vertebra—and/or their subparts—for example, “transverse processes”—many times display trends toward less similarity while in many others display trends toward more similarity, that is, one cannot say that there is a clear, overall trend to anisomerism.     

Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.     

Temperature influences the expression of fimbriae and flagella in <Emphasis Type="Italic">Hafnia alvei</Emphasis> strains: an immunofluorescence study

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.