Novel microsatellite loci for Sebaea aurea (Gentianaceae) and cross-amplification in related species.

[Premise of the study] Microsatellite loci were developed in Sebaea aurea (Gentianaceae) to investigate the functional role of diplostigmaty (i.e., the presence of additional stigmas along the style).[Methods and Results] One hundred seventy-four and 180 microsatellite loci were isolated through 454 shotgun sequencing of genomic and microsatellite-enriched DNA libraries, respectively. Sixteen polymorphic microsatellite loci were characterized, and 12 of them were selected to genotype individuals from two populations. Microsatellite amplifi cation was conducted in two multiplex groups, each containing six microsatellite loci. Cross-species amplifi cation was tested in seven other species of Sebaea . The 12 novel microsatellite loci amplifi ed only in the two most closely related species to S. aurea (i.e., S. ambigua and S. minutifl ora ) and were also polymorphic in these two species.[Conclusions] These results demonstrate the usefulness of this set of newly developed microsatellite loci to investigate the mating system and population genetic structure in S. aurea and related species.We acknowledge grants to J.K. from the Swiss National Science Foundation (PA00P3_129140) and the Velux Stiftung (project no. 679) and to J.G.S.-M. from a postdoctoral research contract “Ramón y Cajal” from the Ministerio de Ciencia e Innovación (MICINN), Spain.Peer Reviewe

Evolutionary factors affecting the cross-species utility of newly developed microsatellite markers in seabirds

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio-temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased non-amplification, or disruptions that may lead to decreased polymorphism in non-target species. Furthermore, high mutation rates and constraints on allele size may also lead, with evolutionary time, to an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next-generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub-Antarctic seabird, the thin-billed prion (Pachyptila belcheri), that we tested for cross-species amplification in other Pachyptila and related sub-Antarctic species. We found that heterozygosity decreased and the proportion of non-amplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific FST provided better approximations of mtDNA relationships among the studied species than those estimated using DC, even though FST was more affected by null alleles. We observed a significantly non-linear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross-species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa

Anti-HIV-1 Response Elicited in Rabbits by Anti-Idiotype Monoclonal Antibodies Mimicking the CD4-Binding Site

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies

NGS shotgun data from Pachytila belcheri

These are the 454 sequencing reads generated from two Pachyptila belcheri individuals (Kerguelen Archepelago) from which the 26 microsatellite loci used in this study were isolated

Prions_Msat_genotypes_by_Species_Final

These are the genotypes for the 25 unlinked microsatellite loci analysed in this paper. The Excel file is in "Covert" format, divided into species populations for easy conversion into any other data format

Tree files and pairwise distance matrices

These are the MEGA input distance matrices for Fst and DC distances calculated from 25 microsatellite loci. Also included are the newick format tree files for each matric. These are the trees presented in Figure 2A and B

Cytochrome B ALignment and Tree

Cytochrome B ALignment and Tre

Imaging Techniques for Postmortem Forensic Radiology

Postmortem forensic radiology aims to acquisition, interpretation, and reporting of radiologic images for the purpose of forensic investigations, in the living as well as the deceased. Conventional radiology still remains the most common modality used in the forensic setting and the gold standard method for many forensic challenges. X-rays are commonly used for visualization and localization of foreign bodies, and for body identification and identities confirmation. Computed tomography (PMCT) is the most frequent imaging tool in forensic pathology besides X-ray. Indications of PMCT are especially focused on cases of unnatural deaths: traumatic events such as bone fractures and nonaccidental injury in children; gunshot injuries; hanging, strangulation, and drowning cases; putrefied, carbonized, and badly damaged bodies. In order to visualize the soft tissue, especially organs, MRI can be used. Although this technique has the potential to overcome the limitations of PCMT, it is only rarely used in forensic imaging as it is a complex technology requiring specific training, expensive, and with some complication in execution due to body size, artifact, and protocols. MRI is of special significance for the diagnosis of natural death, especially related to diseases of the cardiovascular or central nervous system, and for investigations concerning neonatal and perinatal deaths. Comparing the results of postmortem imaging with subsequent autopsies, rates of major discrepancies between cause of death identified by radiology and autopsy of 32% for PMCT, 43% for PMMR, and 30% for PMCT + PMMRI have been reported. Vice versa, different studies have demonstrated that PMCT or PMMRI in conjunction with conventional postmortem examinations can augment the value of postmortem examinations, providing more information than either examination alone