Improving baculovirus recombination

Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation.

First contribution of mites (Acari) to the forensic analysis of hanged corpses: a case study from Spain

This case study from North Spain, highlights the importance of the collection of mites in addition to insects, from crime scenes or corpses subjected to environmental constraints that reduce or minimise insect activity, such as hanged corpses. In addition, this analysis highlights the relevance of arthropods’ collection in the field, even after the corpse has been moved away for autopsy. Four species of mites, phoretic on carrion (Silphidae) and rove (Staphylinidae) beetles, complemented and reinforced the autopsy analysis as well as the scarce information provided by insect activity. Poecilochirus carabi Canestrini & Canestrini, 1882 and Poecilochirus (Physoparasitus) davydovae Hyatt, 1980 (Mesostigmata: Parasitidae) were found in association with two Silphidae, Nicrophorus Fabricius, 1775 and Necrodes Leach, 1815, only when sampled in the autopsy room; this is suggestive of host-switching of mites and was likely due to the lack of availability of specific carriers in the field. The interpretation of the activity of Parasitidae mites both in the field and the autopsy room allows a better understanding of the timing and circumstances of decomposition. Phoretic deutonymphs of Pelzneria Scheucher 1957 (Astigmata: Histiostomatidae) were highly abundant, mostly P. crenulata Oudemans, 1909 and are reported for the first time on a Staphylinidae rove beetle, Creophilus maxillosus (L., 1758). Surprisingly, in this case study no Pelzneria were associated with the Silphidae found, which are their most common hosts, such as Necrodes littoralis (L., 1758) and Nicrophorus interruptus (Stephens, 1830). All histiostomatids were removed from the staphylinid (rove beetle) collected from the soil, at the scene of death, suggesting a recent arrival of the beetle. The occurrence of Staphylinidae beetles and their associated mites, such as Parasitidae and Pelzneria, and the information they provided would have been easily overlooked or lost if only the autopsy sampling would have been considered in the analysis of the case. The four mite species are reported for the first time for the Iberian Peninsula.

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.