Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

Autographa Californica Multiple Nucleopolyhedrovirus P48(Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nudeopolyhedrovirus(AcMNPV)p48(acl03)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs).However,the exact role of p48 in the morphogenesis of ODVs remains unknown.In this study,we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles.To further understand its functional role in intranuclear microvesicle formation,we characterized the distribution of the P48 protein,which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs.In AcMNPV-infected cells,P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs,with a limited but discernible distribution in the plasma membrane,nuclear envelope,intranuclear microvesicles,and ODV envelope.Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation,F48 associated with Ac93 in the absence of viral infection.

Nerve transfer with 3D-printed branch nerve conduits

Background:Nerve transfer is an important clinical surgical procedure for nerve repair by the coaptation of a healthy donor nerve to an injured nerve.Usually,nerve transfer is performed in an end-to-end manner,which will lead to functional loss of the donor nerve.In this study,we aimed to evaluate the efficacy of 3D-printed branch nerve conduits in nerve transfer.Methods:Customized branch conduits were constructed using gelatine-methacryloyl by 3D print-ing.The nerve conduits were characterized both in vitro and in vivo.The efficacy of 3D-printed branch nerve conduits in nerve transfer was evaluated in rats through electrophysiology testing and histological evaluation.Results:The results obtained showed that a single nerve stump could form a complex nerve network in the 3D-printed multibranch conduit.A two-branch conduit was 3D printed for transferring the tibial nerve to the peroneal nerve in rats.In this process,the two branches were connected to the distal tibial nerve and peroneal nerve.It was found that the two nerves were successfully repaired with functional recovery.Conclusions:It is implied that the two-branch conduit could not only repair the peroneal nerve but also preserve partial function of the donor tibial nerve.This work demonstrated that 3D-printed branch nerve conduits provide a potential method for nerve transfer.

3D printing of functional nerve guide conduits

Nerve guide conduits(NGCs),as alternatives to nerve autografts and allografts,have been widely explored as an advanced tool for the treatment of peripheral nerve injury.However,the repairing efficiency of NGCs still needs significant improvements.Functional NGCs that provide a more favorable microenvironment for promoting axonal elongation and myelination are of great importance.In recent years,3D printing technologies have been widely applied in the fabrication of customized and complex constructs,exhibiting great potential for tissue engineering applications,especially for the construction of functional NGCs.In this review,we introduce the 3D printing technologies for manufacturing functional NGCs,including inkjet printing,extrusion printing,stereolithographybased printing and indirect printing.Further,we summarize the current methods and strategies for constructing functional NGCs,such as designing special conduit architectures,using appropriate materials and co-printing with different biological cues.Finally,the challenges and prospects for construction of functional NGCs are also presented.