Significance Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.
One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 mug/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.
Abstract In the face of new emerging respiratory viruses, such as SARS-CoV2, vaccines, and drug therapies are not immediately available to curb the spread of the infection. Non-pharmaceutical interventions, such as mask-wearing and social distance, can slow the transmission. However, both mask and social distance are not 100% effective at preventing the spread of respiratory viruses, such as SARS-CoV2 and influenza viruses. There is an urgent need to develop an intervention that could reduce the spread of respiratory viruses. Rhamnolipids are environmentally friendly and biologically safe surfactants that can kill enveloped viruses. Two rhamnolipid products, 222A and 222B, were investigated in this study to determine their ability to inactivate two enveloped viruses, bovine coronavirus and herpes simplex virus 1. We found that 222B at 0.005%, which has no toxicity to cells, can inactivate 10 5 PFU/ml enveloped viruses in 3-5 min. Moreover, 50-100µl of 222B at 0.005% on 1 cm 2 mask fabrics can inactivate ~ 10 3 PFU /10 µl in 3-5 min. These results suggest that 222B can be coated on masks to prevent or reduce the spread of enveloped viruses.
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