Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well.

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low‐cost production, although low yields remain the major challenge. The studies on scaling‐up to pilot‐scale bioreactors, especially in conventional stainless‐steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled‐up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40‐L pilot‐scale STB. Employing cyclical operation together with a simplified‐process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab‐scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46–58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot‐scale STB to be equivalent to the lab‐scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L−1 day−1, respectively, which are comparable to the lab‐scale culture. Here, we demonstrate pilot‐scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long‐term, reproducible, and sustained production of rrBChE.

Malaria is a devastating disease that resulted in an estimated 627,000 deaths in 2020. About 80% of those deaths were among children under the age of five. Our approach is to develop small molecule inhibitors against cytoskeletal targets that are vital components of parasite function, essential at multiple stages of parasite infection, can be targeted with high specificity, and are highly druggable. Here we describe KNX-115, which inhibits purified Plasmodium falciparum myosin A (PfMyoA) actin-activated ATPase with a potency in the 10s of nanomolar range and >50-fold selectivity against cardiac, skeletal, and smooth muscle myosins. KNX-115 inhibits the blood and liver stages of Plasmodium with an EC50 of about 100 nanomolar, with negligible liver cell toxicity. In addition, KNX-115 inhibits sporozoite cell traversal and blocks the gametocyte to oocyst conversion in the mosquito. KNX-115 displays a similar killing profile to pyrimethamine and parasites are totally killed after 96 hours of treatment. In line with its novel mechanism of action, KNX-115 is equally effective at inhibiting a panel of Plasmodium strains resistant to experimental and marketed antimalarials. In vitro evolution data likely suggests a refractory potential of KNX-115 in developing parasite resistance.

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low-cost production, although low yields remain the major challenge. The studies on scaling-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically-regulated transgenic rice cell suspension cultures in a 40-L pilot-scale STB. Employing cyclical operation together with a simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale (5-L) bioreactor studies, we found consistent maximum total active rrBChE production level of 46-58 μg/g fresh weight in four cycles over 82 days of continuous operation.Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot-scale STB to be equivalent to the lab-scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 μg/g fresh weight and 387 μg L-1 day-1, respectively, which are comparable to the lab-scale culture. Here, we demonstrate pilot scale bioreactor performance using a metabolically-regulated transgenic rice cell culture for long-term, reproducible, and sustained production of rrBChE.

Highly detailed steered Molecular Dynamics simulations are performed on differently glycosylated receptor binding domains of the SARS-CoV-2 spike protein. The binding strength and the binding range increases with glycosylation. The interaction energy rises very quickly with pulling the proteins apart and only slowly drops at larger distances. We see a catch slip type behavior where interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode are hydrogen bonds but Lennard-Jones and electrostatic interactions are relevant as well.