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The aggregation and spread of alpha-synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the establishment of a safe and effective disease-modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB-Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat-labile Escherichia coli enterotoxin [LTB] as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB-Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB-Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 μg/mL. Furthermore, the purified soluble rLTB-Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB-Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies., (© 2023 American Institute of Chemical Engineers.)

2,7'-Cyclolignans constitute a group of compounds regularly employed in clinical contexts due to their antiviral and anticancer properties as expressed in a number of already available designed drugs. Possibilities for other therapeutic developments are indicated. All commercial preparations actually originate from a single compound, podophyllotoxin, currently extracted from a few limited-in-abundance species of Podophyllum. This supply problem has stimulated considerable interest in the development of alternative strategies offering greater sustainable availability of the compound at affordable costs. Approaches such as plant breeding and micropropagation of the high-producing Podophyllum species have been explored with the aim of establishing production of these compounds. Since 20 years of exploration of the total chemical synthesis of podophyllotoxin has not proved economically viable, extensive research has now been undertaken to find methods for stimulating the accumulation of 2,7'-cyclolignans using tissue cultures. Both undifferentiated and differentiated cell cultures, mainly from Linum, Podophyllum, Juniperus, Callitris, Anthriscus and Forsynthia genera, have been reported. Although considerable progress has been made concerning the concentration and productivity of certain strains of Linum album, no economically viable production system has been established. Exploration and development of biosynthetical pathways leading to 2,7'-cyclolignans may open paths for future metabolic engineering, such as the bioconversion of deoxypodophyllotoxin to epipodophyllotoxin employing a human hepatic enzyme heterologously expressed in Escherichia coli.

In vitro growth of Solanum chrysotrichum hairy roots was carried out in three different types of reactors: shake flasks, a glass-draught internal-loop 2-L basic design airlift reactor (BDR), and a novel modified mesh-draught with wire-helixes 2-L reactor (MR). In each of them, the growth patterns were different, as well as some of the dynamic parameters. The specific growth rates were 0.08, 0.067, and 0.112 d(-1) for shake flasks, BDR, and MR, respectively. In shake flasks and in the MR, growth followed first-order kinetics. In the MR without roots, superficial liquid velocity in the riser and downcomer ranged from 2.1 to 2.7 and 1.4 to 1.7 cm s(-1), respectively (nearly the same as the BDR values). After 42 days in culture, tissue density in the MR was twice that found in the BDR and about the same as that found in the shake flasks. At the tissue densities reached at 42 days, superficial liquid velocities in the MR and BDR downcomers were 4-5 and 7-8 times lower, and mixing times were 11 and 18 times longer than those observed without roots. Tissue densities measured at three points in the MR's downcomer and riser ranged from 10.21 to 12.17 and 4.94 to 5.24 gDW L(-1) respectively. Dynamic gas hold-up dropped faster when roots grew radially in the mesh-draught. In addition, root cultures were scaled-up in a 10-L MR reactor in which some geometric relations were maintained, such as the Q/V radio. Growth in 10-L MR followed first-order kinetics, but despite this, specific growth velocity was 0.09 d(-1) and overall tissue density diminished slightly with respect to that of the 2-L MR. Tissue inoculation, distribution, and harvest were more easily accomplished in the MRs.