From δ-aminolevulinic acid to chlorophylls and every step in between: in memory of Constantin (Tino) A. Rebeiz, 1936–2019

Photosynthesis Research - Constantin A. (Tino) Rebeiz, a pioneer in the field of chlorophyll biosynthesis, and a longtime member of the University of Illinois community of plant biologists, passed...     

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Translocation for conservation: Neonates are less suitable than adults

Animal behaviour can affect the outcome of conservation translocations. It is important to understand the behaviour of the species being considered for translocation and how its behaviour varies over life stages. There may be uncertainty about what life stages are best as founders for release back into wild populations. A technique called head‐starting whereby juvenile life stages are raised in captivity and then released is one potential pre‐release strategy. However, juveniles of many species have a dispersive role in the life cycle, potentially raising difficulties for establishing new populations due to dispersal from the intended habitat following release. For this study, we compared aspects of the behaviour of captive adult and neonate pygmy bluetongue lizards (Tiliqua adelaidensis) – an endangered species for which translocation is likely to be an important management strategy – to determine if neonate behavioural characteristics are appropriate for their translocation. We filmed adult and neonate pygmy bluetongue lizards and compared their behaviour. We also filmed adults over an activity season to compare seasonal behaviour. Behavioural parameters measured included basking time, burrow exits, burrows occupied and walking the perimeter wall. Neonates basked significantly more than adults in summer and autumn. Neonates are likely to be basking more than adults because they are in a stage of rapid growth and need to gain body mass before the winter inactivity period. Neonates exited burrows more often than adults and used a greater number of burrows. These results indicate neonate lizards are actively exploring their habitat. Neonates are unlikely to be as suitable for translocation as they are actively moving about and more likely to be predated upon or disperse from the translocation site. Our finding can be applied to other species that have active juvenile life stages and are at particular risk of predation due to their small size.