Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

• Premise of the study: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations.• Methods and Results: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143.• Conclusions: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

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ObjectiveThe objectives of this study were to evaluate whether dose to the vasculature is associated with local control after surgery in patients with borderline resectable (BLR) and resectable pancreatic cancer (PCA) receiving neoadjuvant radiation therapy (RT) and to identify a dose threshold for clinical use.MethodsPatients with BLR and resectable PCA treated with neoadjuvant RT were retrospectively reviewed. During this period, the institutional paradigm shifted from standard fractionation to hypofractionation/stereotactic body radiation therapy (SBRT). A vasculature clinical target volume (Vasc CTV) was contoured for each patient and defined as a 5-mm margin around the superior mesenteric artery (SMA) from its origin to the pancreatic head, the celiac artery from its origin to the level of the trifurcation and any involved vein. The Vasc CTV D95 was normalized to a 2-Gy equivalent dose to determine the optimal dose associated with optimal local failure-free survival (LFFS).ResultsForty-seven patients were included in the analysis. A Vasc CTV D95 of 32.7 Gy was the optimal cutoff for LFFS. Patients with Vasc CTV D95 Equivalent dose in 2 Gy per fraction (EQD2) >32.7 Gy had significantly longer LFFS compared to patients with Vasc CTV D95 EQD2 ≤32.7 Gy at 12 months (91% vs. 51%, respectively) and 24 months (86% vs. 12%, respectively). The median disease-free survival (DFS) for patients with EQD2 >32.7 Gy was 30.4 months compared to 14.0 months in patients with EQD2 ≤32.7 Gy (p = 0.01). There was no significant difference in overall survival (OS) between the two groups.ConclusionsDuring neoadjuvant treatment, dose to the Vasc CTV is associated with durability of local control (LC) after resection and should be intentionally included in the treatment volume with an EQD2 goal of 31–33 Gy.

Background Patients with rectal cancer undergo preoperative neoadjuvant chemoradiation with approximately 70% exhibiting pathologic down-staging in response to treatment. There is currently no accurate test to predict patients who are likely to be complete responders to therapy. 5-Fluorouracil (5-FU) is regularly used in the neoadjuvant treatment of rectal cancer. Genetic polymorphisms affect the activity of thymidylate synthase (TS), an enzyme involved in 5-FU metabolism. This may account for observed differences in response to neoadjuvant treatment between patients. Detection of such polymorphisms might identify patients likely to have a complete response to neoadjuvant therapy and perhaps allow them to avoid surgery. Methods DNA was isolated from whole blood taken from patients with newly diagnosed rectal cancer who received neoadjuvant therapy (n=50). Response to therapy was calculated with a tumor regression score, based upon histology, from the time of surgery. PCR was performed targeting the promoter region of TS. PCR products were separated using electrophoresis to determine whether patients were homozygous for a double-tandem repeat (2R), a triple-tandem repeat (3R), or heterozygous (2R/3R). A single nucleotide polymorphism (SNP), 3G or 3C, may also be present in the second repeat unit of the 3R allele. Restriction fragment length polymorphism assays were performed in patients with at least one 3R allele using HaeIII. Results Patients with at least one TS 3G allele were more likely to have complete or partial pathological response to 5-FU neoadjuvant therapy (OR 10.4 95% CI 1.3–81.6) (p=0.01) than those without at least one 3G allele. Conclusions Identification of patients with specific genetic polymorphisms in enzymes involved in 5-FU metabolism appears to predict the likelihood of complete or partial pathologic response of rectal cancer to preoperative neoadjuvant therapy.

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