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Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

• Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. • We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. • SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single ‘A-type’ granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. • We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Accumulating evidence supports pharmacological roles for the plant stilbene, resveratrol in aging and disease contexts. Pterostilbene, a derivative of resveratrol, has been shown to have higher bioavailability and bio-efficacy than resveratrol, but is not abundant in natural sources. Previous work demonstrated that expression of the stilbene synthase gene, isolated from grapevine Vitis vinifera, under the control of a plant-wide promoter, led to a variety of growth and fertility problems in the transformed plants. To circumvent this problem two transgenic tomato lines were developed in this thesis to produce resveratrol and pterostilbene specifically in the fruit of tomato. An ex vivo full thickness human skin explant model was used to investigate the biological effects of aqueous tomato juices on normal human skin, and in an inflammatory disease-like context by treatment with inflammatory cytokines. Microarray analysis and qRT-PCR validation identified the differential regulation of several genes in human skin with tomato juice treatment, including a significant downregulation in expression of the aging-associated matrix metalloproteinase, MMP-12, observed with both wild type and resveratrol-enriched tomato extracts. MMP-12 protein could be detected in human skin explant media but regulation at this level proved elusive. Analysis of elastin fibres, a crucial structural component of skin, revealed subtle effects of tomato extracts. The effect of tomato extracts on reepithelialisation aspects of wound-healing was assessed using a full thickness skin explant model, in addition to scratch-wounds of keratinocyte and fibroblast skin cell lines. Initial studies of resveratrol- and pterostilbene-enriched tomato extracts indicated a greater biological activity associated with pterostilbene, and a potential inhibitory effect on migration of cells. Overall novel tomato lines have been generated and their fruit showed effects which could be relevant in the prevention of skin aging and in overcoming the consequences of inflammation.

The spatial and temporal patterns by which starch granules initiate vary greatly between species and organs, but molecular factors that contribute to these diverse patterns are poorly understood. We reveal distinct organ-specific roles of the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) in regulating granule initiation in the endosperm and leaves of wheat. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homeolog, which we showed to be the only active homeolog in tetraploid wheat due to a disruption of the B-genome homeolog. Wheat endosperm contains both large A-type granules initiated during early grain development, and small B-type granules that initiate about 10 - 15 days later. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild-type, they appeared as early as 10 dpa in the mrc-1 mutant, suggesting a role for MRC in suppressing B-type granule initiation during early grain development. By contrast, MRC promotes granule initiation in leaves: mutants carrying premature stop mutations in MRC had fewer granules per chloroplast than the wild type. These contrasting roles of MRC among wheat organs provide new insight into functional diversification of granule initiation proteins, and suggest that they may facilitate the diverse patterns of granule initiation observed across species and organs. Competing Interest Statement The authors have declared no competing interest. Footnotes * We have improved readability by changing some of the wording in the manuscript text, and corrected minor typographical errors in the figure legend.

Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. * SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild type initiated single ‘A-type’ granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. * We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation. Competing Interest Statement The authors have declared no competing interest.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107 (20):9222—9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Glycans are multi-branched sugars that are displayed from lipids and proteins. Through their diverse polysaccharide structures they can potentiate a myriad of cellular signaling pathways involved in development, growth, immuno-communication and survival. Not surprisingly, disruption of glycan synthesis is fundamental to various human diseases; including cancer, where aberrant glycosylation drives malignancy. Here, we report the discovery of a novel mannose-binding lectin, ML6, which selectively recognizes and binds to these irregular tumor-specific glycans to elicit potent and rapid cancer cell death. This lectin was engineered from gene models identified in a tropical rainforest tree root transcriptome and is unusual in its six canonical mannose binding domains (QxDxNxVxY), each with a unique amino acid sequence. Remarkably, ML6 displays antitumor activity that is >105 times more potent than standard chemotherapeutics, while being almost completely inactive towards non-transformed, healthy cells. This activity, in combination with results from glycan binding studies, suggests ML6 differentiates healthy and malignant cells by exploiting divergent glycosylation pathways that yield naïve and incomplete cell surface glycans in tumors. Thus, ML6 and other high-valence lectins may serve as novel biochemical tools to elucidate the glycomic signature of different human tumors and aid in the rational design of carbohydrate-directed therapies. Further, understanding how nature evolves proteins, like ML6, to combat the changing defenses of competing microorganisms may allow for fundamental advances in the way we approach combinatorial therapies to fight therapeutic resistance in cancer.

Background Venous thromboembolism (VTE) remains a persistent source of postoperative morbidity despite prevention and mitigation efforts. Cancer, surgery, and chemotherapy are known risk factors for VTE. Existing literature suggests that neoadjuvant therapy (NAT) may contribute to increased VTE risk in the postoperative period, but few authors specifically examine this relationship in distal pancreatic adenocarcinoma (PDAC). In this study, we analyze the association of NAT and postoperative VTE in patients who underwent distal pancreatectomy (DP) for PDAC. Patients and Methods Using the American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP) database, we analyzed the Procedure Targeted files for pancreatectomy from 2014 to 2020. Adults with PDAC who underwent DP were grouped by receipt of NAT. The primary outcome was the rate of deep venous thrombosis (DVT) and the secondary outcome was the rate of pulmonary embolism (PE). We performed univariate and multivariate logistic regression analysis to determine risk factors associated with postoperative DVT. Results There were 4327 patients with PDAC who underwent DP. Of these, 1414 (32.7%) had NAT. Receipt of NAT was significantly associated with postoperative DVT requiring therapy (3.5% vs. 2.3%, p = 0.02), but was not associated with PE ( p = 0.42). On MVA, NAT was associated with a 73% greater chance of developing postoperative DVT [odds ratio (OR) 1.73, 95% CI 1.18–2.55]. Conclusions Patients who receive NAT prior to DP for PDAC are 73% more likely to develop postoperative DVT compared with upfront resection. As NAT becomes more commonplace, these high-risk patients should be prioritized for guideline-recommended extended duration prophylaxis.