Plant breeding is achieved through the controlled self- or cross-pollination of individuals and typically involves isolation of floral parts from selected parental plants. Paper, cellulose or synthetic materials are used to avoid self pollination or cross contamination. Low seed set limits the rate of breeding progress and increases costs. We hypothesized that a novel ‘non-woven’ fabric optimal for both pollination and seed set in multiple plant species could be developed. After determining the baseline pollen characteristics and usage requirements we established iterative three phase development and biological testing. This determined (1) that white fabric gave superior seed return and informed the (2) development of three non-woven materials using different fibre and layering techniques. We tested their performance in selfing and hybridisation experiments recording differences in performance by material type within species. Finally we (3) developed further advanced fabrics with increased air permeability and tested biological performance. An interaction between material type and species was observed and environmental decoupling investigated, showing that the non-woven fabrics had superior water vapour transmission and temperature regulation compared to controls. Overall, non-woven fabrics outperformed existing materials for both pollination and seed set and we found that different materials can optimize species-specific, rather than species-generic performance.

Three pollination control tents (PCTs) made from novel nonwoven synthetic fabrics with more open pore structure (DWB10, DWB23 and DWB24) were compared with the standard DWB01 fabric for pollen proofing in sugar beet (Beta vulgaris L.) at the research station of Lion Seed Ltd Essex, UK in 2019. PCTs of 63.5 x 63.5 cm footprint accommodated single potted plants. A completely randomised trial with five replications including an open pollinated control was conducted using cytoplasmic male sterile line 1TM37. Analysis was computed for (a) full data and (b) excluding three DWB23 defective tents. Differences among treatments were non-significant for all morphological traits except for number of secondary branches in (a) only. There was thus no micro-climatic difference among treatments for morphological traits of the 1TM37 CMS line. Among the seed related traits, 1000-seed weight and 10-day germination (%) were significant between treatments in (a) but only 1000-seed weight in (b). The mean 1000-seed weight was significantly higher for the open control than all other PCT treatment means which did not differ significantly from zero. Therefore, all four fabrics of PCTs were equally pollen proof in preventing pollen contamination. It is concluded for the first time that mini-tents of these novel nonwoven fabrics, engineered for both larger pores for air permeability and fibre architecture to prevent pollen transmission, adequately eliminated cross-pollination while maintaining ambient environmental conditions and are effective for sugar beet breeding. The PCT technology may be equally usefully deployed in other traditional, commercial and fibre crops for hybrid seed production.

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Efficacy of pollination bags made of new nonwoven fabrics was compared with the traditional paper bags in sorghum during 2015 using three cultivars comprising BR007B (red seeded), SC283 (white seeded) and 1167048 hybrid with tannin (brown seeded). The five pollination bag treatments were: no bagging, traditional paper bag, paper bag plus plastic screen bag for extra bird protection, duraweb ® SG2 polypropylene bag and duraweb ® SG1 polyester bag. There was no bird damage on tannin hybrid but birds damaged bags to access grains of the other two varieties. Varieties and bag types differed significantly, and also showed significant interactions for panicle weight (at P<0.06), seed weight and average seed weight per panicle. The tannin hybrid was consistently a better performer for all traits regardless of bag type. The paper bags were the worst for bird damage. Duraweb® SG1 was the best performer for all traits including bird damage followed by duraweb ® SG2. The joint regression analysis showed that BR007B performed consistently under all bag types with average response. On the other hand, SC283 improved its response with the increasing quality of bag type at an above average rate for panicle weight and seed traits. It was concluded that new nonwoven fabric bags could replace paper bags in providing better seed production potential and greater protection against bird damage.

This work investigated the effects of seven pollination bag treatments on three varieties of sorghum for: grain loss to birds; total weight of five panicles (g); total grain weight of five panicles (g); average grain weight per panicle (g); germination per cent; and occurrence of grain mold during 2016. Varieties were: 1167048 hybrid (brown seeded); BR007B (red seeded); and P9401 (white seeded). The bag treatments were: 1. No bagging; 2. Kraft paper; 3. Kraft paper + plastic bag screen; 4 Used duraweb® SG1; 5. Used duraweb® SG2; 6. New duraweb® SG1; 7. New duraweld® SG2. High bird pressure resulted in 100% seed loss on uncovered panicles and 75% under Kraft paper pollination bags. Birds preferred white seeded P9401, which led to no seed recovery under Kraft paper bags. There was virtually no bird damage with all other pollination bags. For panicle and grain yields the varieties performed in the order 1167048>BR007B>P9401. Unprotected panicles and paper bag treatments had the lowest yields. Panicles covered with the new synthetic bags exhibited 195 to 652% higher yields compared to Kraft paper bags. Varieties x bag type interactions were not important as they contributed 4 to 6% to the total sum of squares for yield traits. Germination test under normal and stress conditions showed no significant adverse effect of bag treatments on seed health. Reused bags performed as well as new bags for all of these traits. Varieties differed significantly for the occurrence of five grain mold pathogens, with highest occurrence of Alternaria, up to 40%, on 1167048 hybrid. Of the five pathogens, bag types differed significantly for Phoma with the highest occurrence of 9% on re-used duraweb®SG2 bags. Thus bags require disinfecting and cleaning before re-use. It is concluded that nonwoven synthetic bags are a better choice than the Kraft paper pollination bags for increasing the grain yield and virtually eliminating the bird damage in sorghum.

The practices of using isolation and distance in the seed production of open pollinated crops are fundamental concepts to ensure seed purity. We uniquely examined the effectiveness of replacing isolation plots for seed production and grass breeding with different sizes of novel nonwoven synthetic fabric pollination control tents (PCTs). Two fabrics, DWB10 and DWB24, were used along with multiple genotypes of tall fescue at Ardmore, Burneyville and Gene Autry locations in Oklahoma, USA during 2018 and 2019. Treatment effects were consistently significant in both years, but location differences were more pronounced in 2019. Interactions of treatments with locations or genotypes were not predominant. The two tent fabrics, generally, performed equally well for various traits in both years. Tent performance for both fabrics was particularly superior over control for various traits in 2019 (e.g., DWB10 tent showed a 36% increase for seed yield (SY) over the control). Introduction of fans in tents for increasing pollen flow in 2019 was not advantageous as it reduced the SY by 23%. The average temperature within tents was higher with lower average humidity than the control producing a microclimate for good yield and disease free seeds. The final germination (%) of seeds from tents and controls at 21 days was high and not much different with a minimum overall germination of 89% at Burneyville in 2018. There was no evidence of pollen contamination from tetraploid ryegrass pollen in any of the tent fabrics. Bad weather in 2018 affected the sturdiness of tents, but modifications in 2019 corrected all such mishaps. Further improvements in the structures, design and cover have since been made for field exploitation of technology in grass improvement and seed multiplication.

The impact on full-time carers of children with intellectual disabilities who exhibit challenging behaviour has been well researched (e.g. Lach et al., 2009;Shah et al., 2010;Wodehouse and McGill, 2009), however there is to date no published research into the impact of behaviour that challenges on seasonal carers . Five participants who had been employed in summer playschemes for children and young people (up to the age of 18) were interviewed about their experiences of behaviour that challenges. The transcripts were analysed using Interpretative Phenomenological Analysis (IPA) which revealed six superordinate themes; the belief in and sanctuary of temporary work, emotional impact, personality and gender, strength through knowledge, communication difficulties and the belief in integration. Seasonal workers discussed suppressing their emotions in order to stay in control of a challenging situation, using coping styles developed through experience or based on personal skills; it is suggested that formalised training, particularly regarding non-verbal communication, would support playscheme workers in the management of and adaption to challenging behaviour. Journal of Intellectual Disabilities 0(0)2

Introduction: Generating clinical scale doses of natural killer (NK) cells is currently a challenge for manufacturing CAR-NK cell therapies. Current methods to expand NK cells include irradiated feeder cells that have been engineered to mimic dendritic cells as well as commercially available combinations of antibody & proteins for targeted NK cell expansion. These methods are restrictive due to high costs, scale-up challenges, and licensing restrictions for clinical & commercial applications. We hypothesized that using a dissolvable phase-change hydrogel conjugated with ligands targeting NK cell activation may provide a more efficient and defined alternative for NK manufacturing processes. Additionally, we investigated cytokine culture combinations that optimize the expansion of NK cells. Methods: Hydrogel microspheres (9 µm median diameter) - referred to as Cloudz™ particles - were functionalized with αNKp46 and αCD2 ligands using standard bioconjugation techniques. In this study, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in G-Rex® 24-well plates containing NK cell-functionalized Cloudz™ particles along with various cytokine combinations. Prior to expansion, PBMCs were analyzed for CD3-/CD56+ NK cells and 6x105 NK cells were seeded into each well with a 2:1 ratio of Cloudz™ particles:NK cells. Miltenyi MACS iBeads were used as a control and were prepared according to the manufacturer instructions. During days 0-3, the cells were cultured with IL-2 (40 U/mL) along with combinations of IL-12, IL-18, and IL-21 (10 ng/mL each). After 3 days, the IL-2 concentration was increased to 200 U/mL, while the other cytokine concentrations remained constant. The cytokines and media were refreshed every 3 days with ½ media changes. After 7 days in culture the cells were analyzed by flow cytometry for CD3-/CD56+ NK cell expansion and purity. NK cell purity was defined as the percentage of CD3-CD56+ cells relative to the total population. Results: Cultures using NK cell-functionalized Cloudz™ particles combined with IL-2 resulted in a 69% NK cell purity and 29.9-fold expansion. In comparison, cultures using MACS iBeads combined with IL-2 resulted in 49% NK cell purity and a 6.8-fold expansion, suggesting that Cloudz™-based methods provide better expansion. When IL-12 was added to cultures with Cloudz™ particles, both the purity and expansion decreased (45% and 7.2-fold, respectively). When IL-12 and IL-18 were added, the expansion increased to 33.9-fold and the purity increased to 60%. The best combination was IL-2/IL-12/IL-18/IL-21 which had an 88.9-fold expansion with a 78% purity. IL-12 and IL-18 mimic the activating signals released by dendritic cells and IL-2 and IL-21 mimic the activating signals released by activated T cells. Conclusion: These data demonstrate that Cloudz™ particles functionalized with αNKp46 and αCD2 can efficiently induce NK activation and expansion with high purity and growth. In future studies, we will determine if NK cells expanded using this method show killing ability of cancer cells and persistence in in vivo models. We will also further explore the possibilities of this novel system for rapid expansion and recovery of specific NK cell subtypes. Citation Format: Christopher Johnson, Hannah Senior, Nithya Jesuraj. Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-072.