Seed Collection in Temperate Trees-Clean, Fast, and Effective Extraction of Populus Seeds for Laboratory Use and Long-term Storage.

Seeds ensure the growth of a new generation of plants and are thus central to maintaining plant populations and ecosystem processes. Nevertheless, much remains to be learned about seed biology and responses of germinated seedlings to environmental challenges. Experiments aiming to close these knowledge gaps critically depend on the availability of healthy, viable seeds. Here, we report a protocol for the collection of seeds from plants in the genus Populus. This genus comprises trees with a wide distribution in temperate forests and with economic relevance, used as scientific models for perennial plants. As seed characteristics can vary drastically between taxonomic groups, protocols need to be tailored carefully. Our protocol takes the delicate nature of Populus seeds into account. It uses P. deltoides as an example and provides a template to optimize bulk seed extraction for other Populus species and plants with similar seed characteristics. The protocol is designed to only use items available in most labs and households and that can be sterilized easily. The unique characteristics of this protocol allow for the fast and effective extraction of high-quality seeds. Here, we report on seed collection, extraction, cleaning, storage, and viability tests. Moreover, extracted seeds are well suited for tissue culture and experiments under sterile conditions. Seed material obtained with this protocol can be used to further our understanding of tree seed biology, seedling performance under climate change, or diversity of forest genetic resources. Key features • Populus species produce seeds that are small, delicate, non-dormant, with plenty of seed hair. Collection of seed material needs to be timed properly. • Processing, seed extraction, seed cleaning, and storage using simple, sterilizable laboratory and household items only. Obtained seeds are pure, high quality, close to 100% viability. • Seeds work well in tissue culture and in experiments under sterile conditions. • Extractability, speed, and seed germination were studied and confirmed for Populus deltoides as an example. • Can also serve as template for bulk seed collection from other Populus species and plant groups that produce delicate seeds (with no or little modifications). Graphical overview.

Success Factors for and Barriers to Integration of Electronic Mental Health Screening in Primary Care.

Currently, only a third of primary care providers screen for substance use, which is a growing epidemic. This quality improvement study aimed to improve the screening process by integrating the Drug Abuse Screening Test without information systems support into the electronic health record to increase completed screenings and provider interventions for positive screenings in adult patients at an urban primary care clinic. Electronic drug abuse screening should include a prescreen followed by the Drug Abuse Screening Test, interprofessional approach, comprehensive education, and utilization of generic tools to create new screening forms. Staff participated in a new drug abuse screening process, and chart audits and staff interviews were conducted. There was a 9% increase in completed screenings by medical assistants with electronic versus paper screening (30% vs 21%, respectively; P < .001). There was a 33.4% increase in provider intervention for positive screenings with electronic versus paper screening (55% vs 21%, respectively; P = .1081). Primary care providers can play an increased role in drug abuse treatment by using available technology to overcome barriers to screening independent of information systems support. By adopting the new electronic screening documentation process, this clinic was able to increase its screening outcomes.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools.

PURPOSE: Aluminum-based adjuvants including aluminum phosphate (AlPO4) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO4, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality. METHODS: This study focuses on the compositional analysis of AlPO4 adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO4 was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®). RESULTS: There was no observable difference in size distribution between the intermediate and final stage AlPO4 by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO4 manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO4 manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO4 adjuvant was also monitored by in-line ReactIR probe. CONCLUSION: This study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

Identity, Structure and Compositional Analysis of Aluminum Phosphate Adsorbed Pediatric Quadrivalent and Pentavalent Vaccines.

PURPOSE: The goal of this study is to set an empirical baseline to map the structure-function relation of the antigens from the commercialized vaccine products. METHODS: To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM have been used. RESULTS: All antigens have shown wide range of hydrodynamic diameter from 7 nm to 182 nm. Upon adjuvantation, the size distribution has become narrow, ranging from 10 to 12 µm, and has been driven by the derived diameter of aluminum phosphate (AlPO4) adjuvant. Further to examine size and morphology of adsorbed antigens, SEM has been used. The SEM results have demonstrated that the AlPO4 adjuvant suspension and adsorbed proteins consist of submicron particles that form a continuous porous surface. Diphtheria Toxoid (DT), Tetanus Toxoid (TT), and chemically-modified Filamentous Haemagglutinin (FHA) have shown surface adsorption to AlPO4. Secondary structure alpha-helix and beta-sheet content of DT and TT has increased after adsorption to AlPO4 adjuvant as shown by FTIR, whereas no significant changes were noted for other protein antigens. The results from Intrinsic Fluorescence have shown a structural rearrangement in DT and TT, consistent with the FTIR results. Multivalent vaccine product identity has been determined by FTIR as unique fingerprint spectrum. CONCLUSION: The globular proteins such as DT and TT have shown changes in secondary structure upon adsorption to AlPO4, whereas fibrillar protein FHA has not been affected by adsorption. FTIR can be used as a lean technique to confirm product identity at different manufacturing sites.