Effect of molecular weight of amine end-modified poly(β-amino ester)s on gene delivery efficiency and toxicity

Amine end-modified poly(β-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.Alnylam Pharmaceuticals (Firm)National Institutes of Health (U.S.) (Grant R01-EB000244-27)National Institutes of Health (U.S.) (Grant 5-R01-CA132091-04)National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32-EB011867

ATRP in the design of functional materials for biomedical applications

Atom Transfer Radical Polymerization (ATRP) is an effective technique for the design and preparation of multifunctional, nanostructured materials for a variety of applications in biology and medicine. ATRP enables precise control over macromolecular structure, order, and functionality, which are important considerations for emerging biomedicaldesigns. This article reviews recent advances in the preparation of polymer-based nanomaterials using ATRP, including polymer bioconjugates, block copolymer-based drug delivery systems, cross-linked microgels/nanogels, diagnostic and imaging platforms, tissue engineering hydrogels, and degradable polymers. It is envisioned that precise engineering at the molecular level will translate to tailored macroscopic physical properties, thus enabling control of the key elements for realized biomedicalapplications

Precise let-7 expression levels balance organ regeneration against tumor suppression

The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, and have important implications for let-7 based therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09431.00

Global maps of soil temperature

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km² resolution for 0–5 and 5–15 cm soil depth. These maps were created by calculating the difference (i.e., offset) between in-situ soil temperature measurements, based on time series from over 1200 1-km² pixels (summarized from 8500 unique temperature sensors) across all the world’s major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in-situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Global maps of soil temperature

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km2 resolution for 0–5 and 5–15 cm soil depth. These maps were created by calculating the difference (i.e. offset) between in situ soil temperature measurements, based on time series from over 1200 1-km2 pixels (summarized from 8519 unique temperature sensors) across all the world\u27s major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (−0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Global maps of soil temperature.

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km2 resolution for 0-5 and 5-15 cm soil depth. These maps were created by calculating the difference (i.e. offset) between in situ soil temperature measurements, based on time series from over 1200 1-km2 pixels (summarized from 8519 unique temperature sensors) across all the world's major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Delivery of Tissue-Targeted Scalpels: Opportunities and Challenges for In Vivo CRISPR/Cas-Based Genome Editing

Copyright © 2020 American Chemical Society. CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies

Automated ARGET ATRP Accelerates Catalyst Optimization for the Synthesis of Thiol-Functionalized Polymers

Conventional synthesis of polymers by ATRP is relatively low throughput, involving iterative optimization of conditions in an inert atmosphere. Automated, high-throughput controlled radical polymerization was developed to accelerate catalyst optimization and production of disulfide-functionalized polymers without the need of an inert gas. Using ARGET ATRP, polymerization conditions were rapidly identified for eight different monomers, including the first ARGET ATRP of 2-(diethylamino)­ethyl methacrylate and di­(ethylene glycol) methyl ether methacrylate. In addition, butyl acrylate, oligo­(ethylene glycol) methacrylate 300 and 475, 2-(dimethylamino)­ethyl methacrylate, styrene, and methyl methacrylate were polymerized using bis­(2-hydroxyethyl) disulfide bis­(2-bromo-2-methylpropionate) as the initiator, tris­(2-pyridylmethyl)­amine as the ligand, and tin­(II) 2-ethylhexanoate as the reducing agent. The catalyst and reducing agent concentration was optimized specifically for each monomer, and then a library of polymers was synthesized systematically using the optimized conditions. The disulfide-functionalized chains could be cleaved to two thiol-terminated chains upon exposure to dithiothreitol, which may have utility for the synthesis of polymer bioconjugates. Finally, we demonstrated that these new conditions translated perfectly to conventional batch polymerization. We believe the methods developed here may prove generally useful to accelerate the systematic optimization of a variety of chemical reactions and polymerizations