Species diversity in restoration plantings: Important factors for increasing the diversity of threatened tree species in the restoration of the Araucaria forest ecosystem.

The Araucaria forest ecosystem in southern Brazil is highly threatened: less than one percent of the original forest remains, and what is left is a fragmented agro-mosaic of mostly early-to-late secondary forest patches among high-yield agriculture and timber monocultures. Forest restoration initiatives in this region aim to restore degraded areas, however the limited number of species used in restoration projects represents a missed opportunity for species-rich plantings. High diversity plantings represent a larger number of functional groups and provide a targeted conservation strategy for the high number of threatened species within this ecosystem. This study interviewed nurseries (Ns) and restoration practitioners (RPs) in Paraná and Santa Catarina states to identify what species are being cultivated and planted, and what factors are driving the species selection process. An average of 20 species were reportedly used in restoration plantings, most of which are common, widespread species. Baseline data confirms that Ns and RPs have disproportionately low occurrences of threatened species in their inventories and plantings, supporting findings from previous research. Questionnaire responses reveal that opportunities for seed acquisition are an extremely important factor in order for nurseries to increase their diversity of cultivated species. Results also suggest that facilitating species-rich plantings for restoration practitioners would only be feasible if it did not increase the time required to complete planting projects, as it would minimize their ability to keep costs low. This study proposes solutions for increasing the number of species used in restoration practice-such as developing a comprehensive species list, fostering knowledge-sharing between actors, creating seed sharing programs, and increasing coordination of planting projects. Long-term strategies involve complimenting traditional ex situ approaches with emerging inter-situ and quasi in situ conservation strategies which simultaneously provide long-term preservation of genetic diversity and increase seed production of target species.

Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics.

Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.

RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction.

Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE(-/-) mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)-targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.