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Neutralizing the RNA phosphodiester backbone enables delivery of siRNA across cell membranes. RNA interference (RNAi) has great potential to treat human disease 1 , 2 , 3 . However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle 4 , 5 , 6 , 7 , 8 , 9 . Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates 9 . Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson's Disease. However, before siRNAs can become drugs, they must overcome a billion years of evolutionary defenses designed to keep invading RNAs on the outside cells from getting to the inside of cells. Not surprisingly, significant effort has been placed in developing a wide array of delivery technologies. Foremost of these has been the development of N -acetylgalactosamine (GalNAc) siRNA conjugates for delivery to liver. Tris-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs enter the cytoplasm to induce robust, target selective RNAi responses in vivo . Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, are currently underway by three biotech companies to treat a wide variety of diseases. GalNAc-siRNA conjugates are a simple solution to the siRNA delivery problem for liver hepatocytes and have shown the RNAi (and antisense oligonucleotide) field the path forward for targeting other tissue types.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Key message Significant introgression-by-environment interactions are observed for traits throughout development from small introgressed segments of the genome.Relatively small genomic introgressions containing quantitative trait loci can have significant impacts on the phenotype of an individual plant. However, the magnitude of phenotypic effects for the same introgression can vary quite substantially in different environments due to introgression-by-environment interactions. To study potential patterns of introgression-by-environment interactions, fifteen near-isogenic lines (NILs) with > 90% B73 genetic background and multiple Mo17 introgressions were grown in 16 different environments. These environments included five geographical locations with multiple planting dates and multiple planting densities. The phenotypic impact of the introgressions was evaluated for up to 26 traits that span different growth stages in each environment to assess introgression-by-environment interactions. Results from this study showed that small portions of the genome can drive significant genotype-by-environment interaction across a wide range of vegetative and reproductive traits, and the magnitude of the introgression-by-environment interaction varies across traits. Some introgressed segments were more prone to introgression-by-environment interaction than others when evaluating the interaction on a whole plant basis throughout developmental time, indicating variation in phenotypic plasticity throughout the genome. Understanding the profile of introgression-by-environment interaction in NILs is useful in consideration of how small introgressions of QTL or transgene containing regions might be expected to impact traits in diverse environments.

The first documented human leptospirosis cases in the U.S. Virgin Islands (USVI) occurred following 2017 Hurricanes Irma and Maria. We conducted a representative serosurvey in USVI to estimate the seroprevalence and distribution of human leptospirosis and evaluate local risk factors associated with seropositivity. A stratified, two-stage cluster sampling design was used and consisted of three island strata and random selection of census blocks and then households. All eligible members of selected households were invited to participate (≥5 years old, resided in USVI ≥6 months and ≥6 months/year). Household and individual-level questionnaires were completed, and serum collected from each enrolled individual. Microscopic agglutination test serology was conducted, and bivariate and logistic regression analyses completed to identify risk factors for seropositivity. In March 2019, 1,161 individuals were enrolled from 918 households in St. Croix, St. Thomas, and St. John. The territory-wide weighted seroprevalence was 4.0% (95% CI:2.3-5.7). Characteristics/exposures independently associated with seropositivity using logistic regression included contact with cows (OR: 39.5; 95% CI: 9.0-172.7), seeing rodents/rodent evidence or contact with rodents (OR: 2.6; 95% CI: 1.1-5.9), and increasing age (OR: 1.02; 95% CI: 1.002-1.04); full or partial Caucasian/White race was negatively correlated with seropositivity (OR: 0.02, 95% CI: 0.04-0.7). Bivariate analysis showed self-reported jaundice since the 2017 hurricanes (pRR: 5.7; 95% CI: 1.0-33.4) was associated with seropositivity and using a cover/lid on cisterns/rainwater collection containers (pRR: 0.3; 95% CI: 0.08-0.8) was protective against seropositivity. Leptospirosis seropositivity of 4% across USVI demonstrates an important human disease that was previously unrecognized and emphasizes the importance of continued leptospirosis surveillance and investigation. Local risk factors identified may help guide future human and animal leptospirosis studies in USVI, strengthen leptospirosis public health surveillance and treatment timeliness, and inform targeted education, prevention, and control efforts.

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

Circulating lymphocytes gain access to lymph nodes owing to their ability to initiate rolling along specialized high endothelial venules (HEVs). One mechanism of rolling involves L-selectin binding to peripheral node addressin (PNAd) on HEVs. Activated platelets are shown to bind to circulating lymphocytes and to mediate rolling in HEVs, in vivo, through another molecule, P-selectin, which also interacts with PNAd. In vitro, activated platelets enhanced tethering of lymphocytes to PNAd and sustained lymphocyte rolling, even in the absence of functional L-selectin. Thus, a platelet pathway operating through P-selectin provides a second mechanism for lymphocyte delivery to HEVs.

Objectives Crop improvement relies on analysis of phenotypic, genotypic, and environmental data. Given large, well-integrated, multi-year datasets, diverse queries can be made: Which lines perform best in hot, dry environments? Which alleles of specific genes are required for optimal performance in each environment? Such datasets also can be leveraged to predict cultivar performance, even in uncharacterized environments. The maize Genomes to Fields (G2F) Initiative is a multi-institutional organization of scientists working to generate and analyze such datasets from existing, publicly available inbred lines and hybrids. G2F’s genotype by environment project has released 2014 and 2015 datasets to the public, with 2016 and 2017 collected and soon to be made available. Data description Datasets include DNA sequences; traditional phenotype descriptions, as well as detailed ear, cob, and kernel phenotypes quantified by image analysis; weather station measurements; and soil characterizations by site. Data are released as comma separated value spreadsheets accompanied by extensive README text descriptions. For genotypic and phenotypic data, both raw data and a version with outliers removed are reported. For weather data, two versions are reported: a full dataset calibrated against nearby National Weather Service sites and a second calibrated set with outliers and apparent artifacts removed.

Seeding native plants is an increasingly common management technique for promoting plant diversity and stabilizing soil on restoration and post-fire rehabilitation projects. The effectiveness of the technique is uncertain, however, especially in semiarid southwestern forests, as very few studies have been conducted to monitor the results. Land managers in the Mount Trumbull area of the Grand Canyon-Parashant National Monument (GCPNM) in northwestern Arizona have been seeding with natives since 1996 as part of ponderosa pine (Pinus ponderosa) forest ecological restoration treatments. Early research on the effects of seeding treatments there (Springer et al. 2001, Springer and Laughlin 2004) showed an increase in overall species richness when seeding with a diverse mix of native grasses, forbs, and shrubs, but did not conclusively detect an increase in cover of the seeded species. Subse-quendy, managers with the Bureau of Land Management (BLM, which co-administers the GCPNM along with the National Park Service) have trimmed their seed mix to a smaller assortment of grasses and forbs, and we have refined our monitoring methods to better detect changes in species cover at multiple scales.