Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean.

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

MicroRNA and gene expression changes in unruptured human cerebral aneurysms.

OBJECTIVE The molecular mechanisms behind cerebral aneurysm formation and rupture remain poorly understood. In the past decade, microRNAs (miRNAs) have been shown to be key regulators in a host of biological processes. They are noncoding RNA molecules, approximately 21 nucleotides long, that posttranscriptionally inhibit mRNAs by attenuating protein translation and promoting mRNA degradation. The miRNA and mRNA interactions and expression levels in cerebral aneurysm tissue from human subjects were profiled. METHODS A prospective case-control study was performed on human subjects to characterize the differential expression of mRNA and miRNA in unruptured cerebral aneurysms in comparison with control tissue (healthy superficial temporal arteries [STA]). Ion Torrent was used for deep RNA sequencing. Affymetrix miRNA microarrays were used to analyze miRNA expression, whereas NanoString nCounter technology was used for validation of the identified targets. RESULTS Overall, 7 unruptured cerebral aneurysm and 10 STA specimens were collected. Several differentially expressed genes were identified in aneurysm tissue, with MMP-13 (fold change 7.21) and various collagen genes (COL1A1, COL5A1, COL5A2) being among the most upregulated. In addition, multiple miRNAs were significantly differentially expressed, with miR-21 (fold change 16.97) being the most upregulated, and miR-143-5p (fold change -11.14) being the most downregulated. From these, miR-21, miR-143, and miR-145 had several significantly anticorrelated target genes in the cohort that are associated with smooth muscle cell function, extracellular matrix remodeling, inflammation signaling, and lipid accumulation. All these processes are crucial to the pathophysiology of cerebral aneurysms. CONCLUSIONS This analysis identified differentially expressed genes and miRNAs in unruptured human cerebral aneurysms, suggesting the possibility of a role for miRNAs in aneurysm formation. Further investigation for their importance as therapeutic targets is needed.