Biosearch Technologies (United States)

Genomic selection (GS) has potential to accelerate the genetic gain in crop plants. This study was undertaken to assess the accuracy and potential of GS in faba bean [Vicia faba (L.)] and to enhance its application in breeding programs. A set of 118 diverse faba bean accessions were phenotyped for key agronomic traits under herbicide and heat stress across 16 environments in Morocco, Lebanon, Sudan, and the United States. These accessions were genotyped, revealing 170 single nucleotide polymorphisms (SNPs) strongly associated with target traits. kompetitive allele‐specific PCR (KASP) markers were subsequently designed and validated on 4512 diverse breeding lines. Prediction accuracy (PA) was assessed using the reproducing kernel Hilbert space model, with and without genotype‐by‐environment interactions and taking into consideration two cross‐validation (CV) strategies: CV1 (predicting new lines) and CV2 (predicting complete records from unbalanced data). Additionally, 75 KASP markers with known association with heat tolerance traits were prioritized to estimate the PA of the models. The results showed a comparable PA between the two models, with CV1 outperforming CV2. This highlighted the difficulty in predicting the performance of untested lines in tested environments compared to lines evaluated in some environments but not others. Moreover, SNP subset size and composition significantly impacted PA, especially under heat stress. The highest accuracies were observed for days to flowering and plant height under heat stress and for plant height and grain yield under herbicide environments, indicating that these traits are ideal for training population selection. Optimizing the size and composition of the training population will enhance the effectiveness of GS in faba bean breeding.

Abstract Environmental DNA (eDNA) detections of imperiled species have the potential to inform conservation action, but this requires the acceptance of new technologies by decision‐makers. Here we describe how engaging stakeholders into a collaborative process led to the successful translation of new eDNA findings into conservation outcomes. We characterized the distribution of three federally listed pond‐breeding amphibians across nearly 200 wetlands in coastal California using both traditional field surveys and eDNA sampling; the latter had greater detection rates overall. Regulatory agency staff gained trust in the rigor and effectiveness of eDNA data by joining traditional surveys and through the collaborative development of recommendations for the adoption of eDNA methods. Extensive outreach to the local community within the range of the highly endangered Santa Cruz long‐toed salamander resulted in invitations to sample previously unsurveyed wetlands on private property and the detection of new breeding sites. Conservation organizations and resource management agencies were integrated into our core team from the start, and ultimately shaped wetland management actions, siting of new wetlands, and land acquisition priorities informed by the data generated. Thus, this project serves as a model for actionable eDNA science directly affecting conservation.

Single molecule RNA FISH (smRNA FISH) is an imaging method that labels individual mRNA molecules in cells to facilitate localization and quantitative studies. Here we present a modified protocol for mRNA labelling in Arabidopsis root meristem cells that retains GFP fluorescence. Although the processing steps impact GFP intensity, this protocol demonstrates visualization of RNA together with highly expressed protein. This protocol is presented together with simplified image analysis steps that aim to support inexperienced cell biologists through from experimental set up to mRNA quantification.

Single molecule RNA FISH (smRNA FISH) is an imaging method that labels individual mRNA molecules in cells to facilitate localization and quantitative studies. Here we present a modified protocol for mRNA labelling in Arabidopsis root meristem cells that retains GFP fluorescence. Although the processing steps impact GFP intensity, this protocol demonstrates visualization of RNA together with highly expressed protein. This protocol is presented together with simplified image analysis steps that aim to support inexperienced cell biologists through from experimental set up to mRNA quantification.

ABSTRACT The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we found 2,700 genes with interdependent alternative transcription, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules. The analysis of three human primary tissues revealed similar patterns of interdependency between transcription and mRNA processing events. We predict thousands of novel Open Reading Frames from the sequence of full-length mRNAs and obtained evidence for their translation by shotgun proteomics. The mapping database rescued 358 previously unassigned peptides and improved the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improved proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription and mRNA processing.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.

Abstract Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein. Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH. Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell. Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant. The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3’ end processing in normal and aberrant cells. Citation Format: Hans E. Johansson, Raymund H. Yin, Swati Mandal, Myung-Hee Park. Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1998.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70–90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

Probiotics have been used as alternative therapies in intestinal inflammatory disorders. Many studies have shown that different bacterial probiotic strains possess immuno-modulatory and anti-inflammatory properties. However, there is an increasing interest in the use of non-viable bacteria to reduce the risk of microbial translocation and infection. The aim of this study was to evaluate whether the viability of L. fermentum CECT5716 is essential to exert its intestinal anti-inflammatory effect. We compared the preventative effects of viable and non-viable probiotic in the TNBS model of rat colitis. In vitro studies were also performed in Caco-2 and RAW 264.7 cells to evaluate the probiotic effects on IL-8, IL-1β and nitrite production, and p44/42 and p38 MAP kinase protein expressions. In vitro results revealed a decrease in the stimulated production of pro-inflammatory mediators regardless of the viability of the probiotic. Likewise, both forms of the probiotic administered to colitic rats produced a significant reduction of IL-1β and TNF-α levels and colonic iNOS expression. In conclusion, both live and dead L. fermentum CECT5716 have been demonstrated to attenuate the inflammatory process and diminish the production of some of the inflammatory mediators. In fact, the viability of this probiotic did not affect its immuno-modulatory and anti-inflammatory properties.

A set of Stellaris FISH Probes comprises up to 48 singly labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. Please see the Biosearch