Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates towards the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C4 maize (Zea mays L.) plants. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which abBS cells provide the main route for apoplasmic sucrose transfer towards the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell (SE/CC) complex, as in Arabidopsis thaliana. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of abBS cells and support a modification to the canonical phloem loading pathway in maize.

Background Amyotrophic lateral sclerosis (ALS) is a debilitating disease with few treatment options. Progress towards new therapies requires validated disease biomarkers, but there is no consensus on which fluid-based measures are most informative. Methods This study analyzed microarray data derived from blood samples of patients with ALS ( n = 396), ALS mimic diseases ( n = 75), and healthy controls ( n = 645). Goals were to provide in-depth analysis of differentially expressed genes (DEGs), characterize patient-to-patient heterogeneity, and identify candidate biomarkers. Results We identified 752 ALS-increased and 764 ALS-decreased DEGs (FDR < 0.10 with > 10% expression change). Gene expression shifts in ALS blood broadly resembled acute high altitude stress responses. ALS-increased DEGs had high exosome expression, were neutrophil-specific, associated with translation, and overlapped significantly with genes near ALS susceptibility loci (e.g., IFRD1 , TBK1 , CREB5 ). ALS-decreased DEGs, in contrast, had low exosome expression, were erythroid lineage-specific, and associated with anemia and blood disorders. Genes encoding neurofilament proteins ( NEFH , NEFL ) had poor diagnostic accuracy (50–53%). However, support vector machines distinguished ALS patients from ALS mimics and controls with 87% accuracy (sensitivity: 86%, specificity: 87%). Expression profiles were heterogeneous among patients and we identified two subgroups: (i) patients with higher expression of IL6R and myeloid lineage-specific genes and (ii) patients with higher expression of IL23A and lymphoid-specific genes. The gene encoding copper chaperone for superoxide dismutase ( CCS ) was most strongly associated with survival (HR = 0.77; P = 1.84e−05) and other survival-associated genes were linked to mitochondrial respiration. We identify a 61 gene signature that significantly improves survival prediction when added to Cox proportional hazard models with baseline clinical data (i.e., age at onset, site of onset and sex). Predicted median survival differed 2-fold between patients with favorable and risk-associated gene expression signatures. Conclusions Peripheral blood analysis informs our understanding of ALS disease mechanisms and genetic association signals. Our findings are consistent with low-grade neutrophilia and hypoxia as ALS phenotypes, with heterogeneity among patients partly driven by differences in myeloid and lymphoid cell abundance. Biomarkers identified in this study require further validation but may provide new tools for research and clinical practice. Electronic supplementary material The online version of...

Background: Understanding of gravity sensing and response is critical to long-term human habitation in space and can provide new advantages for terrestrial agriculture. To this end, the altered gene expression profile induced by microgravity has been repeatedly queried by microarray and RNA-seq experiments to understand gravitropism. However, the quantification of altered protein abundance in space has been minimally investigated. Results: Proteomic (iTRAQ-labelled LC-MS/MS) and transcriptomic (RNA-seq) analyses simultaneously quantified protein and transcript differential expression of three-day old, etiolated Arabidopsis thaliana seedlings grown aboard the International Space Station along with their ground control counterparts. Protein extracts were fractionated to isolate soluble and membrane proteins and analyzed to detect differentially phosphorylated peptides. In total, 968 RNAs, 107 soluble proteins, and 103 membrane proteins were identified as differentially expressed. In addition, the proteomic analyses identified 16 differential phosphorylation events. Proteomic data delivered novel insights and simultaneously provided new context to previously made observations of gene expression in microgravity. There is a sweeping shift in post-transcriptional mechanisms of gene regulation including RNA-decapping protein DCP5, the splicing factors GRP7 and GRP8, and AGO4,. These data also indicate AHA2 and FERONIA as well as CESA1 and SHOU4 as central to the cell wall adaptations seen in spaceflight. Patterns of tubulin-α 1, 3,4 and 6 phosphorylation further reveal an interaction of microtubule and redox homeostasis that mirrors osmotic response signaling elements. The absence of gravity also results in a seemingly wasteful dysregulation of plastid gene transcription.

Tissue preservation is a minimal requirement for the success of plant RNA and protein expression studies. The standard of snap-freezing in liquid nitrogen is not always practical or possible. RNAlater, a concentrated solution of ammonium and cesium sulfates, has become a standard preservative in the absence of liquid nitrogen. Here, we demonstrate the effectiveness of RNAlater in preserving both RNA and proteins in Arabidopsis thaliana tissues for use in RNAseq and LC-MS/MS analysis of proteins. While successful in preserving plant material, a transcriptomic and proteomic response is evident. Specifically, 5770 gene transcripts, 84 soluble proteins, and 120 membrane-bound proteins were found to be differentially expressed at a log-fold change of ±1 (P ≤ 0.05). This response is mirrored in the abundance of post-translational modifications, with 23 of the 108 (21.3%) phosphorylated proteins showing altered abundance at a log-fold change of ±1 (P ≤ 0.05). While RNAlater is effective in preserving biological information, our findings warrant caution in its use for transcriptomic and proteomic experiments.

The Biological Research in Canisters (BRIC) hardware has been used to house many biology experiments on both the Space Transport System (STS, commonly known as the space shuttle) and the International Space Station (ISS). However, microscopic examination of Arabidopsis seedlings by Johnson et al. (2015) indicated the hardware itself may affect cell morphology. The experiment herein was designed to assess the effects of the BRIC-Petri Dish Fixation Units (BRIC-PDFU) hardware on the transcriptome and proteome of Arabidopsis seedlings. To our knowledge, this is the first transcriptomic and proteomic comparison of Arabidopsis seedlings grown with and without hardware. Arabidopsis thaliana wild-type Columbia (Col-0) seeds were sterilized and bulk plated on forty-four 60 mm Petri plates, of which 22 were integrated into the BRIC-PDFU hardware and 22 were maintained in closed containers at Ohio University. Seedlings were grown for approximately 3 days, fixed with RNAlater and stored at -80 °C prior to RNA and protein extraction, with proteins separated into membrane and soluble fractions prior to analysis. The RNAseq analysis identified 1651 differentially expressed genes; MS/MS analysis identified 598 soluble and 589 membrane proteins differentially abundant both at p < .05. Fold enrichment analysis of gene ontology terms related to differentially expressed transcripts and proteins highlighted a variety of stress responses. Some of these genes and proteins have been previously identified in spaceflight experiments, indicating that these genes and proteins may be perturbed by both conditions.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH–GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

Summary With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab.

Viola is a large genus with worldwide distribution and many traits not currently exemplified in model plants including unique breeding systems and the production of cyclotides. Here we report de novo genome assembly and transcriptomic analyses of the non-model species Viola pubescens using short-read DNA sequencing data and RNA-Seq from eight diverse tissues. First, V. pubescens genome size was estimated through flow cytometry, resulting in an approximate haploid genome of 455 Mbp. Next, the draft V. pubescens genome was sequenced and assembled resulting in 264,035,065 read pairs and 161,038 contigs with an N50 length of 3,455 base pairs (bp). RNA-Seq data were then assembled into tissue-specific transcripts. Together, the DNA and transcript data generated 38,081 ab initio gene models which were functionally annotated based on homology to Arabidopsis thaliana genes and Pfam domains. Gene expression was visualized for each tissue via principal component analysis and hierarchical clustering, and gene co-expression analysis identified 20 modules of tissue-specific transcriptional networks. Some of these modules highlight genetic differences between chasmogamous and cleistogamous flowers and may provide insight into V. pubescens’ mixed breeding system. Orthologous clustering with the proteomes of A. thaliana and Populus trichocarpa revealed 8,531 sequences unique to V. pubescens , including 81 novel cyclotide precursor sequences. Cyclotides are plant peptides characterized by a stable, cyclic cystine knot motif, making them strong candidates for drug scaffolding and protein engineering. Analysis of the RNA-Seq data for these cyclotide transcripts revealed diverse expression patterns both between transcripts and tissues. The diversity of these cyclotides was also highlighted in a maximum likelihood protein cladogram containing V. pubescens cyclotides and published cyclotide sequences from other Violaceae and Rubiaceae species. Collectively, this work provides the most comprehensive sequence resource for Viola , offers valuable transcriptomic insight into V. pubescens , and will facilitate future functional genomics research in Viola and other diverse plant groups.