Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Activation of the JAK1 / STAT1 Signaling Pathway is Associated with Prdx6 Expression Levels in Human Epididymis Epithelial Cells

I. Background: Peroxiredoxin 6 (Prdx6) is widely expressed in mammalian tissues. Our previous study demonstrated that Prdx6 was expressed in human epididymis and spermatozoa, and the protective role of Prdx6 in human spermatozoa was also reported. In this study, we demonstrate the potential role and mechanism of Prdx6 in human epididymis epithelial cells (HEECs).II. Methods and Results: Western blotting was used to measure expression levels of key proteins in the JAK / STAT signaling pathway. Digital gene expression analysis (DGE) was used to identify gene expression patterns in control HECs and in HECs after Prdx6-RNA interference (P6-RNAi). The DGE analysis identified 589 up-regulated and 314 down-regulated genes (including Prdx6) in Prdx6-RNAi (P6-RNAi) HEECs. Thirteen significantly different pathways were identified between the two groups, with the majority different expressed genes belonging to the CCL, CXCL, IL, and IFIT families. In particular, the expression levels of IL6, IL6ST, and eighteen IFN related genes were significantly increased in the condition of the down-regulated expression of Prdx6. Compared to control HEECs, the expression levels of JAK1, STAT1, phosphorylated JAK1 and STAT1 were significantly increased, while the expression levels of SOCS3 was significantly decreased in P6-RNAi HEECs. The Malondialdehyde (MDA) level and total antioxidant capacity in P6-RNAi HEECs were significantly increased and decreased compared to that of control, respectively. III. Conclusions: We speculated that knockdown of Prdx6 resulted in higher levels of ROS in HEECs, which in turn, activated the JAK1 / STAT1 signaling pathway induced by IL-6 receptor and IFN.

Dipeptidase PEPDA is required for the conidiation pattern shift in Metarhizium acridum

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involved a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum , which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in Δ pepdA mutant. The defect of MC in Δ pepdA can be rescued when nonpolar amino acids, α-alanine, β-alanine or proline, were added into s ucrose y east extract a gar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum . IMPORTANCE Conidia, as the asexual propagules in many fungi, are start and end of fungal lifecycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the Δ pepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

Translation of a Circulating microRNA Signature of Melanoma to a Novel Solid-Tissue Biomarker to Improve Diagnostic Accuracy and Reproducibility.

Background Successful treatment of cutaneous melanoma depends on early and accurate diagnosis of clinically suspicious melanocytic skin lesions. Currently, histopathology examination of excised skin lesions is considered the ‘gold standard’ for diagnosis of melanoma. Multiple studies have shown the low accuracy and reproducibility of this method, underscoring the urgent need for new diagnostic tools, including disease-specific biomarkers. Previously, a 38-microRNA signature of melanoma (‘Mel38’) was previously identified in plasma and validated as novel circulating biomarker. In this study, Mel38 expression in solid biopsy tissue is examined to determine its ability to contribute to accurate and reproducible melanoma diagnoses.Methods Nanostring digital gene expression profiling was used to apply the Mel38 signature in a cohort of 308 formalin fixed paraffin embedded skin biopsies (‘Mel38’). Genomic data were interrogated using hierarchical clustering, univariate and multivariate statistical approaches. Mel38 classification scores (range 0 to 10) were compared to consensus histopathology results, including MPATH-DX class, AJCC disease stage, histological subtype as well as technical assay factors.Results The Mel38 score can identify high-risk melanomas (MPATH-Dx Class IV) from less-malignant forms of the disease with an area-under-the curve of 0.96 (P < 0.001). The genomic score ranges from 0 to 10 and is positively correlated with the melanoma progression, from benign naevi to metastatic disease (intraclass correlation coefficient: 0.85). Using a score threshold of > 2.3 identifies higher-risk melanomas, associated with poorer outcomes and more intensive suggested clinical actions. Multivariate analysis showed the score to be a significant predictor of malignancy, independent of technical and clinical covariates. Analysis of the Mel38 signature in spitz naevi reveal an intra-subtype profile, in common to both benign and malignant conditions.Conclusion Melanoma-specific circulating microRNAs maintain their association with malignancy when measured in the hypothesized tissue of origin. The Mel38 signature is an accurate and reproducible metric of melanoma status, based on changes in microRNA expression that occur as the disease develops and spreads. Inclusion of the Mel38 score into routine practice would give physicians a genomic assessment of a patient’s disease status. Combining molecular biomarker data with conventional histopathology data may improve diagnostic accuracy, reproducibility, and patient outcomes.

Digital Gene Expression Analysis of Epithelioid and Sarcomatoid Mesothelioma Reveals Differences in Immunogenicity

Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with asbestos exposure. Median survival ranges from 14 to 20 months after initial diagnosis. As of November 2020, the FDA approved a combination of immune checkpoint inhibitors after promising intermediate results. Nonetheless, responses remain unsatisfying. Adequate patient stratification to improve response rates is still lacking. This retrospective study analyzed formalin fixed paraffin embedded specimens from a cohort of 22 MPM. Twelve of those samples showed sarcomatoid, ten epithelioid differentiation. Complete follow-up, including radiological assessment of response by modRECIST and time to death, was available with reported deaths of all patients. RNA of all samples was isolated and subjected to digital gene expression pattern analysis. Our study revealed a notable difference between epithelioid and sarcomatoid mesothelioma, showing differential gene expression for 304/698 expressed genes. Whereas antigen processing and presentation to resident cytotoxic T cells as well as phagocytosis is highly affected in sarcomatoid mesothelioma, cell–cell interaction via cytokines seems to be of greater importance in epithelioid cases. Our work reveals the specific role of the immune system within the different histologic subtypes of MPM, providing a more detailed background of their immunogenic potential. This is of great interest regarding therapeutic strategies including immunotherapy in mesothelioma.

Digital gene expression analysis of the response to Ralstonia solanacearum between resistant and susceptible tobacco varieties

Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious soil-borne disease of tobacco. However, molecular mechanism information of R. solanacearum resistance is limited to tobacco, hindering better breeding of resistant tobacco. In this study, the expression profiles of the rootstalks of Yunyan87 (susceptible cultivar) and Fandi3 (resistant cultivar) at different stages after R. solanacearum infection were compared to explore molecular mechanisms of tobacco resistance against the bacterium. Findings from gene-expression profiling indicated that the number of upregulated differentially expressed genes (DEGs) at 3 and 7 days post-inoculation (dpi) increased significantly in the resistant cultivar. WRKY6 and WRKY11 family genes in WRKY transcription factors, ERF5 and ERF15 family genes in ERFs transcription factors, and genes encoding PR5 were significantly upregulated in the resistant cultivar response to the infection. For the first time, WRKY11 and ERF15 were found to be possibly involved in disease-resistance. The Kyoto Encyclopedia of Genes and Genomes analysis demonstrated glutathione metabolism and phenylpropane pathways as primary resistance pathways to R. solanacearum infection. In the resistant cultivar, DEGs encoding CYP450, TCM, CCoAOMT, 4CL, PAL, CCR, CSE, and CADH, involved in the synthesis of plant antitoxins such as flavonoids, stilbenoids, and lignins, enriched in the phenylpropane pathway were upregulated at 3 and 7 dpi. Furthermore, a pot experiment was performed to verify the role of flavonoids in controlling TBW. This study will strongly contribute to a better understanding of molecular interactions between tobacco plants and R. solanacearum.

3’ RNA sequencing for robust and low-cost gene expression profiling

3’seq-RNA Profiling (3’SRP) approach is based on multiplexing samples and molecular indexing mRNA in order to drive genome-wide transcriptional profiling at reasonable cost in comparison to standard RNA-sequencing. The protocol is performed according to the 3′-digital gene expression (3′-DGE) approach developed by the Broad institute. The libraries are prepared from small amounts of total RNA where the mRNA poly(A) tails are tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs). We have improved the fragmentation step by implementing tagmentation based on the activity of a bead-linked transposome. This technique allows sample multiplexing on 96-well plates. Libraries are then sequenced using standard procedures, e.g. on Hiseq2500 or NovaSeq 6000 SP Flow Cells. We have developed a snakemake pipeline including every analysis step from raw fastq de-multiplexing to functional annotation of the differentially expressed genes, producing a complete HTML report for end-user.

Acceleration of Carbon Fixation in Chilling-Sensitive Banana under Mild and Moderate Chilling Stresses

Banana is one of the most important food and fruit crops in the world and its growth is ceasing at 10–17 °C. However, the mechanisms determining the tolerance of banana to mild (>15 °C) and moderate chilling (10–15 °C) are elusive. Furthermore, the biochemical controls over the photosynthesis in tropical plant species at low temperatures above 10 °C is not well understood. The purpose of this research was to reveal the response of chilling-sensitive banana to mild (16 °C) and moderate chilling stress (10 °C) at the molecular (transcripts, proteins) and physiological levels. The results showed different transcriptome responses between mild and moderate chilling stresses, especially in pathways of plant hormone signal transduction, ABC transporters, ubiquinone, and other terpenoid-quinone biosynthesis. Interestingly, functions related to carbon fixation were assigned preferentially to upregulated genes/proteins, while photosynthesis and photosynthesis-antenna proteins were downregulated at 10 °C, as revealed by both digital gene expression and proteomic analysis. These results were confirmed by qPCR and immunofluorescence labeling methods. Conclusion: Banana responded to the mild chilling stress dramatically at the molecular level. To compensate for the decreased photosynthesis efficiency caused by mild and moderate chilling stresses, banana accelerated its carbon fixation, mainly through upregulation of phosphoenolpyruvate carboxylases.

Identification of Differentially Expressed Drought-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub]

Drought remains one of the most serious environmental stresses because of the continuous reduction in soil moisture, which requires the improvement of crops with features such as drought tolerance. Guar [Cyamopsis tetragonoloba (L.) Taub], a forage and industrial crop, is a nonthirsty plant. However, the information on the transcriptome changes that occur under drought stress in guar is very limited; therefore, a gene expression analysis is necessary in this context. Here, we studied the differentially expressed genes (DEGs) in response to drought stress and their metabolic pathways. RNA-Seq via an expectation-maximization algorithm was used to estimate gene abundance. Subsequently, an Empirical Analysis of Digital Gene Expression Data in the R Bioconductor package was used to identify DEGs. Blast2GO, InterProScan, and the Kyoto Encyclopedia of Genes and Genomes were used to explore functional annotation, protein analysis, enzymes, and metabolic pathways. Transcription factors were identified using the PlantTFDB database. Our study identified 499 upregulated and 191 downregulated genes in response to drought stress. Of those, 32 upregulated and six downregulated genes were deemed as novel genes exclusive to guar. An aggregate of 137 protein families, 306 domains, 12 repeats, and two sites were upregulated. The proton-dependent oligopeptide transporter family and transferase, aquaporin transporter, calcium/calmodulin-dependent/calcium-dependent protein kinase, aspartic peptidase A1 family, UDP-glucuronosyl/UDP-glucosyltransferase, and major intrinsic protein were the most upregulated protein families. The upregulated unigenes were associated with 88 enzymes and 77 KEGG pathways. Finally, the MYB-related, MYB, and ERF transcription factor families were upregulated. These data may be useful for understanding the plant molecular response to drought stress.