Absolute Expression Research Articles

Effect of E-beam treatment on expression of virulence and stress-response genes of Listeria monocytogenes in dry-cured ham

Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.

Expression and Sympathy in Walter Pater, Oscar Wilde, and Kenneth Burke

ABSTRACT As corrective to rhetorical theorists who disparage “expression,” the following article analyzes Walter Pater, Oscar Wilde, and Kenneth Burke on “expression” and its communicative counterpart “sympathy.” Pater viewed ideal style as a unity of expression and sympathy. Wilde saw Christ as the singular representative of absolute expression and sympathy. Burke resolved both expression and sympathy into the “compromise” of the symbol. I advocate for a return to expression and sympathy as rhetorical values in the twenty-first century.

Measuring lncRNA Expression by Real-Time PCR.

Long noncoding RNAs are defined as transcripts longer than 200nt with no protein coding potential. Most lncRNAs are expressed in a tissue-specific manner and barring a few, their absolute expression is lower compared to most coding transcripts. Differential expression studies have contributed the most to the functional characterisation of the lncRNAs we know. Sensitive and specific quantification of lncRNA expression is crucial for such studies. SYBR Green dye based real time quantitative PCR is a simple and affordable method of quantitative PCR, wherein the specific binding of the dye to double stranded DNA amplicon emits fluorescence proportionate to the amount of PCR products. Here we describe a detailed protocol for successful lncRNA quantitation by reverse transcription followed by SYBR Green chemistry-based real-time PCR.

Buckwheat FeNramp5 Mediates High Manganese Uptake in Roots.

Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.

Multiple imputation and direct estimation for qPCR data with non-detects

BackgroundQuantitative real-time PCR (qPCR) is one of the most widely used methods to measure gene expression. An important aspect of qPCR data that has been largely ignored is the presence of non-detects: reactions failing to exceed the quantification threshold and therefore lacking a measurement of expression. While most current software replaces these non-detects with a value representing the limit of detection, this introduces substantial bias in the estimation of both absolute and differential expression. Single imputation procedures, while an improvement on previously used methods, underestimate residual variance, which can lead to anti-conservative inference.ResultsWe propose to treat non-detects as non-random missing data, model the missing data mechanism, and use this model to impute missing values or obtain direct estimates of model parameters. To account for the uncertainty inherent in the imputation, we propose a multiple imputation procedure, which provides a set of plausible values for each non-detect. We assess the proposed methods via simulation studies and demonstrate the applicability of these methods to three experimental data sets. We compare our methods to mean imputation, single imputation, and a penalized EM algorithm incorporating non-random missingness (PEMM). The developed methods are implemented in the R/Bioconductor package nondetects.ConclusionsThe statistical methods introduced here reduce discrepancies in gene expression values derived from qPCR experiments in the presence of non-detects, providing increased confidence in downstream analyses.

The evaluation of the balance of standard rates within the program on state guarantees to deliver free medical care to the citizens of the Russian Federation

Aim. The evaluation of absolute and relative difference in the growth of standard rates and financing of medical help based on the calculation for Program on State Guarantees to Deliver Free Medical Care to the Citizens of the Russian Federation (PSG) by the parameters approved by the Decree of the Government for 2019 and 2020.Materials and methods. The evaluation was performed by the methods of mathematic modeling. Mathematic modeling of the calculation of the volumes of medical help (MH) and financial costs in the subjects of the RF was based on the Decrees of the Government for 2019 and 2020 and the method of calculation of the standard rates specified in the Letter on the economic grounds for territorial PSG. Modeling for each region was used to compare the absolute and relative difference in the growth of standard rates for the obtained values in the absolute and relative expression of the calculated planned parameters of PSG for 2019 and 2020 according to the described method in terms of types, conditions, and profiles of medical help.Results. It was revealed that the existing standard rates for financial costs did not provide a uniform growth of costs by all the conditions and types of MH. The growth of financing by types and conditions varied from 1.43 to 9.59% of the 2019 baseline in the RF. Certain “specialized” (profile) standard rates within the conditions of MH showed a significant growth at a slower growth of financing of the rest of MH types (3.86% for all-day inpatient service and -0.6% (decrease in financing) for day-time inpatient service). The calculation of the model for the subjects of the RF showed even greater fluctuations: from –0.11% to –3.03% for day-time inpatient service and from 1.35% to 7.27% for all-day inpatients service.Conclusion. Despite the annual growth of the financing of PSG, redistribution of the budget within the conditions of medical service provision is uneven. This situation provides background for the improvement of the mechanisms of planning of MH within PSG using obligatory modeling and balanced standard rates for the subjects of the RF.

Ikaros Expression in the Graft Is Associated with Chronic Graft Versus Host Disease

Introduction: Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment for several diseases. Immune reconstitution post HSCT is a complex and extremely variable process. Ikaros transcription factor has an important role in hematopoiesis of several cell lines, especially in the lymphoid compartment. We hypothesized that Ikaros expression, both in the graft and in the recipient after transplant, might influence immune reconstitution and, consequently, the risk of opportunistic infections, relapse, and graft versus host disease (GVHD). Objectives: To correlate Ikaros expression both in the graft and in the recipient's peripheral blood (PB) after engraftment with the risk of GVHD after allogeneic HSCT outcomes. Patients and methods: 51 patients were included. Median age was 51 years (19-80), 53% of patients were male, and 57% of them had acute leukemia. Donor were haploidentical in 45% of cases, related identical in 29% and unrelated identical in 26%. Most patients (82%) received reduced-intensity conditioning regimens. Median follow-up was 20 months (10-40 months). Samples were collected from the graft and from the PB of the recipient 3 weeks after neutrophil recovery. Real time polymerase chain reaction (RT-PCR) was performed for analysis of absolute and relative Ikaros expression. Results: There was no association between Ikaros expression and the risk of acute GVHD, relapse or mortality. However, a significant association was observed in the risk of chronic GVHD. Cumulative incidence (CI) of chronic GVHD and of moderate / severe chronic GVHD (according to National Institute of Health - NIH classification) in two years were 49% and 28%, respectively. Higher Ikaros expression in the graft was associated with a significantly higher risk of moderate/severe chronic GVHD (54% vs. 15%, respectively, P=0.01). Higher Ikaros expression in the recipient's PB after engraftment was also associated with a significantly higher risk of moderate/severe chronic GVHD (65% vs. 11%, respectively, P=0.01). Conclusions: Ikaros expression in the graft and in the PB of the recipient after transplant seems to be correlated with a higher risk of moderate/severe chronic GVHD and might be evaluated in larger and prospective trials as a potential biomarker. Disclosures No relevant conflicts of interest to declare.

Characterization and absolute quantification of the Cellulose synthase-like F6 homoeologs in oats

The Cellulose synthase-like F6 (CslF6) gene has been reported as a key factor for β-glucan synthesis in grasses. However, the action of this gene on oat β-glucan content is poorly understood. The objectives of this study were: (i) to quantify the absolute expression of CslF6 transcripts in oats under field conditions; and (ii) to characterize the CslF6 homoeologs in oat genotypes with different levels of β-glucan content. Developing kernels of five oat genotypes were collected under field conditions in two planting dates. The CslF6 absolute quantification was performed using droplet digital PCR considering global and homoeolog-specific expressions. The third exon of the CslF6 gene, which covers approximately 62.2% of the coding region, was sequenced in each homoeolog. The CslF6 expression was highly influenced by planting date, kernel developmental stage, and genotype. High CslF6 transcript number was not associated with high β-glucan content, indicating the action of a complex biosynthesis pathway controlling this trait in oats. Under stressful conditions, the CslF6 expression was more pronounced and associated with β-glucan content. The CslF6 motif has 1767 bases pair and is highly conserved between A and D homoeologs. Single nucleotide polymorphisms were not identified among the assessed genotypes, regardless of the homoeolog. The results presented in this study may contribute for a better understanding of the CslF6 influence on oat β-glucan content.

Multiomic Analysis of Subtype Evolution and Heterogeneity in High-Grade Serous Ovarian Carcinoma.

Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but their interpretation and translation are complicated by tumor evolution and polyclonality accompanied by extensive accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. In this study, we examined the chronology of HGSOC subtype evolution in the context of these factors using a novel integrative analysis of absolute copy-number analysis and gene expression in The Cancer Genome Atlas complemented by single-cell analysis of six independent tumors. Tumor purity, ploidy, and subclonality were reliably inferred from different genomic platforms, and these characteristics displayed marked differences between subtypes. Genomic lesions associated with HGSOC subtypes tended to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations was evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors were characterized by largely intact genome integrity, high immune infiltration, and younger patient age. Single-cell sequencing of 42,000 tumor cells revealed widespread heterogeneity in tumor cell type composition that drove bulk subtypes but demonstrated a lack of intrinsic subtypes among tumor epithelial cells. Our findings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tumor stage and tissue of origin, and evolution between properties previously associated with discrete subtypes. SIGNIFICANCE: This study infers whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are, therefore, appropriate targets for therapy and demonstrates that this is not the case for HGSOC.

TOP2A and CENPF are synergistic master regulators activated in cervical cancer

BackgroundIdentification of master regulators (MRs) using transcriptome data in cervical cancer (CC) could help us to develop biomarkers and find novel drug targets to fight this disease.MethodsWe performed differential expression (DE) analyses of public microarray and RNA-seq transcriptome data of CC and normal cervical tissues (N). Virtual Inference of Protein activity by Enriched Regulon analysis (VIPER) was used to convert the DE outcomes to differential activity (DA) signature for MRs. Synergy analysis was conducted to study synergistic effect of MR-pairs. TCGA and microarray data were used to test the association of expression of a MR and a clinical feature or a molecular feature (e.g. somatic mutations). Various bioinformatic tools/websites (DAVID, GEPIA2, Oncomine, cBioPortal) were used to analyze the expression of the top MRs and their regulons.ResultsTen DE and 10 DA signatures were generated for CC. Two MRs, DNA topoisomerase II alpha (TOP2A) and centromere protein F (CENPF) were found to be up-regulated, activated and synergistic in CC compared to N across the 10 datasets. The two MRs activate a common set of genes (regulons) with functions in cell cycle, chromosome, DNA damage etc. Higher expression of CENPF was associated with metastasis. High expression of both MRs is associated with somatic mutation of a set of genes including tumor suppressors (TP53, MSH2, RB1) and genes involved in cancer pathways, cell cycle, DNA damage and repair. The magnitude of up-regulation and the absolute expression level of both MRs in CC are significantly higher compared to many other cancer types.ConclusionTOP2A and CENPF are a synergistic pair of MRs that are overexpressed and activated in CC. Their high expression is correlated with some prognosis features (e.g. metastasis) and molecular features (e.g. somatic mutations) and distinctly high in CC vs. many other cancer types. They may be good biomarkers and anticancer drug targets for CC.

Development of a serum miRNA panel for detection of early stage non-small cell lung cancer.

Minimally invasive testing for early detection of lung cancer to improve patient survival is a major unmet clinical need. This study aimed to develop and validate a serum multi-microRNA (multimiR) panel as a minimally invasive test for early detection of nonsmall cell lung cancer (NSCLC) regardless of smoking status, gender, and ethnicity. Our study included 744 NSCLC cases and 944 matched controls, including smokers and nonsmokers, male and female, with Asian and Caucasian subjects. Using RT-qPCR and a tightly controlled workflow, we quantified the absolute expression of 520 circulating microRNAs (miRNAs) in a Chinese cohort of 180 early stage NSCLC cases and 216 healthy controls (male smokers). Candidate biomarkers were verified in two case-control cohorts of 432 Chinese and 218 Caucasians, respectively (including females and nonsmokers). A multimiR panel for NSCLC detection was developed using a twofold cross-validation and validated in three additional Asian cohorts comprising 642 subjects. We discovered 35 candidate miRNA biomarkers, verified 22 of them, and developed a five-miR panel that detected NSCLC with area under curve (AUC) of 0.936-0.984 in the discovery and verification cohorts. The panel was validated in three independent cohorts with AUCs of 0.973, 0.916, and 0.917. The sensitivity of five-miR test was 81.3% for all stages, 82.9% for stages I and II, and 83.0% for stage I NSCLC, when the specificity is at 90.7%. We developed a minimally invasive five-miR serum test for detecting early stage NSCLC and validated its performance in multiple patient cohorts independent of smoking status, gender, and ethnicity.

Changes in MicroRNA Expression during Rabbit Hemorrhagic Disease Virus (RHDV) Infection.

Current knowledge on the role of microRNAs (miRNAs) in rabbit hemorrhagic disease virus (RHDV) infection and the pathogenesis of rabbit hemorrhagic disease (RHD) is still limited. RHDV replicates in the liver, causing hepatic necrosis and liver failure. MiRNAs are a class of short RNA molecules, and their expression profiles vary over the course of diseases, both in the tissue environment and in the bloodstream. This paper evaluates the expression of miRNAs in the liver tissue (ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p) and serum (ocu-miR-122-5p) of rabbits experimentally infected with RHDV. The expression levels of ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p in liver tissue were determined using reverse transcription quantitative real-time PCR (RT-qPCR), and the expression level of circulating ocu-miR-122-5p was established using droplet digital PCR (ddPCR). The expression levels of ocu-miR-155-5p and ocu-miR-16b-5p were significantly higher in the infected rabbits compared to the healthy rabbits (a fold-change of 5.8 and 2.5, respectively). The expression of ocu-miR-122-5p was not significantly different in the liver tissue from the infected rabbits compared to the healthy rabbits (p = 0.990), while the absolute expression level of the circulating ocu-miR-122-5p was significantly higher in the infected rabbits than in the healthy rabbits (p < 0.0001). Furthermore, a functional analysis showed that ocu-miR-155-5p, ocu-miR-16b-5p, and ocu-miR-122-5p can regulate the expression of genes involved in processes correlated with acute liver failure (ALF) in rabbits. Search tool for the retrieval of interacting genes/proteins (STRING) analysis showed that the potential target genes of the three selected miRNAs may interact with each other in different pathways. The results indicate the roles of these miRNAs in RHDV infection and over the course of RHD and may reflect hepatic inflammation and impairment/dysfunction in RHD.

Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 106 copy/μg total RNA), AKR1C3 (1.3 × 106), AKR1C1 (1.3 × 106), CBR1 (9.7 × 105), and HSD11B1 (1.1 × 106) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.

Noninvasive Monitoring of Glycemia-Induced Regulation of GLP-1R Expression in Murine and Human Islets of Langerhans.

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify β-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and β-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in β-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

Abstract 5432: Cancer of unknown primary site-of-origin: An enigma ready to be miR-solved

Abstract Background Cancer of Unknown Primary is a rare syndrome of metastatic cancers for which the primary site cannot be recognized after detailed physical examinations, blood analyses, imaging and immunohistochemical (IHC) testing. CUPs make up 3-5% of newly diagnosed cancers worldwide and represent an important diagnostic and clinical problem. Treatment failure to empirical chemotherapy is common, resulting in progressive disease and poor prognosis. We and others proposed that a molecular inference of the tissue of origin could be feasible for most CUP tumors (Ferracin et al. J Pathol, 2011), thus giving access to more effective therapeutic agents and potentially extending CUP patient life-expectancy. Methods We optimized a cancer-type classifier based on microRNA (miRNA) expression for the prediction of CUP primary tumor site (Laprovitera et al., Front Oncol. 2018). Specifically, we designed a molecular assay to quantify the absolute copy number of 89 miRNAs in formalin-fixed paraffin-embedded (FFPE) samples using EvaGreen-based Droplet Digital PCR (BioRad). We tested 153 samples from three groups: primary tumors (N=95), metastases of known origin (N=10) and cancers of unknown origin (N=48). CUP diagnosis was obtained after a detailed histo-pathological investigation as per CUP diagnostic guidelines (NCCN occult primary v.2-2019 and ESMO guidelines). The study was approved by the local ethical committee. Results We investigated a pre-determined set of 89 miRNAs to infer the site-of origin of metastatic cancers of unknown or uncertain primary. Primary tumors from 16 different sites (lung, pancreas, liver, bile duct, kidney, bowel, testis, ovary, endometrium, stomach, bladder, breast, prostate, melanoma, gastrointestinal neuroendocrine, head and neck) were tested for absolute miRNA expression and used to train our classifier (Tibshirani et al. PNAS, 2002). We used the metastases of known origin as a test-set for our molecular prediction. We applied the shrunken centroids approach as predictive algorithm to obtain the most probable CUP site(s)-of- origin. Our results were judged against the hypothesized primary site suggested by standard diagnostic workup and pathological assessment. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification (with a probability &amp;gt; 50%) for all samples, potentially within a one-week time frame from sample inclusion. Conclusions Our study demonstrated that miRNA expression profiling in CUP samples have the potential to be employed in a clinical setting. Our molecular analysis can be performed on-request, concomitantly with the standard diagnostic workup and in association with genetic profiling, to offer valuable indication about the possible primary site thereby supporting treatment decisions. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Elisa Porcellini, Mattia Riefolo, Elisabetta Broseghini, Ingrid Garajova, Silvia Sabbioni, Martin Pichler, Andrea Ardizzoni, Antonia D'Errico, Manuela Ferracin. Cancer of unknown primary site-of-origin: An enigma ready to be miR-solved [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5432.

Digital Absolute Gene Expression Analysis of Essential Starch-Related Genes in a Radiation Developed Amaranthus cruentus L. Variety in Comparison with Real-Time PCR

We investigated the expression pattern of four major starch genes at different seed developmental stages in the radiation-bred amaranth variety “Pribina” (Amaranthus cruentus L.) and corresponding control genotype “Ficha” (Amaranthus cruentus L.). Two platforms were used and compared for the gene expression analysis of GBSSI, SSSI, SBE, and DBE amaranth genes, including a standard quantitative real-time PCR (qPCR) technique and relatively novel droplet digital PCR (ddPCR) assay. In our conditions, both methods showed great accuracy and revealed higher expression of the investigated genes in the mutant variety than in the control genotype. Here we report for the first time, a ddPCR gene expression assay for the cultivated grain amaranth, as the most important group of the species in the genus Amaranthus.

Daphnia magna modifies its gene expression extensively in response to caloric restriction revealing a novel effect on haemoglobin isoform preference.

Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro-RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6,000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three haem-binding erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of superoxide dismutase (SOD) in normally fed individuals, which contrasts with observations of high SOD levels under CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between haemoglobin and CR occurs in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

ABSTRACT Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be ‘hot spots’ for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

Promoter polymorphisms of the PCA3 gene are not associated with its overexpression in prostate cancer patients

In male, the prostate cancer (PCa) is one of the most frequent neoplasias and the second cause of cancer deaths worldwide. In 2015, more than 6000 men died in Mexico due to this disease. In this regard, prostate cancer associated gene 3 (PCA3) has become an interesting target in PCa as is found highly overexpressed. Moreover, TAAA tandem repeats have been suggested to be associated with the regulation of PCA3 expression and, in turn, to be related with the development of the disease. The aim of the study was to understand thegenetic basis of the disease in search for a better diagnosis. Expression levels of PCA3 gene were analysed in tissue of 13 patients diagnosed with PCa and six patients diagnosed with a benign prostatic disease (BPD). The absolute expression of PCA3 was quantified by real-time PCR. Genotype for TAAA tandem repeats was measured using automatic sequencing and the results were analysed to determine whether an association existed between them. We identified three alleles: 4, 5, 6 and four genotypes: 4/5, 5/5, 5/6, 6/6. Our analysis identified amutation in the nucleotide 76764237 of the PCA3 gene that generates an extra TAAA tandem repeat. The nucleotide mutation is present in 61.53% of PCa and 66.66% of BPD patients. Our study revealed the presence of a mutation in the PCA3 gene that generates an extra TAAA tandem. We observed no association between the absolute expression of PCA3 messenger and the number of TAAA repetitions.

Novel target discovery in pembrolizumab-resistant gastric cancer using a comprehensive RNA-seq analysis pipeline.

e16541 Background: Immune checkpoint inhibition (ICI) has made significant breakthroughs in several tumor types including gastric cancer (GC) in recent years. We recently showed that single agent pembrolizumab demonstrated remarkable and durable response in MSI and EBV GC. However, the response to ICI remains low in MSS and most patients progress after initial response. We explore novel targets in ICI-resistant GC patients by analyzing pre- and post-resistant expression. Methods: Of the 61 patients who were enrolled onto our previously reported phase II pembrolizumab trial (NCT#02589496), whole transcriptome RNA-seq analysis of 10 paired freshly collected tissue samples (all from primary gastric tumors) was performed using TruSeq. All biopsies were performed at progression following stable disease (SD) or partial response (PR) to pembrolizumab. All patients had a MSI status of MSS and EBV negative. Molecular features were extracted using the validated Ocean Genomics, Inc. gene expression analysis pipeline, which trims reads, computes transcript- and gene-level expression, predicts structural variants, assembles novel isoforms, and computes per-sample quality control metrics, among other analyses. Samples that passed quality control, with mapping rates &gt; 88%, were selected for analysis. Differentially expressed (DE) genes between resistant and pre-resistant samples were identified using a statistical test with a study design that accounted for the pairing of samples for each patient. Results: 16 genes (GENCODE v31) had absolute log2-fold expression changes (L2FC) &gt; 2, P-value &lt; 10−5 and FDR-adjusted P-value &lt; 0.05. Because sex was only partially controlled for, we excluded genes on the X and Y chromosomes. We also excluded non-protein encoding genes and pseudogenes. The 7 remaining genes are in the table. PDL-1 (CD274) was not identified as significantly DE (FDR-adjusted P-value &gt; 0.9999). Conclusions: This is the first study to identify novel targets in pembrolizumab-resistant GC using RNA-seq algorithms beyond PDL-1. [Table: see text]