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Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation‐transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non‐myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate‐dependent fashion. Mechanistically, several calcium‐handling proteins, including the sodium‐calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin‐dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX‐dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX.

How diversity in growth thermo-responsiveness is generated for local adaptation is a long-standing biological question. We investigated molecular genetic basis of natural variations in thermo-responsiveness of plant architecture in Arabidopsis thaliana. We measured the extent of rosette architecture at 22°C and 28°C in a set of 69 natural accessions and determined their thermo-responsiveness of plant architecture. A genome-wide association study was performed to identify major loci for variations in thermo-responsiveness. The SAUR26 subfamily, a new subfamily of SAUR genes, was identified as a major locus for the thermo-responsive architecture variations. The expression of SAUR26/27/28 is modulated by temperature and PIF4. Extensive natural polymorphisms in these genes affect their RNA expression levels and protein activities and influence the thermo-responsiveness of plant architecture. In addition, the SAUR26 subfamily genes exhibit a high variation frequency and their variations are associated with the local temperature climate. This study reveals that the SAUR26 subfamily is a key variation for thermo-responsive architecture and suggests a preference for generating diversity for local adaptation through signaling connectors.

Tracking the expression of RNA in a cell-specific manner is a major challenge in basic and disease research. Herein we outline the current state of employing chemical approaches for cell-specific RNA expression studies. We define the utility of metabolic labels for tracking RNA synthesis, the approaches for characterizing metabolic incorporation and enrichment of labeled RNAs, and finally outline how these approaches have been used to study biological systems by providing mechanistic insights into transcriptional dynamics. Further efforts on this front will be the continued development of novel chemical handles for RNA enrichment and profiling as well as innovative approaches to control cell-specific incorporation of chemically modified metabolic probes. These advancements in RNA metabolic labeling techniques permit sensitive detection of RNA expression dynamics within relatively small subsets of cells in living tissues and organisms that are critical to performing complex developmental and pathological processes. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Evolution and Genomics > Ribonomics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.

MicroRNAs (miRNAs) are short non-coding single-stranded RNAs, which play significant roles in the regulation of a myriad of biological processes. Overwhelmingly increasing high-impact research has also deepened our understanding about the central role of miRNAs in cancer development, metastatic spread, and development of resistance against various drugs. Recent studies have identified miRNAs that regulate RNA expression/processing and posttranscriptional expression of important oncogenes and tumor suppressors. Rapidly emerging experimentally verified data have started to shed light on the significance of miRNAs as biomarkers for diagnostic, prognostic, and monitoring purposes. Next-generation sequencing and DNA microarray technologies have helped us tremendously in the identification of miRNA and mRNA signatures in different cancers and their subtypes on a genome-wide scale. It is being increasing realized that miRNAs have diametrically opposite roles in different cancers. miR-410 is context-dependently involved in positive and negative regulation of cancers. miR-410 negatively regulates BAK1, CETN3, and BRD7 to promote cancer. However, miR-410 effectively targetes c-MET, AGTR1, and SNAIL to suppress cancer. In this review, we will comprehensively summarize most recent evidence available related to the "split personality" of miR-410 in different cancers.

Single-cell RNA sequencing on circulating tumor cells (CTCs) proves useful to study mechanisms of tumor heterogeneity, metastasis, and drug resistance. Currently, single-cell RNA sequencing of CTCs usually takes three prerequisite steps: enrichment of CTCs from whole blood, characterization of captured cells by immunostaining and microscopic imaging, and single-cell isolation through micromanipulation. However, multiple pipetting and transferring steps can easily cause the loss of rare CTCs. To address this issue, a novel integrated microfluidic chip for sequential enrichment, isolation, and characterization of CTCs at single-cell level, is developed. And, single CTC lysis is achieved on the same chip. The microfluidic chip includes functions of blood clot filtration, single-cell isolation, identification, and target single-cell lysate collection. By spiking tumor cells into whole blood, it is validated that this microfluidic chip can effectively conduct single-cell CTCs RNA sequencing. The approach lays a solid foundation for the analysis of RNA expression profiling of single-cell CTCs.

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

Raman spectroscopy is a molecular spectroscopic technique that can measure the molecular composition of tissue samples within seconds without any extraction processes or dyes. In microbiology, Raman spectroscopy is used to identify bacteriae. In glioblastoma tissue, it was reported that necrosis, normal brain and tumor can be discriminated using Raman spectroscopy. Therefore, we hypothesized that Raman spectroscopy could discriminate glioblastoma tissue from different glioma subtypes defined by RNA expression profiling. We analyzed 20 glioma samples from two distinct molecular subtypes. Both subtypes consisted of glioblastoma samples showing a variety in glioma grading and typing. The Raman spectroscopic results could be grouped in two distinct clusters in an unsupervised cluster analysis. Further analysis of these clusters showed that they were fully congruent with the two clusters as defined by RNA expression profiling. Conclusion: our results demonstrate that Raman spectroscopy can discriminate between different molecular subtypes of glioma and, therefore, may prove to be a valuable tool in in vitro cancer research. Copyright © 2013 John Wiley & Sons, Ltd.

The use of growth-promoting agents in food-producing animals is forbidden in the European Union (EU). Therefore a strict control programme has been developed, detecting residues of all known growth-promoting agents using chromatographical methods in combination with mass spectrometry or immunoassays. New designed xenobiotic substances or hormone cocktails are difficult to identify with these methods and therefore the development of new sensitive test methods is important. A promising indirect approach is the detection of physiological effects of the administered growth promoters on the molecular level using ‘omic’ technologies. The analysis of the transcriptome on mRNA and miRNA level and thereby identifying biomarkers for the use of anabolic agents is one possible strategy for developing a new screening method. This paper describes the technologies available for gene expression profiling and summarizes the efforts made in the analysis of the transcriptome in order to identify potential gene expression biomarkers for the use of growth promoters in cattle. © 2012 John Wiley & Sons, Ltd.

High throughput molecular biology began around the mid-1990s with the introduction of microarrays – a technology that enabled investigators to quantify the cellular expression levels of tens of thousands of mRNA transcripts simultaneously. To date, a large number of microarray experiments have been performed in the investigation of RNA expression signatures in normal and pathological tissues. This review focuses on a next generation tool in high throughput investigation: RNA sequencing or RNA-Seq, highlighting its advantages over traditional microarray investigation and discussing its utility in investigative dermatology. In contrast with the results obtained from microarray experiments, RNA-Seq generates mRNA abundance counts, can identify novel transcripts and splice variants, and provides sequence resolution at the level of single base-pairs. Implementing RNA-Seq in the investigation of skin disease will yield novel insights into the pathogenesis of disease, will facilitate the discovery of new diseases and new mechanisms of disease, and will allow researchers to probe genetic disease in high resolution and with unprecedented efficiency.

Background and Objectives Analysis of survivin RNA expression in peripheral blood as a non-invasive molecular predictor of response to neoadjuvant radiochemotherapy in patients with locally advanced cancer of the esophagus. Material and Methods Blood samples were drawn from 29 patients with esophageal cancer prior to neoadjuvant radiochemotherapy. After extraction of cellular tumor-RNA from blood samples, quantitative expression analysis of survivin was done by quantitative real-time RT-PCR. Results Twenty of 29 (69%) of patients showed a minor histopathological response and 9 of 29 (31%) showed a major-response to neadjuvant radiochemotherapy. RNA expression in blood of patients was detectable for survivin in 27.6%, and in 100% for β-actin. The mean survivin expression was not significantly different between minor- and major-responders. No significant associations were detected between survivin expression levels and patients clinical variables. A high expression level for survivin was significantly associated with a minor-response to neoadjuvant treatment (P = 0.042). Relative survivin expression levels above 0.15 were not associated with major histopathological response (sensitivity: 35%; specificity: 100%). Conclusion Minor-response to the applied therapy was significantly associated with a high survivin RNA expression level in patient's blood. Survivin appears to be a specific non-invasive predictor of response to neoadjuvant therapy in esophageal cancer. J. Surg. Oncol. 2009;100:447–451. © 2009 Wiley-Liss, Inc.

Background Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high-throughput platforms. But these mainly chip-based methods can only generate biologically relevant data if the samples investigated consist of homogenous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results. Methods Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser-assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available. Results These laser-assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high-throughput RNA expression profiling by microdissecting and processing even single-cell samples. Conclusions Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient-specific drugs tailored to the requirements assessed by assaying only a few biopsy cells. © 2004 Wiley-Liss, Inc.

Although aquaporins (AQP) are known to be involved in water movement, the role of AQP3 in the barrier dysfunction that characterizes inflammatory bowel diseases remains unknown. We hypothesized tha...

Summary: We have developed a strategy to individually analyse large numbers of small tissue samples by RNA in situ hybridisation. Samples of approximately 0.4 mm × 0.5 mm are processed in rectangular capillary tubes fitted with nylon mesh and glass beads using standard protocols. Eighteen samples can be assayed simultaneously without loss, and background is low. Specifically, mouse Sox2 RNA expression is examined in the chorion of extraembryonic tissue of 7.5 days post-coitum embryos. This technique works equally well for double RNA labelling and could potentially be used for antibody staining of proteins. genesis 27:76–80, 2000. © 2000 Wiley-Liss, Inc.

Cells with procollagen α1(I) transcripts were demonstrated at the interface of natural coral mineral implanted in the femur of rats after 7, 14, 21 and 28 days by in situ hybridization with digoxigenin-labelled RNA probes. Procollagen α1(I) transcripts were detectable in osteoblasts between 7 and 28 days after implantation. As early as 7 days after implantation, procollagen α1(I) RNA expression was also demonstrated in fibroblasts; the transcript steady state levels decreased until the 28th day, when they subsided completely. The development of bone was significant during the 4 weeks of implantation. Inhibition of bone development by the coralline material could not be recorded.

A vast amount of DNA sequence data, protein three-dimensional (3D) structure data, and RNA expression data have been produced by the efforts of genome sequencing, structural genomics, and omics projects, and we are at the stage where comprehensive views of cell activity and molecular mechanisms of life can be deduced. But in reality, we are inundated with massive amounts of data and are still in the process of finding ways to fully utilize the data. In this report, I would like to present our observations on the growth of protein 3D structure data and our effort to deduce the functions from the 3D structures. We found that the 3D structure of quite a high proportion of proteins derived from genome sequences can be now predicted and methods to predict the functions from 3D structures are in high demand. The methods we have developed can be used to predict some functions, namely RNA and ligand interfaces, based on those 3D structures and DNA sequences with relatively high accuracy. The methods enable predictions that are accurate enough to help with deducing the atomic structures of the complexes.

The mechanism of modulation of sodium channel α-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K+ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K+ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K+ equilibrium potential (EK), (2)decreased in slope conductance near EK, (3) was dependent on [K+]o and (4) was blocked by external Ba2+ and Cs+. These properties were similar to those of the inwardly rectifying K+ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K+ channels.

Anal carcinomas were tested for the presence of human papillomavirus (HPV) RNA transcripts by in situ hybridization using 125I-labeled RNA probes. The HPV transcripts were detected in 73.2% of 41 nonglandular anal carcinomas but in none of six anal or 11 rectal adenocarcinomas. In anal nonglandular carcinomas, no difference in the percentage of tumors that were HPV RNA positive was observed between tumors classed as basaloid carcinomas or squamous cell carcinomas. Patients with HPV RNA-negative tumors were significantly older (9.2 years) than those with HPV RNA-positive tumors. Anal intraepithelial neoplasia Grade 3 in the surrounding epithelium was seen in 14 of 25 assessable HPV RNA-positive tumors and in none of nine assessable HPV RNA-negative tumors. Anal tumors may include two separate clinical and biologic groups distinguished by their dependence, or lack of dependence, on HPV RNA expression for maintenance of the neoplastic state.

The multiprobe RNase protection assay enables investigators to monitor RNA expression of 8-12 genes with as little as 1 microg of total RNA. The commercial availability of numerous multi-gene template sets makes this assay practical for all basic research programs.