Quantification noise in single cell experiments

In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample

Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018

All living organisms secrete molecules for intercellular communication. Recent research has revealed that extracellular vesicles (EVs) play an important role in inter-organismal cell-to-cell communication by transporting diverse messenger molecules, including RNA, DNA, lipids and proteins. These discoveries have raised fundamental questions regarding EV biology. How are EVs biosynthesized and loaded with messenger/cargo molecules? How are EVs secreted into the extracellular matrix? What are the EV uptake mechanisms of recipient cells? As EVs are produced by all kind of organisms, from unicellular bacteria and protists, filamentous fungi and oomycetes, to complex multicellular life forms such as plants and animals, basic research in diverse model systems is urgently needed to shed light on the multifaceted biology of EVs and their role in inter-organismal communications. To help catalyse progress in this emerging field, a mini-symposium was held in Munich, Germany in August 2018. This report highlights recent progress and major questions being pursued across a very diverse group of model systems, all united by the question of how EVs contribute to inter-organismal communication

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model

Abstract Background Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone. Methods Quadrilateral tendon lesions were induced in six horses by mechanical tissue disruption combined with collagenase application 3 weeks before treatment. Adipose-derived MSC suspended in serum or serum alone were then injected intralesionally. Clinical examinations, ultrasound and magnetic resonance imaging were performed over 24 weeks. Tendon biopsies for histological assessment were taken from the hindlimbs 3 weeks after treatment. Horses were sacrificed after 24 weeks and forelimb tendons were subjected to macroscopic and histological examination as well as analysis of musculoskeletal marker expression. Results Tendons injected with MSC showed a transient increase in inflammation and lesion size, as indicated by clinical and imaging parameters between week 3 and 6 (p < 0.05). Thereafter, symptoms decreased in both groups and, except that in MSC-treated tendons, mean lesion signal intensity as seen in T2w magnetic resonance imaging and cellularity as seen in the histology (p < 0.05) were lower, no major differences could be found at week 24. Conclusions These data suggest that MSC have influenced the inflammatory reaction in a way not described in tendinopathy studies before. However, at the endpoint of the current study, 24 weeks after treatment, no distinct improvement was observed in MSC-treated tendons compared to the serum-injected controls. Future studies are necessary to elucidate whether and under which conditions MSC are beneficial for tendon healing before translation into human medicine

Insights into the regulation of DMSP synthesis in the diatom Thalassiosira pseudonana through APR activity, proteomics and gene expression analyses on cells acclimating to changes in salinity, light and nitrogen

Despite the importance of dimethylsulphoniopropionate (DMSP) in the global sulphur cycle and climate regulation, the biological pathways underpinning its synthesis in marine phytoplankton remain poorly understood. The intracellular concentration of DMSP increases with increased salinity, increased light intensity and nitrogen starvation in the diatom Thalassiosira pseudonana. We used these conditions to investigate DMSP synthesis at the cellular level via analysis of enzyme activity, gene expression and proteome comparison. The activity of the key sulphur assimilatory enzyme, adenosine 5′- phosphosulphate reductase was not coordinated with increasing intracellular DMSP concentration. Under all three treatments coordination in the expression of sulphur assimilation genes was limited to increases in sulphite reductase transcripts. Similarly, proteomic 2D gel analysis only revealed an increase in phosphoenolpyruvate carboxylase following increases in DMSP concentration. Our findings suggest that increased sulphur assimilation might not be required for increased DMSP synthesis, instead the availability of carbon and nitrogen substrates may be important in the regulation of this pathway. This contrasts with the regulation of sulphur metabolism in higher plants, which generally involves upregulation of several sulphur assimilatory enzymes. In T. pseudonana changes relating to sulphur metabolism were specific to the individual treatments and, given that little coordination was seen in transcript and protein responses across the three growth conditions, different patterns of regulation might be responsible for the increase in DMSP concentration seen under each treatment

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1-2, d 3-4, and d 5-7 (early CL, eCL), d 8-12 (mid CL, mCL), d 13-16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, -2898, -96, -7-5p, -183-5p, -182, and -202 showed drastic up-regulation with a fold-change of >= 2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility