Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

AbstractCHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation

Ethics of clinical trials from bayesian perspective: medical decision making should use posteriors, not priors.

<p><b>A-B. Analysis of stimulation-induced changes in muscle fiber diameter (MFD) of TAM (A) and PCAM (B).</b> MFD values were grouped in 10 μm bins and are presented as percentage of total fibers <b>3C. Analysis of stimulation-induced changes in fiber type distribution</b>. Relative percentages of type 1 and type 2 fibres for both muscles.</p

MicroRNA Functions in Brite/Brown Fat — Novel Perspectives towards Anti-Obesity Strategies

Current anti-obesity strategies are aiming at restricting energy uptake, but still, obesity treatment is far from being satisfactory. The discovery of active brown adipose tissue (BAT) in adult humans currently opens new avenues to combat obesity and follow-up complications as it tackles the other site of the energy balance: energy expenditure via non-shivering thermogenesis. This process of energy dissipation in the adipose tissue is tightly controlled, and the elucidation of its regulatory network is a key plank for therapeutic applications. MicroRNAs (miRNAs) belong to a novel class of regulatory determinants which are small non-coding RNAs with vital roles in regulating gene expression that also play a role in many human diseases. In this review we summarize miRNAs which have been shown to govern thermogenic, i.e. brite or brown, adipocyte recruitment and physiology. Notably, most miRNAs in this context have so far been characterized solely in mice, revealing a great demand for more human studies. As in the context of other diseases, RNA-based therapeutics have meanwhile entered clinical trials, further exploring the functions of miRNAs in brown and white adipose tissues could result in novel therapeutic approaches to treat obesity and its follow-up complications

Hunting the Needle in the Haystack: A Guide to Obtain Biologically Meaningful MicroRNA Targets

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of ~23 nucleotides in length that form up a novel class of regulatory determinants, with a large set of target mRNAs postulated for every single miRNA. Thousands of miRNAs have been discovered so far, with hundreds of them shown to govern biological processes with impact on disease. However, very little is known about how they specifically interfere with biological pathways and disease mechanisms. To investigate this interaction, the hunt for direct miRNA targets that mediate the miRNA effects—the “needle in the haystack”—is an essential step. In this review we provide a comprehensive workflow of successfully applied methods starting from the identification of putative miRNA-target pairs, followed by validation of direct miRNA–mRNA interactions, and finally presenting methods that dissect the impact of particular miRNA-target pairs on a biological process or disease. This guide allows the way to be paved for obtaining biologically meaningful miRNA targets

Development of a Triton X‐100 replacement for effective virus inactivation in biotechnology processes

After the transmission of human viruses through plasma derivatives had been recognized in the early 1980s, virus inactivation and then removal steps have been implemented into their manufacturing processes. These measures have kept plasma derivatives safe ever since and have also served as a barrier against more recently emerging viruses. Given the success of these interventions, they have also been embedded into the manufacturing processes for cell‐derived biological medicinal products. The most effective inactivation process for lipid‐enveloped viruses is treatment by detergents or combinations of solvents and detergents, and thus, these processes have been almost universally adopted. One of the most widely used detergents, Triton X‐100, has recently raised environmental concerns because one of its degradation products possesses hormone‐like (estrogen‐mimetic) activity that may act on wildlife. Consequently, use of the chemical in the European Union will ultimately be prohibited. The current study was conducted to establish an environmentally friendly detergent alternative to Triton X‐100 with fully equivalent efficacy in biotechnological use. A newly synthesized compound, named Nereid, as well as Triton X‐100 reduced, seem to satisfy these requirements, and thus may be suitable replacements for Triton X‐100

Expression Profiling of a Heterogeneous Population of ncRNAs Employing a Mixed DNA/LNA Microarray

Mammalian transcriptomes mainly consist of non protein coding RNAs. These ncRNAs play various roles in all cells and are involved in multiple regulation pathways. More recently, ncRNAs have also been described as valuable diagnostic tools. While RNA-seq approaches progressively replace microarray-based technologies for high-throughput expression profiling, they are still not routinely used in diagnostic. Microarrays, on the other hand, are more widely used for diagnostic profiling, especially for very small ncRNA (e.g., miRNAs), employing locked nucleic acid (LNA) arrays. However, LNA microarrays are quite expensive for high-throughput studies targeting longer ncRNAs, while DNA arrays do not provide satisfying results for the analysis of small RNAs. Here, we describe a mixed DNA/LNA microarray platform, where directly labeled small and longer ncRNAs are hybridized on LNA probes or custom DNA probes, respectively, enabling sensitive and specific analysis of a complex RNA population on a unique array in one single experiment. The DNA/LNA system, requiring relatively low amounts of total RNA, which complies with diagnostic references, was successfully applied to the analysis of differential ncRNA expression in mouse embryonic stem cells and adult brain cells

Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC

For decades, the ability of detergents to solubilize biological membranes has been utilized in biotechnological manufacturing to disrupt the lipid envelope of potentially contaminating viruses and thus enhance the safety margins of plasma- and cell-derived drugs. This ability has been linked to detergent micelles, which are formed if the concentration of detergent molecules exceeds the critical micelle concentration (CMC). Traditionally, the CMC of detergents is determined in deionized water (ddH2O), i.e., a situation considerably different from the actual situation of biotechnological manufacturing. This study compared, for five distinct detergents, the CMC in ddH2O side-by-side with two biopharmaceutical process intermediates relevant to plasma-derived (Immunoglobulin) and cell-derived (monoclonal antibody) products, respectively. Depending on the matrix, the CMC of detergents changed by a factor of up to ~4-fold. Further, the CMC in biotechnological matrices did not correlate with antiviral potency, as Triton X-100 (TX-100) and similar detergents had comparatively higher CMCs than polysorbate-based detergents, which are known to be less potent in terms of virus inactivation. Finally, it was demonstrated that TX-100 and similar detergents also have virus-inactivating properties if applied below the CMC. Thus, the presence of detergent micelles might not be an absolute prerequisite for the disruption of virus envelopes

Development and validation of a novel phonomimetic bioreactor.

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold's lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device