Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.

The cell-free transcription–translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis.

Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, we investigated a series of ribosome binding sites (RBSs). Although RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including the GC content of the coding sequence. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.

Background Understanding cell membrane-like lipid bilayers is crucial for studying fundamental biological mechanisms. Giant Unilamellar Vesicles (GUVs) are key tools for this investigation and have applications in both synthetic biology and, more recently, in microrobotics. The effects of cholesterol, a key component of cellular membranes, on synthetic phospholipid membrane models like GUVs are however not fully understood, as they may vary with lipid composition and production method. Methods We examined the size distribution, temporal stability and deformability of GUVs prepared with the droplet transfer method using different Dioleoylphosphatidylcholine (DOPC) to cholesterol ratios in the oil phase (namely 100:0, 85:15, 71:29, 60:40). Phase-contrast microscopy assessed size and stability, while deformability was tested by loading the GUVs with an aqueous ferrofluid and applying a uniform magnetic field to induce their elongation. Image analysis was conducted using Fiji and a custom Julia script. Results The median diameters increased with the content of cholesterol, together with the dimensional distribution. In terms of stability, cholesterol generally reduced GUV median diameter over time, while it varyingly influenced the number of vesicles. As for deformability, beyond the expected elongation dependent on the intensity of the applied magnetic field, there were no statistically significant differences in GUV deformability in the presence or absence of cholesterol. Conclusions Our findings suggest that cholesterol can lead to increased average diameter of GUVs made with DOPC through droplet transfer, while varyingly affecting their time-stability and not affecting their deformability. This study shows how small adjustments on a straightforward protocol like the droplet transfer method, provide a simple and effective way of tailoring GUV properties. Edits in the oil phase enable precise tuning of GUV membranes providing a tool for both fundamental studies and applications such as artificial cells or microrobots.