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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2440 KEY WORDShESCs, pluripotency related genes, epigenetic background, methylation ACKNOWLEDGEMENTSWe are very grateful to Dr.Suzanne Kadereit for critical reading the manuscript and helpful discussions. Report Diverse Epigenetic Profile of Novel Human Embryonic Stem Cell Lines ABSTRACTHuman embryonic stem cells (hESCs) are a promising model for studying mechanisms of regulation of early development and differentiation. OCT4, NANOG, OCT4-related genes and some others were recently described to be important in pluripotency maintenance. Lesser is known about molecular mechanisms involved in their regulation. Apart from genetic regulation of gene expression epigenetic events, particularly methylation, play an important role in early development. Using RT-PCR we studied the expression of pluripotency-related genes OCT4, NANOG, DPPA3 and DPPA5 during hESCs differentiation to embryoid bodies. Analysis of methylation profiles of promoter or putative regulatory regions of the indicated genes demonstrated that expression of the pluripotency-maintaining genes correlated with their methylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. We propose that DNA methylation underlies the developmental stage-specific mechanisms of pluripotency-related genes expression and reactivation and may have an impact on differentiation potential of hESC lines.

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.

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The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.