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mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.

The ability to more faithfully mimic the tumor microenvironment (TME) of cancer patients offers a wide range of possibilities for non-clinical evaluation of anti-cancer therapeutics, including guidance for patient selection, dose, and combinations. We have established a patient-derived tumor explant (PDE) model to monitor immune activation within a structurally preserved TME, following ex vivo incubation with immune check point inhibitors (ICI). When testing anti-PD-1 agents, such as dostarlimab and pembrolizumab, in a cohort of NSCLC explants from stage I-III treatment-naïve patients, we observed that approximately half demonstrate release of inflammatory cytokines after two days incubation. Interestingly, responsive tumors presented with a high level of PD-L1 expression (high TPS), had elevated TMB, and were highly infiltrated by CD8+ T cells at baseline, in line with tumor baseline characteristics predictive of clinical response to anti-PD-1. When testing a dose-range of dostarlimab and pembrolizumab comparable potencies were observed when measuring cytokine release after treatment of the NSCLC explants. In conclusion, we describe here an in vitro patient-derived NSCLC model that mirrors the heterogeneity of patients’ response for anti-PD-1 agents observed in clinical settings, representing a valuable tool for comparative analysis of novel ICI assets and combinations, such as the CD226 axis members. Importantly, this system is also amenable to extensive multi-omics and genetics-based analyses, providing the opportunity to better identify predictive or pharmacodynamic characteristics within patient tumors. Citation Format: Jessica Perrin, Frank Wippich, Valeriia Sherina, Dmytro Dvornikov, Katharina Lupar, Murad M. Melhem, Patrick Hanafin, Jeremy Waight, Jong Yu, Sapna Yadavilli, Daniel Poeckel, Giovanna Bergamini. Dostarlimab shows dose-dependent immune activation of the tumor microenvironment in a patient-derived NSCLC explant model similar to pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6647.

Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling—including conjugation, enzymatic synthesis, and product purification—can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

Arrays of singly-labelled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single molecule microscopy analysis and high efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that 1) all steps of the oligonucleotide labelling – including conjugation, enzymatic synthesis and product purification – can be performed in a standard biology laboratory, 2) the process yields >90 %, often >95 % labeled product with minimal carry-over of impurities and 3) the oligonucleotides can be labeled with different dyes or biotin, allowing single molecule FISH or RNA affinity purification to be performed.Graphical abstract

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.

The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.

Measuring translation in space and time The ribosome translates the information contained within messenger RNAs (mRNAs) into proteins. When and where ribosomes encounter mRNAs can regulate gene expression. Halstead et al. developed an RNA biosensor that allows single molecules of mRNAs that have never been translated to be distinguished from ones that have undergone translation by the ribosome in living cells (see the Perspective by Popp and Maquat). The authors demonstrated the utility of their technique by examining the spatial and temporal regulation of translation in single cells and in Drosophila oocytes during development. Science , this issue p. 1367 ; see also p. 1316

Throughout the literature, Open Innovation is lauded for yielding reduced time-to-market, higher R&D efficiencies and additional revenue throughout-licensing or spin-offs. Nevertheless, the Open Innovation model bears challenges: Managing an increasing number of partnerships and capturing the full potential from external technology providers (Fetterhoff & Voelkel, 2006). How can managers and team members of Open Innovation projects effectively respond to these challenges beyond the widely cited “Not-Invented-Here” syndrome? The topic of leadership is an area unexplored in the context of Open Innovation (Chesbrough, 2010a). This study provides such an exploration and presents a flexible leadership model that demands a joint application of goal-oriented, involving and engaging elements: establishing clear contractual agreements through tight partnership involvement and inspiring the prospects of a joint ecosystem. Throughout the partnership interaction, leadership in Open Innovation becomes mainstream, so that everyone involved in the value creation process needs to exhibit skills along the proposed flexible leadership framework.