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We report an approach for in situ detection of genomic DNA sequences, where transiently opening DNA duplexes are captured by circularizing DNA strands - padlock probes - that lock in place in a sequence-specific manner through the action of a DNA ligase. Reacted probes, wound around their target strands, are then replicated by rolling-circle amplification for localized fluorescence detection. The technique serves to shorten assay time and enables detection of shorter specific DNA sequences compared to standard fluorescence in situ hybridization, FISH. Genomic sequences with thousands of locally repeated copies were detected in human leukocytes with greater than 99% efficiency and less than 0.15% false positives in just a few hours. Using a longer variant of the protocol targets of as little as 36 or 112 nt were visualized, albeit at lower efficiency and with a higher false positive rate. The technique of targeting sequences in duplex DNA using padlock probes is promising for both research and clinical diagnostics.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and  proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.

Cell migration and cell proliferation are the basic principles that make up a living organism, and both biologically and medically. In order to understand living organism and biological phenomena, it is essential to track the migration, proliferation, and fate of cells in living cells and animals and to clarify the properties and molecular expression of cells. Recent developments in novel fluorescent proteins have made it possible to observe cell migration and proliferation as the cell cycle at the single-cell level in living individuals and tissues. Here, we introduce cell cycle visualization of living cells and animals by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system and in situ cell labeling of cells and tracking cell migration by photoactivatable and photoconvertible proteins. In addition, we will present our established methods as an example of combines above tools with single-cell molecular expression analysis to reveal the fate of migrating cells at single cell level.

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEVSVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29-and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation. (C) 2020 The Authors. Published by Elsevier Inc.

Regulatory T cells (Tregs) migrate between lymphoid and peripheral tissues for maintaining immune homeostasis. Tissue-specific function and functional heterogeneity of Tregs have been suggested, however, correlation between them and inter-tissue movement remain unknown. We used a contact hypersensitivity model of mice expressing a photoconvertible protein for tracking migratory cells. After marking cells in skin, we purified Tregs exhibiting a different migration pattern [Tregs recruiting to or remaining in the skin and emigrating from the skin to draining lymph nodes (dLNs) within half a day] and examined single-cell gene and protein expression profiles. Correlation and unsupervised clustering analyses revealed that Tregs in both skin and dLNs comprised two subpopulations, one highly expressing Nrp1 with variable CD25, Granzyme B, and/or CTLA-4 expression and another with 3 subsets strongly expressing CD25, Granzyme B, or CTLA-4 together with CD39. Characteristic subsets of Tregs remaining in the skin displayed higher CD25 and CD39 expression and lower Granzyme B and CTLA-4 expression compared with Tregs migrating to the skin. In addition, CCR5 expression in Tregs in skin was positively and negatively correlated with CD39 and Nrp-1 expression, respectively. To assess the predictive value of these data for immunotherapy, we blocked CCR5 signaling and found modest downregulation of CD39 and modest upregulation of Nrp1 expression in skin Tregs. Our data reveal a high functional diversity of Tregs in skin that is strongly related to trafficking behavior, particularly skin retention. Modulation of tissue-specific trafficking and function is a promising clinical strategy against autoimmune, infectious, and neoplastic diseases.

Gastric cancer (GC) is the third leading cause of cancer death. Early detection and efficient monitoring of tumor dynamics are prerequisites for reducing disease burden and mortality. Blood-based biomarker assays for the detection of early-stage GC could be of great relevance for population-wide or risk-group-based screening programs. Extracellular vesicles (EVs) are nano-sized membrane-enclosed particles released from all cells, which play an essential role as mediators of intercellular communication and are a promising class of biomarkers. We aimed to determine the putative panel of protein targets by collecting EVs from GC cell lines for future diagnostic assays. We measured levels of over 430 proteins using highly sensitive and high-throughput proximity extension assays (PEA). The protein network and function of each protein were analyzed. In conclusion, despite a similarity between GC EVs and EVs from the prostate as a control, we identified a set of 39 proteins in GC EVs that are involved in different cellular pathways and are not expressed or have low expression in exosomes from the prostate.