Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling

Tissue-specific somatic stem cells are characterized by their ability to reside in a state of prolonged reversible cell cycle arrest, referred to as quiescence. Maintenance of a balance between cell quiescence and division is critical for tissue homeostasis at the cellular level and is dynamically regulated by numerous extrinsic and intrinsic factors. Analysis of the activation of quiescent stem cells has been challenging because of a lack of methods for direct detection of de novo dividing cells. Here, we present and experimentally verify a novel method based on double labeling with thymidine analogues to detect de novo dividing stem cells in situ. In a proof of concept for the method, we show that memantine, a drug widely used for Alzheimer’s disease therapy and a known strong inducer of adult hippocampal neurogenesis, increases the recruitment into the division cycle of quiescent radial glia-like stem cells—primary precursors of the adult-born neurons in the hippocampus. Our method could be applied to assess the effects of aging, pathology, or drug treatments on the quiescent stem cells in stem cell compartments in developing and adult tissues

Nitric Oxide Synthase Blockade Impairs Spontaneous Calcium Activity in Mouse Primary Hippocampal Culture Cells

Oscillation of intracellular calcium concentration is a stable phenomenon that affects cellular function throughout the lifetime of both electrically excitable and non-excitable cells. Nitric oxide, a gaseous secondary messenger and the product of nitric oxide synthase (NOS), affects intracellular calcium dynamics. Using mouse hippocampal primary cultures, we recorded the effect of NOS blockade on neuronal spontaneous calcium activity. There was a correlation between the amplitude of spontaneous calcium events and the number of action potentials (APs) (Spearman R = 0.94). There was a linear rise of DAF-FM fluorescent emission showing an increase in NO concentration with time in neurons (11.9 ± 1.0%). There is correlation between the integral of the signal from DAF-FM and the integral of the spontaneous calcium event signal from Oregon Green 488 (Spearman R = 0.58). Blockade of NOS affected the parameters of the spontaneous calcium events studied (amplitude, frequency, integral, rise slope and decay slope). NOS blockade by Nw-Nitro-L-arginine suppressed the amplitude and frequency of spontaneous calcium events. The NOS blocker 3-Bromo-7-Nitroindazole reduced the frequency but not the amplitude of spontaneous calcium activity. Blockade of the well-known regulator of NOS, calcineurin with cyclosporine A reduced the integral of calcium activity in neurons. The differences and similarities in the effects on the parameters of spontaneous calcium effects caused by different blockades of NO production help to improve understanding of how NO synthesis affects calcium dynamics in neurons

YTnC2, an improved genetically encoded green calcium indicator based on toadfish troponin C

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best‐suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7‐fold higher molecular brightness and 6.4‐fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches

Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.Correction in: BMC GENOMICS, Volume: 21, Issue: 1, Article Number: 503, DOI: 10.1186/s12864-020-06897-0</p