RNAscope Multiplex Fluorescent Assay

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth.

Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development.

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity.

The relaxin peptide signaling system is involved in diverse physiological processes, but its possible roles in the brain, including nociception, are largely unexplored.In light of abundant expression of relaxin receptor (RXFP1) mRNA/protein in brain regions involved in pain processing, we investigated the effects of central RXFP1 activation on nociceptive behavior in a mouse model of inflammatory pain and examined the neurochemical phenotype and connectivity of relaxin and RXFP1 mRNA-positive neurons.Mice were injected with Complete Freund Adjuvant (CFA) into a hind paw.

Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins1-4. It is unclear whether neurexin-cerebellin-GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling.

Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay.

Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types.

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose.

The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STI). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue resident memory T cells (TRM) in human FRT is lacking. We have taken single-cell RNA sequencing approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There are significantly more CD8 than CD4 T cells.

Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage.