Probes for INS

Deletion of endoplasmic reticulum (ER) resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared to non-recombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared to Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of glioblastoma (GB) patients are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GB (n=64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment naïve GB PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2x coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with Temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% versus 24%) and allowed detection in cerebrospinal fluid (CSF) and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GB where current methods have largely failed.

Pericytes are mural cells of blood microvessels which play a crucial role at the neurovascular interface of the central nervous system. They are involved in the regulation of blood-brain barrier integrity, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, and neuroinflammation, and they demonstrate stem cell activity. Morphological and molecular studies to characterize brain pericytes recently pointed out some heterogeneity in pericyte population. Nevertheless, a clear definition of pericyte subtypes is still lacking. Here, we demonstrate that a fraction of brain pericytes express Connexin 30 (Cx30), a gap junction protein, which, in the brain parenchyma, was thought to be exclusively found in astrocytes. Cx30 could thus be a candidate protein in the composition of the gap junction channels already described between endothelial cells and pericytes. It could also form hemichannels or acts in a channel-independent manner to regulate pericyte morphology, as already observed in astrocytes. Altogether, our results suggest that Cx30 defines a novel brain pericyte subtype.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains, making them an excellent in vitro model system for studying human intestinal development and disease. However, hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine, with the mechanism of maturation remaining elusive. Here, we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs, and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers, as well as increased intestine-specific functional activities. Even after in vivo engraftment, in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro, thereby circumventing the need for animal models and in vivo maturation.

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.