Probes for INS

Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8dim and CD8bright memory T cells, some of which expressed granzyme B, and fewer CD11c+ and CD68+ cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.

Excess caloric intake results in increased fat accumulation and an increase in energy expenditure via diet-induced adaptive thermogenesis; however, the underlying mechanisms controlling these processes are unclear. Here we identify the neuropeptide FF receptor-2 (NPFFR2) as a critical regulator of diet-induced thermogenesis and bone homoeostasis. Npffr2-/- mice exhibit a stronger bone phenotype and when fed a HFD display exacerbated obesity associated with a failure in activating brown adipose tissue (BAT) thermogenic response to energy excess, whereas the activation of cold-induced BAT thermogenesis is unaffected. NPFFR2 signalling is required to maintain basal arcuate nucleus NPY mRNA expression. Lack of NPFFR2 signalling leads to a decrease in BAT thermogenesis under HFD conditions with significantly lower UCP-1 and PGC-1α levels in the BAT. Together, these data demonstrate that NPFFR2 signalling promotes diet-induced thermogenesis via a novel hypothalamic NPY-dependent circuitry thereby coupling energy homoeostasis with energy partitioning to adipose and bone tissue.

Parathyroid adenomas are slow growing benign neoplasms associated with hypercalcemia, while atypical parathyroid adenomas and parathyroid carcinomas are uncommon tumors and their histologic features may overlap with parathyroid adenomas. LncRNAs participate in transcription and in epigenetic or post-transcriptional regulation of gene expression, and probably contribute to carcinogenesis. We analyzed a group of normal, hyperplastic, and neoplastic parathyroid lesions to determine the best immunohistochemical markers to characterize these lesions and to determine the role of selected lncRNAs in tumor progression. A tissue microarray consisting of 111 cases of normal parathyroid (n = 14), primary hyperplasia (n = 15), secondary hyperplasia (n = 10), tertiary hyperplasia (n = 11), adenomas (n = 50), atypical adenomas (n = 7), and carcinomas (n = 4) was used. Immunohistochemical staining with antibodies against chromogranin A, synaptophysin, parathyroid hormone, and insulinoma-associated protein 1(INSM1) was used. Expression of lncRNAs including metastasis-associated lung adenocarcinoma transcript one (MALAT1), HOX transcript antisense intergenic RNA (HOTAIR), and long intergenic non-protein coding regulator of reprograming (Linc-ROR or ROR) was also analyzed by in situ hybridization and RT-PCR. All of the parathyroid tissues were positive for parathyroid hormone, while most cases were positive for chromogranin A (98%). Synaptophysin was expressed in only 12 cases (11%) and INMS1 was negative in all cases. ROR was significantly downregulated during progression from normal, hyperplastic, and adenomatous parathyroid to parathyroid carcinomas. These results show that parathyroid hormone and chromogranin A are useful markers for parathyroid neoplasms, while synaptophysin and INSM1 are not very sensitive broad-spectrum markers for these neoplasms. LincRNA ROR may function as a tumor suppressor during parathyroid tumor progression.