Probes for INS

Abstract: Objective: To investigate the potential involvement of secreted protein acidic and rich in cysteine (SPARC) in the progression of the breast tumor and to determine its association with outcome variables and matrix metalloproteinases (MMPs) expression in patients with breast carcinoma (BC). Methods: SPARC expression was examined in 8 pairs of BC tissues and surrounding normal tissues at mRNA and protein levels by qRT-PCR, RNAscope in situ hybridization (ISH), Western blotting, and immunohistochemistry techniques. Immunohistochemical staining of SPARC was done in 26 normal breasts, 76 ductal carcinoma in situ (DCIS), and 198 BC samples. In addition, immunohistochemical staining was performed for MMP-2 and MMP-9 in BC. Results: SPARC expression at mRNA and protein levels was significantly increased in BC tissues compared to the surrounding normal tissues (P < 0.05 and P < 0.01, respectively). RNAscope ISH and immunohistochemistry of SPARC confirmed an increase in SPARC expression in BC tissues compared with the normal tissues. Epithelial SPARC expression increased continuously from normal breast through DCIS to BC (P < 0.001). In patients with BC, high epithelial SPARC expression was associated with worse disease-free survival and overall survival (P = 0.002 and P = 0.048, respectively) and independently predicted worse disease-free survival (P = 0.002). Epithelial SPARC expression was significantly correlated with MMP-2 expression (P < 0.05). Conclusion: Up-regulation of SPARC contributes to breast tumor progression. SPARC expression may be a useful biomarker for the prognostic prediction in patients with BC. SPARC can control extracellular matrix degradation through up-regulation of MMP-2.

Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper- phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down- regulation of apoptosis.

Type 2 diabetes develops when beta cells are not able to fulfill insulin needs. The role of the endoplasmic reticulum-mitochondria junction in coordinating the functions of these two organelles throughout the natural history of type 2 diabetes is determinant and may explain the alterations of insulin biosynthesis. Our goal was to study endoplasmic reticulum and mitochondrial interactions in human beta cells from organ donors with type 2 diabetes. Pancreas samples were obtained via the network for pancreatic organ donors with diabetes (nPOD) based on disease status with 12 subjects with type 2 diabetes and 9 non-diabetic controls. We examined pancreatic specimens by immunofluorescence, in situ hybridization and in situ proximity ligation assay and compared the results to an in vitro model of beta-cell dysfunction. Expression of proteins that enable tethering and exchanges between endoplasmic reticulum (ER) and mitochondria and quantification of interconnection through mitochondria associated membranes (MAM) was investigated. In beta cells from type 2 diabetic cases as compared to controls, there was a significant increase in reticular expression of inositol triphosphate receptor-2 (IP3R2) both at the protein and mRNA levels, no difference in mitochondrial transit peptide receptor TOM20 and mitofusin-2 expressions, and a decrease in the expression of voltage-dependent anion channel-1 (VDAC-1). The number of IP3R2-VDAC-1 complexes identified by in situ proximity ligation assay was significantly lower in diabetic islets and in beta cells of diabetics as compared to controls. Treatment of Min6-B1 cells with palmitate altered glucose-stimulated insulin secretion, increased ER stress and significantly reduced ER-mitochondrial interactions. We can conclude that specific changes in reticular and mitochondrial beta cell proteins characterize human type 2 diabetes with reduction in organelle interactions. This finding opens new targets of intervention.

 Background: ASPM is a newly reported stem cell marker and plays important roles in mitosis, cell cycle and tumorigenesis. It links with poor clinical prognosis in various tumors. However, the clinical significance of ASPM in colonic adenocarcinoma (CA) has not been fully studied. The purpose of this study was to investigate if ASPM is correlated with the clinicopathological features of CA. Methods: Primary CA tissue, adenoma and the matched normal mucosa from 99 patients, were detected using immunohistochemical analysis by primary antibodies against ASPM. Meanwhile, 20 CAs and 20 liver metastatic cases were examined by RNA in situ hybridization (RNAscope). To assess the clinical relevance of ASPM, we analyzed the survival follow-up information. Results: ASPM was found only in single cells in the base of normal colon mucosal crypts. But the expression of ASPM was detected high in colonic adenomas (49.5%, 49/99), and significantly higher in CA (56.6%, 56/ 99, P<0.001). In CAs, ASPM expression was more intense in stage III and IV than II and I stage patients (P=0.03), and positively correlated with lymph node metastasis (P=0.03), but not with the age at diagnosis, gender and histological grade (P>0.05). We also analyzed the survival follow-up information, the data showed that ASPM-positive expression was correlated with a shorter disease-free survival (DFS) time, the average DFS time of patients with ASPM positive and negative expression was 62.79±2.32 months and 71.30±2.72 months, respectively, and there was no statistical significance between the two groups (P>0.05). The results of ASPM mRNA measurement by RNAscope revealed ASPM mRNA expression was higher in primary CA than that in metastatic liver CA (P<0.001). Conclusions: ASPM might play an important role in colonic carcinogenesis and be a potential marker in predicting prognosis of CA. 

Pharmacological inhibition or genetic loss of function defects of the renin angiotensin aldosterone system (RAAS) causes compensatory renin cell hyperplasia and hyperreninemia. The triggers for the compensatory stimulation of renin synthesis and secretion in this situation may be multimodal. Since cyclooxygenase-2 (COX-2) expression in the macula densa is frequently increased in states of a defective RAAS, we have investigated a potential role of COX-2 and its derived prostaglandins for renin expression and secretion in aldosterone synthase-deficient mice (AS-/-) as a model for a genetic defect of the RAAS. In comparison with wild-type mice (WT), AS-/- mice had 9-fold and 30-fold increases of renin mRNA and of plasma renin concentrations (PRC), respectively. Renin immunoreactivity in the kidney cortex of AS-/- mice was 10-fold higher than in WT. Macula densa COX-2 expression was 5-fold increased in AS-/- kidneys relative to WT kidneys. Treatment of AS-/- mice with the COX-2 inhibitor SC-236 for 1 week lowered both renal renin mRNA and PRC by 70%. Hyperplastic renin cells in AS-/-kidneys were found to express the prostaglandin E2 receptors EP2 and EP4. Global deletion of EP2 receptors did not alter renin mRNA nor PRC values in AS-/- mice. Renin cell-specific inducible deletion of the EP4 receptor lowered renin mRNA and PRC by 25% in AS-/- mice. Renin cell-specific inducible deletion of the EP4 receptor in combination with global deletion of the EP2 receptor lowered renin mRNA and PRC by 70-75% in AS-/- mice. Lineage tracing of renin-expressing cells revealed that deletion of EP2 and EP4 leads to a preferential downregulation of perivascular renin expression. Our findings suggest that increased macula densa COX-2 activity in AS-/- mice triggers perivascular renin expression and secretion via prostaglandin E2.

Sleep loss affects many aspects of cognition, and memory consolidation processes occurring in the hippocampus seem particularly vulnerable to sleep loss. The immediate-early gene Arc plays an essential role in both synaptic plasticity and memory formation, and its expression is altered by sleep. Here, using a variety of techniques, we have characterized the effects of brief (3-h) periods of sleep vs. sleep deprivation (SD) on the expression of Arc mRNA and Arc protein in the mouse hippocampus and cortex. By comparing the relative abundance of mature Arc mRNA with unspliced pre-mRNA, we see evidence that during SD, increases in Arc across the cortex, but not hippocampus, reflect de novo transcription. Arc increases in the hippocampus during SD are not accompanied by changes in pre-mRNA levels, suggesting that increases in mRNA stability, not transcription, drives this change. Using in situ hybridization (together with behavioral observation to quantify sleep amounts), we find that in the dorsal hippocampus, SD minimally affects Arc mRNA expression, and decreases the number of dentate gyrus (DG) granule cells expressing Arc. This is in contrast to neighboring cortical areas, which show large increases in neuronal Arc expression after SD. Using immunohistochemistry, we find that Arc protein expression is also differentially affected in the cortex and DG with SD - while larger numbers of cortical neurons are Arc+, fewer DG granule cells are Arc+, relative to the same regions in sleeping mice. These data suggest that with regard to expression of plasticity-regulating genes, sleep (and SD) can have differential effects in hippocampal and cortical areas. This may provide a clue regarding the susceptibility of performance on hippocampus-dependent tasks to deficits following even brief periods of sleep loss.

Melanoma is a highly immunogenic tumor with a good response to treatment with immune checkpoint inhibitors. Tumor-associated macrophages (TAMs) play an important immunosuppressive role in such tumors and have therefore been identified as possible future therapeutic targets in oncology. The aim of this study was to identify novel immunoregulatory receptors specifically expressed on TAM. Expression of Slamf9, a member of the signaling lymphocytic-activating molecule (Slam) immunoreceptor family, was found to be upregulated in a gene expression analysis of murine bone marrow-derived macrophages (BMDM) stimulated with tumor-conditioned medium of B16F1 melanoma cells. SLAMF9+ macrophages were identified in human and murine melanomas by using self-generated antibodies against human and murine SLAMF9. A comprehensive immunohistochemical analysis of tissue microarrays detected SLAMF9+ TAM in 73.3% of human melanomas, but also in 95.5% of naevi of melanoma patients and in 50% of naevi from healthy controls. In addition, 20% of melanomas and 2.3% of naevi from melanoma patients displayed a positive SLAMF9 expression also in melanocytic cells. No SLAMF9 expression was detected in naevus cells of healthy donors. Although SLAMF9 has no intracellular signaling motif, a comprehensive functional analysis revealed that the molecule was able to significantly enhance TNF-α secretion after LPS-stimulation. In addition, SLAMF9 delayed the wound closure of RAW 264.7 cells in a scratch assay, while proliferation and cell death were not affected. Taken together, SLAMF9 is a novel type-I-transmembrane receptor with immunomodulatory properties in macrophages. Further studies are required to evaluate whether SLAMF9 classifies as a promising future therapeutic target in melanoma.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.