Probes for INS

Background Oropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+). Methods Patients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(−) data, survival analysis for HPV(−) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1. Results The patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(−) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(−) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(−) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(−) HNSCC. Conclusions High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(−) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(−) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.

Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and β2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-β2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

Introduction: Inverted papillomas (IPs) are rare, benign, sinonasal tumors with the ability to undergo malignant transformation. While rare, they are the most common type of papilloma within the sinonasal cavity and represent up to 5% of primary nasal cavity tumors. There have been many studies attempting to define a causal link between HPV and malignant transformation of IPs with mixed results. Additionally, these tumors have a high recurrence rate, and their malignant transformation potential has spurred significant investigation into their etiology, disease course, and treatment. Prior meta-analyses of HPV-mediated transformation of IPs have suggested a nearly 50% prevalence of HPV in IPSCC and strong bias toward the high-risk virus types, HPV16 and HPV18, in IP malignant transformation. In this study, we have identified a large, retrospective cohort of benign IPs, IP-SCC, and control sinonasal polyp tissues that have been tested for high-risk HPV types to determine the prevalence in both benign and malignant IPs. Methods: A total of 94 IP tumors, 22 IP-SCC, and 13 sinonasal polyps were stained with HPV16/18 RNAscope and imaged with fluorescence to determine HPV status. Formalin-fixed slides were processed via standard antigen retrieval protocols and anti-HPV RNA staining was performed. Imaging was performed via confocal and bright-field microscopy. Results: We demonstrated significant HPV-positivity in IP-SCC versus benign IP tumors (p 

Early-life trauma (ELT) is a risk factor for binge eating and obesity later in life, yet the neural circuits that underlie this association have not been addressed. Here, we show in mice that downregulation of the leptin receptor (Lepr) in the lateral hypothalamus (LH) and its effect on neural activity is crucial in causing ELT-induced binge-like eating and obesity upon high-fat diet exposure. We also found that the increased activity of Lepr-expressing LH (LHLepr) neurons encodes sustained binge-like eating in ELT mice. Inhibition of LHLepr neurons projecting to the ventrolateral periaqueductal gray normalizes these behavioral features of ELT mice. Furthermore, activation of proenkephalin-expressing ventrolateral periaqueductal gray neurons, which receive inhibitory inputs from LHLepr neurons, rescues ELT-induced maladaptive eating habits. Our results identify a circuit pathway that mediates ELT-induced maladaptive eating and may lead to the identification of novel therapeutic targets for binge eating and obesity.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6% of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7% of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.