Ketamine has emerged as a widespread treatment for a variety of psychiatric disorders when used at sub-anesthetic doses, but the neural mechanisms underlying its acute action remain unclear. Here, we identified NMDA receptors containing the 2A subunit (GluN2A) on parvalbumin (PV)-expressing inhibitory interneurons as a pivotal target of low-dose ketamine. Genetically deleting GluN2A receptors globally or selectively from PV interneurons abolished the rapid enhancement of visual cortical responses and gamma-band oscillations by ketamine. Moreover, during the follicular phase of the estrous cycle in female mice, the ketamine response was transiently attenuated along with a concomitant decrease of grin2A mRNA expression within PV interneurons. Thus, GluN2A receptors on PV interneurons mediate the immediate actions of low-dose ketamine treatment, and fluctuations in receptor expression across the estrous cycle may underlie sex-differences in drug efficacy.

Juvenile eastern water dragons ( Intellagama lesueurii lesueurii) are highly susceptible to infection with Bohle iridovirus (BIV), a species of ranavirus first isolated from ornate burrowing frogs in Townsville, Australia. To investigate the progression of BIV infection in eastern water dragons, 11 captive-bred juveniles were orally inoculated with a dose of 104.33 TCID50 and euthanized at 3, 6, 8, 10, 12, and 14 days postinfection (dpi). Viral DNA was detected via polymerase chain reaction (PCR) in the liver, kidney, and cloacal swabs at 3 dpi. Mild lymphocytic infiltration was observed in the submucosa and mucosa of the tongue and liver at 3 dpi. Immunohistochemistry (IHC) first identified viral antigen in foci of splenic necrosis and in hepatocytes with intracytoplasmic inclusion or rare single-cell necrosis at 6 dpi. By 14 dpi, positive IHC labeling was found in association with lesions in multiple tissues. Selected tissues from an individual euthanized at 14 dpi were probed using in situ hybridization (ISH). The ISH labeling matched the location and pattern detected by IHC. The progression of BIV infection in eastern water dragons, based on lesion severity and virus detection, appears to start in the spleen, followed by the liver, then other organs such as the kidney, pancreas, oral mucosa, and skin. The early detection of ranaviral DNA in cloacal swabs and liver and kidney tissue samples suggests these to be a reliable source of diagnostic samples in the early stage of disease before the appearance of clinical signs, as well as throughout the infection.

The anterior lateral bed nucleus of the stria terminalis (alBST) expresses receptors for glucagon-like peptide-1 (GLP1R) and receives input from caudal brainstem GLP1 neurons. GLP1 administered centrally reduces food intake and increases anxiety-like behavior and plasma corticosterone (cort) in rats, whereas central GLP1R antagonism has opposite effects. Anxiogenic threats and other stressors robustly activate c-fos expression in both GLP1-producing neurons and also in neurons within alBST subregions expressing GLP1R. To examine the functional role of GLP1R signaling within the alBST, adult male Sprague-Dawley rats received bilateral alBST-targeted injections of an adeno-associated viral vector (AAV) expressing shRNA to knock down translation of GLP1R mRNA (GLP1R-KD rats), or similar injections of a control AAV (CTRL rats). In situ hybridization revealed that GLP1R mRNA is expressed in a subset of GABAergic alBST neurons, and qRT-PCR confirmed that GLP1R-KD rats displayed a significant 60% reduction in translatable GLP1R mRNA. Compared to CTRL rats, GLP1R-KD rats gained more body weight over time and displayed less anxiety-like behavior, including a loss of light-enhanced acoustic startle, and less stress-induced hypophagia. Conversely, while baseline plasma cort levels were similar in GLP1R-KD and CTRL rats, GLP1R-KD rats displayed a prolonged stress-induced elevation of plasma cort. GLP1R-KD and CTRL rats displayed similar home cage food intake and a similar hypophagic response to systemic Exendin-4, a GLP1R agonist that crosses the blood-brain barrier. We conclude that GLP1R expressed within the alBST contributes to multiple behavioral responses to anxiogenic threats, yet also serves to limit the plasma cort response to acute stress.SIGNIFICANCE STATEMENTAnxiety is an affective and physiological state that supports threat avoidance. Identifying the neural bases of anxiety-like behaviors in animal models is essential for understanding mechanisms that contribute to normative and pathological anxiety in humans. In rats, anxiety/avoidance behaviors can be elicited or enhanced by visceral or cognitive threats that increase GLP1 signaling from the caudal brainstem to the hypothalamus and limbic forebrain. Data reported here support a role for limbic GLP1R signaling to enhance anxiety-like behavior and to attenuate stress-induced elevations in plasma cort levels in rats. Improved understanding of central GLP1 neural pathways that impact emotional responses to stress could expand potential therapeutic options for anxiety and other stress-related disorders in humans.

Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis1, which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles2-6. Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react 'on demand' to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness.

We previously showed that NADPH oxidase organizer 1 (Noxo1), a component of NADPH oxidase 1 (NOX1), is a TNF-α-induced tumor-promoting factor in gastric tumorigenesis. However, the mechanism of NOX1-induced reactive oxygen species (ROS) signaling for the gastric tumorigenesis has not been understood. Here, we showed that expression of NOX1 complex components, including Noxo1, but not other NOX family members was significantly upregulated in both mouse models for gastritis and gastric tumors, which was associated with increased ROS levels. We also found that NF-κB directly regulated NOXO1 expression in TNF-α-stimulated gastric cancer cells, suggesting that inflammation induces NOX1 complex activation through TNF-α/NF-κB pathway. Notably, in situ hybridization indicated that Noxo1 mRNA was detected in proliferating cells of gastritis and gastric tumors, and pharmacological inhibition of NOX activity significantly suppressed the proliferation of MKN45 gastric cancer cells and gastric hyperplasia of K19-C2mE mice. These results suggest that NOX1/ROS signaling has an important role in increased proliferation of stomach epithelial cells in the inflamed mucosa. Moreover, we found that expression of SOX2, a marker of gastric epithelial stem cells, was increased by NOX1/ROS signaling. Furthermore, disruption of Noxo1 in K19-C2mE micesignificantly suppressed gastritis-associated metaplastic hyperplasia, a potent preneoplastic lesion, which was associated with decreased number of SOX2-positive cells. These results indicate that inflammation-induced Noxo1 expression is responsible for development of metaplastic hyperplasia in the stomach through an increase in SOX2-expressing undifferentiated epithelial cells. Therefore, inhibition of the NOX1/ROS signaling pathway is a possible strategy for prevention and therapy for gastric cancer development.

Abstract

Objectives

The aim of the present study was to investigate the molecular basis for the use of immune checkpoint inhibitors to treat salivary gland carcinomas (SGC).

Materials and methods

We examined the clinical and prognostic significance of programed death ligands 1 and 2 (PD-L1 and -L2) expression using immunohistochemistry and in situ hybridization, as well as microsatellite instability (MSI) status using polymerase chain reaction, along with tumor-infiltrating lymphocytes (TILs) in 30 cases of SGC.

Results

The SGC cases studied included adenoid cystic carcinoma (AdCC, 36.7%), salivary duct carcinoma (SDC, 26.7%), mucoepidermoid carcinoma (MEC, 23.3%), and carcinoma ex pleomorphic adenoma (CxPA, 13.3%). Either PD-L1 or PD-L2 overexpression was observed in 36.7% patients. PD-L2 expression was associated with reduced disease-specific survival (DSS) and disease-free survival (DFS) (P = 0.0266 and P = 0.0209, respectively). Simultaneous PD-L1 and PD-L2 overexpression was detected in 13.3% of cases, and was correlated with reduced DSS (P = 0.0113). Among non-AdCCs, all cases that developed distant metastasis were positive for PD-L2 (P = 0.001). Cases showing low-TILs that were positive for either PD-L1 or L2 were associated with poor DFS. No MSI was detected in the SGC cases studied.

Conclusion

To our knowledge, this is the first comprehensive study examining PD-L1 and PD-L2 status, MSI status, and TILs in SGC. Our results indicate that the PD-1/PD-L1 or PD-L2 pathway, which is associated with poor clinical outcomes, may provide promising therapeutic targets against SGC in selected patients. Further experimental and clinical studies are encouraged.

During development of pancreatic cancer, alternatively-activated macrophages contribute to fibrogenesis, pancreatic intraepithelial neoplasia (PanIN) lesion growth, and generation of an immunosuppressive environment. Here we show that the immunomodulatory agent pomalidomide depletes pancreatic lesion areas of alternatively-activated macrophage populations. Pomalidomide treatment resulted in downregulation of interferon regulatory factor 4 (IRF4), a transcription factor for M2 macrophage polarization. Pomalidomide-induced absence of alternatively-activated macrophages led to a decrease in fibrosis at PanIN lesions and in syngeneic tumors; this was due to generation of an inflammatory, immune-responsive environment with increased expression of IL-1α and presence of activated (IFNγ positive) CD4+ and CD8+ T cell populations. Our results indicate that pomalidomide could be used to decrease fibrogenesis in pancreatic cancer and may be ideal as a combination treatment with chemotherapeutic drugs or other immunotherapies.

Abstract

OBJECTIVE:

Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function.

DESIGN:

Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 hours in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared spectroscopy.

RESULTS:

Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity.

CONCLUSIONS:

CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.

Objective: To observe the clinicopathologic features of oropharyngeal squamous cell carcinoma associated with human papilloma virus (OPSCC-HPV) and discuss the role and value of different in situ hybridization (ISH) detection methods for HPV in pathologic diagnosis. Methods: Fifteen cases of OPSCC-HPV were collected from Department of Pathology, Beijing Tongren Hospital, Capital Medical University from January 2016 to August 2018. These cases were diagnosed in accordance with the WHO classification of head and neck tumors. The histopathologic features and the clinicopathologic data were retrospectively analyzed. Immunohistochemistry (two-step EnVision method) was done to evaluate the expression of p16, Ki-67 and p53. ISH was used to detect HPV DNA (6/11 and 16/18). RNAscope technology was used to evaluate the presence of HPV mRNAs (16 and 18). Results: The mean age for the 15 patients (8 males, 7 females) was 47 years (range from 30 to 69 years). OPSCC-HPV typically presentedat an advanced clinical stage, six patients had cervical lymphadenopathy (large and cystic), seven had tonsillar swelling, one had tumor at base of tongue, and one had odynophagia. Microscopically the tumors exhibited distinctive non-keratinizing squamous cell carcinoma morphology. Cervical nodal metastases were large and cystic, with thickening of lymph node capsules. OPSCC-HPV raised from crypt epithelium and extended beneath the tonsillar surface epithelial lining as nests and lobules, often with central necrosis. Tumor cells displayed a high N: C ratio, and high mitotic and apoptotic rates. Tumor nests are often embedded within lymphoid stroma, and may be infiltrated by lymphoid cells.Fifteen cases (15/15) were strongly positive for p16; Ki-67 index were 60%-90%; they were focally positive or negative for p53. Ten cases (10/10) were negative for HPV 6/11 DNA, and one case(1/10) was focally positive for HPV16/18 DNA. Eleven cases (11/11) were strongly positive for HPV16 mRNA, one case was focally positive for HPV18 mRNA. Conclusions: OPSCC-HPV is a pathologically and clinically distinct form of head and neck squamous cell carcinoma. OPSCC-HPV is associated with high-risk HPV (type 16) in all cases. Detection of high-risk HPV16 mRNA by RNAscope is of great significance in the final diagnosis and pathogen identification.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.