Cannabis can be rewarding or aversive. Cannabis reward is believed to be mediated by activation of cannabinoid CB1 receptors (CB1Rs) on GABAergic neurons that disinhibit dopaminergic neurons in the ventral tegmental area (VTA). However, little is known about the mechanisms underlying cannabis aversion in rodents. In the present study, CB1Rs are found not only on VTA GABAergic neurons, but also on VTA glutamatergic neurons that express vesicular glutamate transporter 2 (VgluT2). We then used Cre-Loxp transgenic technology to selectively delete CB1Rs in VgluT2-expressing glutamatergic neurons (VgluT2-CB1 -/-) and Cre-dependent viral vector to express light-sensitive channelrhodopsin-2 into VTA glutamatergic neurons. We found that photoactivation of VTA glutamatergic neurons produced robust intracranial self-stimulation (ICSS) behavior, which was dose-dependently blocked by DA receptor antagonists, but enhanced by cocaine. In contrast, Δ9-tetrahydrocannabinol (Δ9-THC), the major psychoactive component of cannabis, produced dose-dependent conditioned place aversion and a reduction in the above optical ICSS in VgluT2-cre control mice, but not in VgluT2-CB1 -/- mice. These findings suggest that activation of CB1Rs in VgluT2-expressing glutamate neurons produces aversive effects that might explain why cannabinoid is not rewarding in rodents and might also account for individual differences in the hedonic effects of cannabis in humans.

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

In the present study, we successfully generated lung cancer stem cell (CSC)-like cells by introducing a small set of transcription factors into a lung cancer cell line. In addition to properties that are conventionally referred to as CSC properties, the lung induced CSCs exhibited the ability to form lung cancer-like tissues in vitro with vascular cells and mesenchymal stem cells, which showed structures and immunohistological patterns that were similar to human lung cancer tissues. We named them "lung cancer organoids". We found that interleukin-6 (IL-6), which was expressed in the lung induced CSCs, facilitates the formation of lung cancer organoids via the conversion of mesenchymal stem cells into alpha-smooth muscle actin (αSMA)-positive cells. Interestingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Furthermore, IL-6 mRNA-positive cancer cells were found in clinical lung cancer samples. These results suggest that IL-6 could be a novel therapeutic target in lung cancer.

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system.

The Hippo pathway plays essential roles in organ size control and cancer prevention via restricting its downstream effector, Yes-associated protein (YAP). Previous studies have revealed an oncogenic function of YAP in reprogramming glucose metabolism, while the underlying mechanism remains to be fully clarified. Accumulating evidence suggests long noncoding RNAs (lncRNAs) as attractive therapeutic targets, given their roles in modulating various cancer-related signaling pathways. In this study, we report that lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) is required for YAP-dependent glycolysis. Mechanistically, YAP promotes the expression of BCAR4, which subsequently coordinates the Hedgehog signaling to enhance the transcription of glycolysis activators HK2 and PFKFB3. Therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 attenuated YAP-dependent glycolysis and tumor growth. The expression levels of BCAR4 and YAP are positively correlated in tissue samples from breast cancer patients, where high expression of both BCAR4 and YAP is associated with poor patient survival outcome. Taken together, our study not only reveals the mechanism by which YAP reprograms glucose metabolism, but also highlights the therapeutic potential of targeting YAP-BCAR4-glycolysis axis for breast cancer treatment.

Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus, and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA), but the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5-/- mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays critical roles in patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells.

Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

Abstract

Purpose Patient-derived xenografts ("xenopatients") of colorectal cancer metastases have been essential to identify genetic determinants of resistance to the anti-EGF Receptor (EGFR) antibody cetuximab, and to explore new therapeutic strategies. From xenopatients, a genetically annotated collection of stem-like cultures ("xenospheres") was generated and characterized for response to targeted therapies.

EXPERIMENTAL DESIGN:

Xenospheres underwent exome-sequencing analysis, gene expression profile and in vitro targeted treatments to assess genetic, biological and pharmacological correspondence with xenopatients, and to investigate non-genetic biomarkers of therapeutic resistance. The outcome of EGFR family inhibition was tested in an NRG1-expressing in vivo model.

RESULTS:

Xenospheres faithfully retained the genetic make-up of their matched xenopatients over in vitro and in vivo passages. Frequent and rare genetic lesions triggering primary resistance to cetuximab through constitutive activation of the RAS signaling pathway were conserved, as well as the vulnerability to their respective targeted treatments. Xenospheres lacking such alterations (RASwt) were highly sensitive to cetuximab, but were protected by ligands activating the EGFR family, mostly NRG1. Upon reconstitution of NRG1 expression, xenospheres displayed increased tumorigenic potential in vivo, generated tumors completely resistant to cetuximab, and sensitive only to comprehensive EGFR family inhibition.

CONCLUSIONS:

Xenospheres are a reliable model to identify both genetic and non-genetic mechanisms of response and resistance to targeted therapies in colorectal cancer. In the absence of RAS pathway mutations, NRG1 and other EGFR ligands can play a major role in conferring primary cetuximab resistance, indicating that comprehensive inhibition of the EGFR family is required to achieve a significant therapeutic response.

A classic response to systemic hypoxia is the increased production of red blood cells due to hypoxia-inducible factor (HIF)-mediated induction of erythropoietin (EPO). EPO is a glycoprotein hormone that is essential for normal erythropoiesis and is predominantly synthesized by peritubular renal interstitial fibroblast-like cells, which express cellular markers characteristic of neuronal cells and pericytes. To investigate whether the ability to synthesize EPO is a general functional feature of pericytes, we used conditional gene targeting to examine the von Hippel-Lindau/prolyl-4-hydroxylase domain (PHD)/HIF axis in cell-expressing neural glial antigen 2, a known molecular marker of pericytes in multiple organs. We found that pericytes in the brain synthesized EPO in mice with genetic HIF activation and were capable of responding to systemic hypoxia with the induction of Epo. Using high-resolution multiplex in situ hybridization, we determined that brain pericytes represent an important cellular source of Epo in the hypoxic brain (up to 70% of all Epo-expressing cells). We furthermore determined that Epo transcription in brain pericytes was HIF-2 dependent and cocontrolled by PHD2 and PHD3, oxygen- and 2-oxoglutarate-dependent prolyl-4-hydroxylases that regulate HIF activity. In summary, our studies provide experimental evidence that pericytes in the brain have the ability to function as oxygen sensors and respond to hypoxia with EPO synthesis. Our findings furthermore suggest that the ability to synthesize EPO may represent a functional feature of pericytes in the brain and kidney.

Abstract

BACKGROUND:

Programmed death receptor and programmed death ligand (PD-L1) are immunoregulatory proteins. Nonsmall cell lung cancer bypasses the immune system through the induction of protumorigenic immunosuppressive changes. The better understanding of immunology and antitumor immune responses has brought the promising development of novel immunotherapy agents like programmed death receptor checkpoint inhibitors. The aim of this study was to investigate the expression of PD-L1 in lung adenocarcinoma (ADC), comparing 2 different technologies: immunohistochemistry (IHC) by 2 methods versus RNA in situ hybridization (RISH).

METHODOLOGY:

In total, 20 cases of ADC of the lung and 4 samples of metastatic colon ADC were selected. Evaluation of PD-L1 expression was performed by IHC and RISH. RISH was performed using RNAscope. Both methods were scored in tumor cells and quantified using combined intensity and proportion scores.

RESULTS:

Eight of 20 (40%) lung ADC and 2 of 4 (50%) colon ADC were positive for PD-L1 with Cell Signaling IHC, and 65% lung ADC were positive by Dako IHC (13/20). All 4 cases of colon ADC were negative. When evaluated by RISH, 12 lung ADC (60%) and 1 colon ADC (25%) were PD-L1 positive.

CONCLUSIONS:

RNAscope probes provide sensitive and specific detection of PD-L1 in lung ADC. Both IHC methods (Cell Signaling and Dako) show PD-L1 expression, with the Dako method more sensitive (40% vs. 65%). This study illustrates the utility of RISH and Cell Signaling IHC as complementary diagnostic tests, and Food and Drug Administration approved Dako IHC as a companion diagnostic test.

Abstract

CONTEXT:

Parathyroid adenomas may be composed of chief cells (conventional or water-clear), oxyphilic cells or a mixture of both cells. The molecular background is rarely studied.

OBJECTIVE:

To molecularly characterize parathyroid adenomas of different cell type composition.

DESIGN:

Chief and oxyphilic cell adenomas were compared in a cohort of 664 sporadic cases. Extensive analyses of parathyroid tissueswere performed in subgroup. Gene expressions of known parathyroid-related genes were quantified by qRT-PCR. Protein expression profiles determined by liquid chromatography - tandem mass spectrometry (LC-MS/MS) were compared between each type of parathyroid adenomas. Selected proteins were analysed by Western blot and immunohistochemistry.

RESULTS:

Patients with oxyphilic cell adenoma were found to be older at the time of operation than chief cell adenoma cases but did not differ in gender, serum calcium or tumor weight. The gene expression of CASR, VDR, FGFR1, CYP27B1, CYP24A1, PTHLH, GCM2, NDUFA13, CDKN1B, MEN1 and CNND1 did not differ between the groups. VDR protein levels were weaker in oxyphilic adenomas. The proteomic studies identified a set of novel dysregulated proteins of interest such as nuclear receptor subfamily 2 group C member 2 (TR4), LIM domain only protein 3 (LMO3) and calcium-binding protein B (S100B). LMO3 and S100B showed higher expression in oxyphilic adenoma and may be involve in parathyroid tumorgenesis through the p53 pathway. TR4 showed different subcellular localisation between adenoma and normal rim.

CONCLUSION:

Chief and oxyphilic cell parathyroid adenomas have partly overlapping but also distinct molecular profiles. The calmodulin-eEF2K, TR4 and p53 pathways may be involved in the tumor development.