In order to characterize these understudied receptors within the liver, we must first understand where they are localized and what activates them. Given the dearth of reliable antibodies, we began by examining expression using RNAscope, a powerful _in situ_ technique that specifically localizes mRNA sequences. GPR108, which has previously been linked to regulation of Toll-Like Receptor signaling, was found within hepatocytes. GPR146, a known C-peptide receptor which has been shown to impact blood cholesterol levels, was highly expressed in hepatocytes, and weakly localized to cholangioctyes and vessels. GPR109a, a highly studied receptor known to respond to short chain fatty acids and a potential therapeutic target for liver disease, was found to be primarily expressed in cholangiocytes, which notably play a key role in modification of bile. Finally, GPR125, an atypical adhesion GPCR, exhibited weak expression in hepatocytes but rather strong staining in select cholangiocytes and the liver vasculature. This was confirmed using both immunohistochemistry and immunofluorescence microscopy where we noted basolateral staining along the small cholangiocytes and hepatocytes. Given its localization and function as an adhesion receptor, GPR125 is well-positioned within the liver to respond to, and to regulate, extracellular matrix proteins and their composition in both health and disease. Efforts are currently underway to deorphanize these receptors using the PRESTO-Tango assay and to better understand their roles in the pathogenesis of metabolic diseases in the liver.

The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell functions including proliferation and differentiation. It is known that certain genes of the polyamine system are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system. In different models of acute and chronic kidney injury expression of genes involved in polyamine homeostasis were analyzed by RT-qPCR and RNAScope. In these models, expression of catabolic enzymes (Aoc1 and Sat1) was upregulated, and the anabolic enzymes (Odc1, Sms) were downregulated. The putrescine-degrading enzyme AOC1 exhibits the most striking changes. Interestingly, it can act together with ODC1 as gatekeepers of the polyamine system. The detected increase of Aoc1 takes place in the injured but regenerating proximal tubules. As a screening for stimuli of increased Aoc1 expression, we used mouse embryonic kidney explants. Here we observed changes of Aoc1expression under hypoxia and hyperosmotic conditions. These changes were further examined in mouse models of hypoxia. However, in vivo, hypoxia did not lead to changes of Aoc1 expression. Hyperosmolarity was confirmed as a stimulus by using the kidney cell lines M15 and 209/MDCT as well as cultured primary proximal tubules. Using reporter gene and RNA-stability assays, we could show that the increase in Aoc1 expression is based on mRNA-stabilization and transcriptional activation of one certain isoform. The activated isoform contains an additional set of 22 amino acids N-terminally that lead to an altered subcellular localization. In conclusion, different models of kidney injury exhibit a similar pattern of dysregulation of the polyamine system with the most striking change being the upregulation of Aoc1 in proximal tubules. Using hyperosmolarity as a stimulus, we provide first insights into the regulation of Aoc1 under harmful conditions.

RNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time-dependent transcriptional shifts in each layer of the TM. From both cross-sectional and whole-mount views, the TM shows a rapid, proliferative response to injury by 18 hours post-injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi-layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar-free structure. We identified a regeneration-induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A _K5Cre-ERT2;Confetti_ mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co-factors. When EGFR is deleted _in vivo_, using a _K5-CreERT2_;_Egfrfl/fl; R26mTmG/mTmG_ mouse model, TMs no longer display proliferation post-injury and cannot repair perforations.

Here we administered Cre-dependent shRNA to knock-down _Scn8a_ (thus Nav1.6) in Cre-expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10-14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used _in situ_ hybridization (RNAscope Fluorescent Assay) to label mRNA of _Scn8a_ and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four-Channel, Upright Confocal Microscope Body Based on Cerna System (ThorLabs), and images were acquired using the ThorImage LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open-source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t-test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non-targeting shRNA sequence).

Neuropathic pain is a chronic disorder resulting from damage to the afferent nerve fibers or central pain pathways and is often a complication in pathological conditions such as diabetes, shingles, multiple sclerosis, and stroke. The opioid epidemic has elucidated the need for more efficacious treatments for neuropathic pain. In 2019 alone, nearly 1.6 million people were diagnosed with an opioid use disorder and 48,000 people died from a synthetic opioid overdose. Despite the addictive properties, opioids are still the most frequently prescribed pain medication, even for chronic neuropathic pain. Heterotrimeric G-proteins consisting of the α, β, and γ subunits convey extracellular signals sensed by G-protein coupled receptors (GPCRs) to intracellular effectors. The Gβ5 subunit is a divergent member of the G-protein β subunit family as it does not bind to traditional γ subunits. Instead, Gβ5 complexes with the R7 subfamily of the regulators of G-protein signaling (R7-RGS) containing 4 members: RGS6, 7, 9 and 11. The Gβ5/R7-RGS complex acts as a GTPase accelerating protein (GAP) for G-protein αi/o (Gαi/o) subunits. Previous studies have established the integral role of R7-RGS proteins in pain transmission via their interactions with Gαi/o-coupled receptors including opioid and metabotropic gamma-aminobutyric acid (GABA-B) receptors. Our lab has shown the lack of Gβ5 in sensory ganglia diminishes mechanical, thermal, and chemical nociception. However, the conditional knockout of Gβ5 in Rgs7 expressing neurons reduces only mechanical nociception. This Gβ5/RGS7-dependent mechanical nociception relies on GABA-B receptor signaling as indicated by the rescue of mechanical nociception in Rgs7-Cre; Gnb5 fl/fl mice after treatment with 2-hydroxysaclofen, a GABA-B antagonist. We also established that Rgs9 expressing neurons regulate thermal nociception via a Gβ5-dependent pathway as assayed by the hotplate test in Rgs9-Cre; Gnb5 fl/fl mice. The purpose of this project has been to understand the molecular role of each R7-RGS member in the regulation of pain transmission. First, we confirmed co-localization between the Gnb5 transcript and all four R7-RGS mRNA transcripts in murine dorsal root ganglia (DRG) using the RNAscope HiPlex assay, a novel in situ hybridization technique. We then established the co-localization patterns between each R7-RGS member and various pain related receptors including Mrgprd, Trpa1, and Trpv1. Our RNAscope results support the behavioral tests since Rgs7 transcripts highly co-express with Mrgprd, a mechanical nociceptor, while Rgs9 transcripts most frequently co-express with Trpv1, a thermosensitive receptor. These results suggest that each R7-RGS member might regulate unique types of nociception. We have also shown that Rgs11 transcripts co-localize with Trpv1 and Trpa1 receptor transcripts which indicates Rgs11 might regulate the chemical nociception as tested by capsaicin and mustard oil administration in the eye-wipe test. Next, we aim to study the possible roles of Rgs6 and Rgs11 in regulating chemical nociception using conditional Gβ5 knockout mice mediated by Rgs6-cre and Rgs11-cre, respectively.

Objective The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS- or bleomycin-induced acute lung injury, sheddases (membrane-bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV-induced shedding of the glycocalyx. Hypothesis We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases. Methods We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP-7, -2, -9 and their inhibitors TIMP-1 and -2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT-qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE-12 mouse lung epithelial cells were infected with viable or heat-inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT-qPCR. Results Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP-7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP-7 is only expressed in cells that are directly infected by IAV. Expression of the MMP-7 inhibitor TIMP-1 is similar to uninfected lungs on day 1, but increases 50-fold on day 3. In contrast, MMP-2 and MMP-9, as well as their inhibitor TIMP-2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat-inactivated IAV fails to upregulate MMP-7. Conclusions Together, these data suggest that localized IAV infection increases MMP-7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP-7 may modulate IAV-induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV-ARDS pathogenesis.

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well-established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X-ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum-formed bone (EFB, Fig.1c1), cartilage-formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum-formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle-shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin-4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx-CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx-CreERT2; R26RDTA (carrying a loxP-flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).

Penile cancer (PC) is an extremely rare malignancy, and the patients at advanced stages have currently limited treatment options with disappointing results. Immune checkpoint inhibitors anti-programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) are currently changing the treatment of several tumors. Furthermore, the microsatellite instability (MSI) and the deficient mismatch repair system (dMMR) proteins represent predictive biomarkers for response to immune checkpoint therapy. Until present, few data have been reported related to PD-L1 expression and MSI in PC. The main aim of our study was the evaluation of PD-L1 expression in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) in immune cells and the analysis of dMMR/MSI status in a large series of PCs.A series of 72 PC, including 65 usual squamous cell carcinoma (USCC), 1 verrucous, 4 basaloid, 1 warty, and 1 mixed (warty-basaloid), was collected. Immunohistochemistry (IHC) was performed to assess PD-L1 expression using two different anti-PD-L1 antibodies (clone SP263 and SP142 Ventana) and MMR proteins expression using anti-MLH1, anti-PMS2, anti-MSH2, and anti-MSH6 antibodies. PCR analysis was performed for the detection of MSI status.Of the 72 PC cases analyzed by IHC, 45 (62.5%) cases were TC positive and 57 (79%) cases were combined positive score (CPS) using PDL1 SP263. In our cohort, TILs were present in 62 out of 72 cases (86.1%), 47 (75.8%) out of 62 cases showed positivity to PDL1 clone SP142. In our series, 59 cases (82%) had pMMR, 12 cases (16.7%) had lo-paMMR, and only 1 case (1.3%) had MMR. PCR results showed that only one case lo-paMMR was MSI-H, and the case dMMR by IHC not confirmed MSI status.Our findings showed that PD-L1 expression and MSI status represent frequent biological events in this tumor suggesting a rationale for a new frontier in the treatment of patients with PC based on the immune checkpoint inhibitors.

microRNA-331-3p (miR-331-3p) has been displayed as an oncogene in pancreatic cancer (PC). The current research set out to elucidate how miR-331-3p in carcinoma-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) facilitated the tumorigenesis in PC. First, a dual-luciferase reporter assay was adopted to investigate the relationship between miR-331-3p and SCARA5. In addition, EVs were isolated normal fibroblasts and CAFs, and these isolated EVs were co-cultured with PC cells. Cell proliferative and migrating/invasive potentials were further evaluated with the help of a CCK-8 and Transwell assays, respectively. Gain- and loss-of-function assays were also implemented to assess the role of miR-331-3p, SCARA5, and FAK pathway in PC cells. Lastly, xenograft nude mice were established to investigate the role of miR-331-3p in vivo. miR-331-3p negatively targeted SCARA5 and was highly expressed in CAFs-derived EVs, which accelerated the proliferative, migrating, and invasive potentials of PC cells. Meanwhile, over-expression of miR-331-3p enhanced the proliferative, migrating, and invasive properties of PC cells and promoted tumor growth in vivo by manipulating SCARA5/FAK axis, whereas SCARA5 countered the oncogenic effects of miR-331-3p. Overall, miR-331-3p in CAFs-derived EVs inhibits SCARA5 expression and activates the FAK pathway, thereby augmenting the progression of PC. Our study provides a potential therapeutic target for the treatment of PC.

During fasting, increased sympatho-adrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of AgRP, a peptidergic co-transmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild type animals, 24 hr fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, RT-PCR experiments confirmed the mouse adrenal medulla contains GPR109A mRNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to co-ordinate the homeostatic response to food deprivation.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.