Probes for INS

Background Corticotropin releasing factor (CRF) neural systems are important stress mechanisms in central amygdala (CeA), bed nucleus of stria terminalis (BNST), nucleus accumbens (NAc) and related structures. CRF-containing neural systems are traditionally posited to generate aversive distress states that motivate over-consumption of rewards and relapse in addiction. However, CRF-containing systems may alternatively promote incentive motivation to increase reward pursuit and consumption, without requiring aversive states. Methods We optogenetically stimulated CRF-expressing neurons in CeA, BNST or NAc, using Crh-Cre+ rats (n=37 female, n=34 male) to investigate roles in incentive motivation versus aversive motivation. We paired CRF-expressing neuronal stimulations with earning sucrose rewards in two-choice and progressive ratio tasks and investigated recruitment of distributed limbic circuitry. We further assessed valence with CRF-containing neuron laser self-stimulation tasks. Results Channelrhodopsin excitation of CRF-containing neurons in CeA and NAc amplified and focused incentive motivation and recruited activation of mesocorticolimbic reward circuitry. CRF systems in both CeA and NAc supported laser self-stimulation, amplified incentive motivation for sucrose in a breakpoint test, and focused ‘wanting’ on laser-paired sucrose over a sucrose alternative in a two-choice test. Conversely, stimulation of CRF-containing neurons in BNST produced negative-valence or aversive effects and recruited distress-related circuitry, as stimulation was avoided and suppressed motivation for sucrose. Conclusions CRF-containing systems in NAc and CeA can promote reward consumption by increasing incentive motivation, without involving aversion. By contrast, stimulation of CRF-containing systems in BNST is aversive but suppresses sucrose reward pursuit and consumption, rather than increase as predicted by traditional hedonic self-medication hypotheses.

Common steps in analysis of microRNA expression levels between different tissues, developmental stages, or disease states is to study microRNA expression levels by several methods as: NGS, microarray analysis, real-time PCR, Northern blots, in situ hybridization, and solution hybridization. Of these techniques, quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR) is one the most sensitive and accurate method. For qRT-PCR applications, the tools include: a) Effective method of microRNA isolation from samples; b) RT-qPCR reagents optimized for microRNA detection; c) Assays specific to the microRNAs of interest, and d) Real-time analytical instruments and reagents validated for microRNA detection. MicroRNAs have also been employed diagnostically, using liquid biopsies. Growing interest and utility of Circulating Cell-Free DNA [cfDNA] and interest in their role in oncology re-search is continue to grow in importance, in order to exploit their role as biomarkers for detecting premalignant and early stage cancers. The field of microRNA-based cancer research has witnessed a remarkable evolution over the last two decades, is the role of microRNAs as disease prognostic biomarkers, as well as recent attempts to exploit their role as therapeutic targets, as their small size and their stability in a variety of body fluids make them attractive substrates for employment as biomarkers. Current approaches for detecting microRNAs in blood and other body fluids is inadequate. The advantage of using microRNA approach is based on concurrently tar-geting multiple effectors of pathways involved in cell differentiation, proliferation, as well as in cell survival. In this review, we have employed regulatory small microRNAs as unifying molecules, which have shown a strong correlation with induction and progression of many human cancers, as they progress from the non- to the invasive stages of various types of human cancers, as detailed in this review below.

The interest and utility of high-plex spatial profiling of RNA and protein biomarkers has increased over the last few years. The implementation of high-plex analyte spatial platforms, such as GeoMx^® Digital Spatial Profiler (DSP), is increasing within discovery and development of biomarkers associated with clinical outcome. The surge in spatial platforms comes with an increased adoption of digital pathology in translational and clinical research studies. The integration of these two workflows has the potential to benefit diagnostic and therapeutic development. This study aims to facilitate the implementation of DSP in tissue analysis workflows helping researchers involved in drug discovery and development efforts to (1) assess platform feasibility for their research, (2) design effective DSP experiments, and (3) enable generation of high-quality, analyzable spatial data from large cohortstudies. The GeoMx DSP Biopharma and CRO Consortium has developed consensus-based best practices incorporating expertise of members from biopharma and contract research organizations (CROs). Best practices guidelines for spatial profiling of tissue biopsies in drug discovery and development using GeoMx stands to advance current standard practices in tissue analysis. These recommendations encompass every step in the implementation of DSP for standard tissue analysis workflows, emphasizing the importance of multidisciplinary stakeholder involvement, defining experimental conditions and testing these prior to execution of large-scale studies, and considerations in assessing assay performance. This document offers a practical reference for optimal implementation of GeoMx DSP in exploratory sample analysis for use in research supporting drug discovery and development. Here we present a practical reference for the optimal implementation of GeoMx DSP in exploratory analysis for drug discovery and development studies. Best practices insights for the application of this technology to breast cancer research have been previously published (1) and should also be taken in consideration when designing relevantspatial studies.

The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.This article is protected by

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It's unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Dorsal root ganglion (DRG) neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of Gal-positive (Gal+) DRG neurons. However, the characteristics and functions of Zcchc12+ neurons are largely unknown. Here, we genetically labelled Zcchc12+ neurons in Zcchc12-CreERT2::Ai9 mice, and verified that Zcchc12 represented a new subpopulation of DRG neurons in both sexes. Zcchc12+ neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Besides, Zcchc12+ neurons also formed circumferential endings surround the hair follicles in hairy skin. Functionally, in vivo calcium imaging in DRGs revealed that Zcchc12+ neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation of Zcchc12+ DRG neurons reduced the sensitivity to noxious heat in mice. Taken together, we identify a new subpopulation of Zcchc12+ nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENT:Zcchc12 represents a new subpopulation of DRG neurons. The characteristics and functions of Zcchc12+ neurons are largely unknown. Here we genetically labelled Zcchc12 neurons, and showed that the fibers of Zcchc12+ DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally, Zcchc12+ DRG neurons responded to noxious mechanical and heat stimuli. Ablation of Zcchc12+ DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulatipn of DRG neurons required for noxious heat sensation.

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia.

The pathogenesis of gastroesophageal reflux disease (GERD) is not fully understood. It involves the activation of mucosal immune-mediated and inflammatory responses. Toll-like receptors (TLR) 2 and TLR4 are pattern-recognition receptors of the innate immune system; they recognize microbial and endogenous ligands. Farnesoid X receptor (FXR) is a bile acid receptor that regulates the inflammatory response. We aimed to evaluate TLR2, TLR4 and FXR expression patterns in GERD. We re-evaluated 84 oesophageal biopsy samples according to the global severity (GS) score, including 26 cases with histologically normal oesophagus, 28 with histologically mild oesophagitis and 30 with severe oesophagitis. We used immunohistochemistry and in situ hybridization to assess the expression patterns of TLR2, TLR4 and FXR in oesophageal squamous cells. Immunohistochemistry showed that nuclear and cytoplasmic TLR2 was expressed predominantly in the basal layer of normal oesophageal epithelium. In oesophagitis, TLR2 expression increased throughout the epithelium, and the superficial expression was significantly more intensive compared to normal epithelium, p <0.01. Nuclear and cytoplasmic TLR4 was expressed throughout the thickness of squamous epithelium, with no change in oesophagitis. FXR was expressed in the nuclei of squamous cells, and the intensity of the expression increased significantly in oesophagitis (p <0.05). FXR expression correlated with basal TLR2. In situ hybridization confirmed the immunohistochemical expression patterns of TLR2 and TLR4. In GERD, TLR2, but not TLR4, expression was upregulated which indicates that innate immunity is activated according to a specific pattern in GERD. FXR expression was increased in GERD and might have a regulatory connection to TLR2.

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.

Background: Orai1 is a plasma membrane Ca2+ channel that is involved in store-operated calcium entry (SOCE). In pulmonary cells, SOCE regulates gene expression and stimulates cytokine, mucin, and protease secretion. Activation of Orai1/SOCE results in recruitment of neutrophils to the lungs. Orai1 activation is also upstream of transcription factors such as nuclear factor of activated T cells, which facilitate onset of inflammation. In cystic fibrosis (CF), the immune response is dysregulated, and the lung is chronically inflamed, but Orai1 expression in the CF lung is poorly understood. Orai1 is a promising target for drug development, so we tested the hypothesis that Orai1 was upregulated in CF lungs. Methods: We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). We imaged sections by confocal and super resolution microscopy and analyzed Orai1 and STIM1 expression, colocalization, and particle size in different pulmonary cell types. Results: Orai1 was broadly expressed throughout the lung, but expression was greatest in immune cells. At messenger RNA and protein levels, there were no consistent trends in expression levels between the three groups. Orai1 must interact with STIM1 to activate SOCE, so we used STIM1/Orai1 colocalization as a marker of Orai1 activity. Using this approach, we found significantly greater colocalization between these proteins in CF and asthma lung epithelia (CF 50%, asthma 54%, normal 15%), interstitia (CF 57%, asthma 49%, normal 16%), and luminal immune cells (CF 66%, asthma 70%, normal 38%). Orai1 also aggregates as part of its interaction process. Using super-resolution microscopy, we found significantly more Orai1 and STIM1 aggregation in immune cells from CF and asthmatic lungs (average Orai1 particle size: CF 52 nm, asthma 63 nm, normal 28 nm; average STIM1 particle size: CF 77 nm, asthma 59 nm, normal 14 nm). We also looked at Orai1 in peripheral blood neutrophils from normal and CF donors (5 per group). All CF subjects took elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), but under baseline conditions, there were significantly bigger puncta in CF neutrophils (CF 10 nm, normal 6 nm), suggesting that these patients continued to have significant inflammation despite taking ELX/TEZ/IVA, and mean percentage predicted forced expiratory volume in 1 second in our CF cohort was 55 ± 22%, indicating that these patients had persistent lung disease. Conclusions: This is the first comprehensive analysis of Orai1 and STIM1 expression in lungs from normal and CF donors. We found evidence that Orai1 was more active in CF than normal lungs. This novel application of super-resolution microscopy has the potential to be used in clinical settings for analysis of ex vivo patient samples and to evaluate inflammation in people with CF. Although traditional biomarkers of inflammation such as serum cytokine levels are useful for rapid detection of systemic inflammation, our technique allows for precise localization of upstream inflammatory signaling biomarkers at the cellular level and in fixed samples. Therefore, these data suggest that Orai1 has a key role in CF lung inflammation and attest to the potential of anti-inflammatory therapeutics that target Orai1.We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). W

Immune cell metabolism, or immunometabolism, has recently become of interest for its role in inflammation and disease. A growing field of research has identified that metabolic rewiring and immune cell activation are intimately connected, however the mechanisms driving these connections have remained poorly understood. The tricarboxylic acid cycle and its intermediates have become recognized as major players in disease and inflammation. The immunometabolite itaconate has been identified as a potent immunomodulator produced in high quantities in activated macrophages. Itaconate is produced by the enzyme aconitate decarboxylase 1 (Acod1), which is highly upregulated in proinflammatory macrophages. Although itaconate and Acod1 have been found to be upregulated in macrophages under stimulated conditions, the potential role of itaconate production in other non-immune cells remains poorly understood. Itaconate and its exogenous derivative forms have been found to be potent mediators of inflammation, and specifically have been found to decrease proinflammatory cytokine production in cultured macrophages. In this dissertation, we sought to identify the role of itaconate in three separate murine models of disease: cerebral ischemia/reperfusion injury, diet-induced obesity, and ulcerative colitis. We hypothesized that deletion of Acod1 would lead to greater disease severity in these models and that macrophages would be the primary cell type responsible. To understand the role of endogenously produced itaconate, mice lacking Acod1 (Acod1-/- ) were used. We demonstrate that global Acod1 deletion leads to significantly worsened disease severity in all three models studied. Specifically, Acod1 deletion leads to increased lesion volume size compared to wild type (WT) mice in a model of ischemia/reperfusion stroke. The observed increased lesion volume did not appear to be caused by increased inflammation, indicating a separate potential mechanism driving these changes. In a model of diet-induced obesity, mice lacking Acod1 showed similar weight gain compared to WT mice, however, Acod1-/- mice had elevated blood glucose levels after 12 weeks of high fat diet. Acod1-/- mice also had elevated inflammatory gene expression. Furthermore, naïve Acod1-/- mice had significant increases in fat deposition when on chow diet at 3 and 6 months of age. Lastly, Acod1-/- mice exposed to an acute ulcerative colitis model induced by dextran sulfate xi sodium (DSS) treatment showed increased disease severity with more severe and sustained body weight loss and increased inflammatory gene expression. Importantly, cell specific knockout of Acod1 in myeloid cells (MyAcod1-/- ) with LysM-Cre did not phenocopy disease severity in any of the three in vivo models. This suggests that myeloid cells, specifically macrophages, are not the primary cell type responsible for the observed phenotypes seen in the global Acod1-/- studies. These data define a novel role for Acod1 in transient ischemia/reperfusion occlusion stroke, diet-induced obesity, and ulcerative colitis. Furthermore, these differences do not appear to be regulated by Acod1 and itaconate in macrophage/myeloid cells. These findings suggest that Acod1 and itaconate are likely working through other cell types. To further explore potential mechanisms driving the observed Acod1-/- phenotypes, we sought to identify if Acod1 and endogenous itaconate were capable of modulating ferroptosis induced cell death. Using bone marrow derived macrophages as a model cell type capable of expressing Acod1, we found that macrophages lacking Acod1 had significantly increased susceptibility to RSL3 induced ferroptosis death compared to WT cells. Further analysis found that Acod1-/- macrophages also showed decreased glutathione levels compared to their WT counterparts. Lastly, we found that supplementing Acod1-/- cells with exogenous itaconate restored protection from RSL3 induced cell death and increased glutathione level to what is observed in WT macrophages. These findings would suggest that Acod1 and endogenous itaconate play a role in ferroptosis protection through sustaining intracellular glutathione levels, and could be a relevant mechanism regarding the protective role of Acod1 from ischemia/reperfusion injury.

Background & Aim: One of the major bottlenecks of cancer cellular therapy development is off-target toxicity. It is caused by activated T-cells that unexpectedly recognize epitopes presented on healthy tissues instead of interacting only with the intended target on cancer cells. This mechanism can lead to severe immuno-toxicity resulting in organ dysfunction or even death. Unfortunately, experimental identification of epitopes that may trigger off-target toxicity is both costly and time consuming. In an attempt to accelerate this process, we created ArdImmune Tox, a computational tool assessing epitopes for their potential toxicity. Methods, Results & Conclusion: ArdImmune Tox builds up on the recent advances in computational immunology and Artificial Intelligence (AI). In the first step ArdImmune Tox mimics an experimental approach (X-scan) by simultaneously modifying multiple amino acids in the target peptide creating many possible off-target epitopes (OTE). Next, the peptide collection is mapped against a curated reference dataset encompassing proteomes from more than 1000 patients. In this step we retain peptides found in proteins expressed in healthy human tissues and keep track of their frequency. In order to establish which peptides are presented by the HLAs on the cell surface we use a dedicated machine-learning model developed in-house, which was trained on mass spectroscopy data of peptide-HLA (pHLA) presentation. These peptides are then scored for similarity to the target peptide based on selected physico-chemical properties. Importantly, only amino acids in positions identified by our approach as interacting with TCRs are considered. We present results of ArdImmune Tox on three cancer immunotherapy cases, where the OTEs were identified experimentally. In all of them ArdImmune Tox correctly identified the OTEs, ranking them among the 3 highest scoring potential off-target peptides, whereas BLAST algorithm-based approaches either positioned OTEs much lower in the ranking or even failed to identify them altogether. In conclusion, we introduce ArdImmuneTox - a novel, effective, in silico approach for the identification of peptide- based off-target toxicity and T-cell cross-reactivity in immunotherapies. Our approach shows superior performance to currently used sequence comparison-based methods. Our results indicate that ArdImmune-Tox can aid in the rapid and cost-effective selection of safe epitope targets for cancer immunotherapies.