Probes for INS

The rapid emergence of spatial multi-omics technologies in recent years has revolutionized biomedical research. Among these, the Digital Spatial Profiler (DSP, commercialized by nanoString) has become one of the dominant technologies in spatial transcriptomics and proteomics and has assisted in deconvoluting complex biological questions. Based on our practical experience in the past 3 years with DSP, we share here a detailed hands-on protocol and key handling notes that will allow the broader community to optimize their work procedure.

Stimulatory coupling of dopamine D1 (D1R) and adenosine A2A receptors (A2AR) to adenylyl cyclase within the striatum is mediated through a specific Gαolfβ2γ7 heterotrimer to ultimately modulate motor behaviors. To dissect the individual roles of the Gαolfβ2γ7 heterotrimer in different populations of medium spiny neurons (MSNs), we produced and characterized conditional mouse models, in which the Gng7 gene was deleted in either the D1R- or A2AR/D2R-expressing MSNs. We show that conditional loss of γ7 disrupts the cell type-specific assembly of the Gαolfβ2γ7 heterotrimer, thereby identifying its circumscribed roles acting downstream of either the D1Rs or A2ARs in coordinating motor behaviors including in vivo responses to psychostimulants. We reveal that Gαolfβ2γ7/cAMP signal in D1R-MSNs does not impact spontaneous and amphetamine-induced locomotor behaviors in male and female mice, while its loss in A2AR/D2R-MSNs results in a hyperlocomotor phenotype and enhanced locomotor response to amphetamine. Additionally, Gαolfβ2γ7/cAMP signal in either D1R- or A2AR/D2R-expressing MSNs is not required for the activation of PKA signaling by amphetamine. Finally, we show that Gαolfβ2γ7 signaling acting downstream of D1Rs is selectively implicated in the acute locomotor-enhancing effects of morphine. Collectively, these results support the general notion that receptors utilize specific Gαβγ proteins to direct the fidelity of downstream signaling pathways and to elicit a diverse repertoire of cellular functions. Specifically, these findings highlight the critical role for the γ7 protein in determining the cellular level, and hence, the function of the Gαolfβ2γ7 heterotrimer in several disease states associated with dysfunctional striatal signaling.SIGNIFICANT STATEMENTDysfunction or imbalance of cAMP signaling in the striatum has been linked to several neurological and neuropsychiatric disorders, including Parkinson's disease, dystonia, schizophrenia and drug addiction. By genetically targeting the γ7 subunit in distinct striatal neuronal subpopulations in mice, we demonstrate that the formation and function of the Gαolfβ2γ7 heterotrimer, which represents the rate-limiting step for cAMP production in the striatum, is selectively disrupted. Furthermore, we reveal cell type-specific roles for Gαolfβ2γ7-mediated cAMP production in the control of spontaneous locomotion as well as behavioral and molecular responses to psychostimulants. Our findings identify the γ7 protein as a novel therapeutic target for disease states associated with dysfunctional striatal cAMP signaling.

Introduction In December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic broke out. The virus rapidly spread globally, resulting in a major world public-health crisis. The major disease manifestation occurs in the respiratory tract. However further studies documented other systemic involvement. This study investigate histopathologic eye changes in post-mortem material of Coronavirus Disease 2019 (COVID-19) patients. Methods Sections of formalin-fixed, paraffin-embedded eyes from 5 patients (10 eyes) who died of COVID-19 at the University Hospital in Basel were included. Gross examination and histological evaluation were performed by three independent ophthalmopathologists. Immunohistochemical staining was performed using antibodies against fibrin, cleaved caspase 3 and ACE-2. Five enucleated eyes of patients not infected with SARS-CoV-2 served as control group. All cases have been studied for presence of SARS-CoV-2 RNA by means of RT-PCR and RNA in situ hybridization. The choroidal vessels of one case were analyzed with electron microscope. Results Ophthalmopathologically, eight eyes from four patients displayed swollen endothelial cells in congested choroidal vessels. No further evidence of specific eye involvement of SARS-CoV-2 was found in any of the patients. In the eight eyes with evidence of changes due to SARS-CoV-2, immunohistochemical staining demonstrated fibrin microthrombi, apoptotic changes of endothelial and inflammatory cells. In control eyes, ACE-2 was detectable in the conjunctiva, cornea, retina and in the choroidea, and displayed significantly lower amounts of stained cells as in COVID-19 eyes. SARS-CoV-2 RNA was detectable in both bulbi of 2/5 patients, yet in situ hybridization failed to visualize viruses. Electron microscopy showed no significant results due to the artifacts. Discussion/Conclusion As already described in other organs of COVID-19 patients, the ophthalmological examination revealed-microthrombi, i.e. hypercoagulation and vasculopathy most probably due to endothelial damage. A possible viral spread to the endothelial cells via ACE-2 provides one pathophysiological explanation. The expression of ACE-2 receptors in the conjunctiva hints towards its susceptibility to infection. To what extend eyes function are disrupted by SARS-CoV-2 is subject to further studies, especially in the clinic.

Purpose: The integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling, is crucial for the development, maturation and homeostasis of articular cartilage. Activation of Hh signalling in adulthood and pathophysiological mechanics, have both been associated with the development of murine and human OA. But, how chondrocytes might transduce and integrate these cues remains unknown. A microtubule-based organelle, the primary cilium, most noted for its crucial role in Hh signalling, is assembled by chondrocytes and possesses a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate chondrocyte helps tune the anabolic matrix response to compression and the response to Hh ligand. In vivo, the primary cilium has been proposed to be a platform for the integration of mechanics and Hh signalling in musculoskeletal tissues. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown. Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;Ift88fl/fl : cKO hereafter). Cre activity was validated by qPCR, RNA scope and a ROSA26tdtomato reporter line. Ift88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks-post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, subchondral bone thickness, and OARSI score by means of immunohistochemistry (IHC). The surgical DMM model, which destabilises the joint, was performed at 10 weeks of age. To explore the role of physiological mechanics, mice were allowed two weeks of voluntary wheel exercise immediately following tamoxifen administration at 8 weeks of age. qPCR was performed on micro dissected articular cartilage at 10 weeks of age in control and cKO. RNAscope was performed on cryosections of articular cartilage from 10 week old mice control and cKO. Means ± S.D are quoted throughout, Mann-Whitney U-test or Fisher’s test were used for statistical comparisons. Results: In our previous OARSI abstract of 2020 we described the phenotype arising in IFT88 cKO mice. Here we outline this in further detail and with an exploration of underlying mechanisms. Tamoxifen treatment of cKO mice resulted in a 50% reduction of Ift88 mRNA in articular cartilage (p=0.02, n=6 control, 14 cKO). Ift88 (cKO) mice had thinner medial articular cartilage (MAC), compared with controls, at all 5 time-points (Fig 1.A) In control mice, MAC thickness increased from 102.57μm (95% CI [94.30, 119.80]) at 8 weeks of age to 108.68 +/- (95% CI [101.32, 116.42]) at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.20 μm (95% CI [90.04, 102.36]), p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MAC thickness in cKO was 90.16μm (95% CI [87.11, 93.22]) compared with 111.60μm (95% CI [104.34, 118.79]) in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MAC had continued to thin to 84.55μm (95% CI [75.43, 93.67]) in cKO, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MAC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score were unaffected. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age and at all time-points thinning was attributable to the relative loss of calcified cartilage implying a failure of calcification. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope. There were no measurable increases in subchondral bone thickness or changes in osteoclastic activity in cKO mice. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO (29.83 +/- 7.69) than control (22.08 +/- 9.30, p< 0.05, n= 15 both groups). Two weeks of voluntary wheel exercise rescued cartilage atrophy in cKO mice (p< 0.0001), whilst no change was observed in controls. RNA isolated from microdissected articular cartilage of 10 week old control and cKO mice, two weeks post tamoxifen, revealed a statistically significant correlation between Ift88 and Tcf7l2 expression after Bonferroni correction (p=0.026). Ctgf, Gli2 and Enpp1 were also positively correlated with Ift88 expression before correction (p=0.002, p=0.0037, and p=0.009 respectively). RNA scope analysis of AC found a statistically significant (p< 0.0001, n=4 in both groups) decrease in Ift88 positive cells in cKO (27.78%) compared with controls (45.18%), whilst also showing an increase in Gli1 positive cells in cKO (50.42%) compared with controls (23.63%) (p< 0.0001, n=4 in both groups). Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued mechanoadaptation of adolescent and adult articular cartilage. Depletion of the ciliary gene Ift88 inhibits medial articular cartilage thickening, leading to atrophy, which then predisposes the joint to spontaneous OA. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of Ift88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of Hh signalling, which is also mechanosensitive. Ift88 expression was correlated with Tcf7l2, previously shown to interact and influence Hh signalling pathways in cartilage. On-going experiments are aiming to dissect the relative roles of IFT, mechanics and Hh in the context of adult cartilage. We conclude that Ift88 is crucial to post-natal articular cartilage homeostasis and chondroprotective against OA.

Fetal responses to adverse pregnancy environments are sex-specific. In fetal guinea pigs (GPs), we assessed morphology and messenger RNA (mRNA) expression in fetal growth-restricted (FGR) tissues at midpregnancy. Female GPs were assigned either an ad libitum diet (C) or 30% restricted diet (R) prior to pregnancy to midpregnancy. At midpregnancy, a subset of R females underwent ultrasound-guided nanoparticle (NP) injection to enhance placental function. Five days later, fetuses were sampled. Fetal brain, heart, and liver were assessed for morphology (hematoxylin and eosin), proliferation (Ki67), and vascularization (CD31), as well as expression of inflammatory markers. R fetuses were 19% lighter with reduced organ weights and evidence of brain sparing compared to controls. No increased necrosis, proliferation, or vascularization was found between C and R nor male or female fetal organs. Sexual dimorphism in mRNA expression of Tgfβ and Ctgf was observed in R but not C fetal brains: increased expression in females. NP treatment increased fetal brain mRNA expression of Tgfβ and Ctgf in R males, abolishing the significant difference observed in untreated R fetuses. Sex-specific differences in mRNA expression in the fetal brain with FGR could impart a potential survival bias and may be useful for the development of treatments for obstetric diseases. Male and female fetuses respond differently to adverse pregnancy environments. Under fetal growth restriction conditions, inflammatory marker mRNA expression in the fetal brain was higher in females compared to males. Differences in gene expression between males and females may confer a selective advantage/disadvantage under adverse conditions. Better characterization of sexual dimorphism in fetal development will aid better development of treatments for obstetric diseases.

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

The identification of additional lipid mediators, enzymes, and receptors revealed an expanded endocannabinoid system (ECS) called the endocannabinoidome (eCBome). Furthermore, eCBome research using wild type and genetically modified mice indicate the involvement of this system in modulating alcohol induced neuroinflammatory alterations associated with behavioral impairments and the release of proinflammatory cytokines. We investigated the role of cannabinoid type 2 receptors (CB2Rs) in modulating behavioral and neuro-immune changes induced by alcohol using conditional knockout (cKO) mice with selective deletion of CB2Rs in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2) cKO mice. We used a battery of behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests to evaluate behavioral changes induced by alcohol. ELISA assay was used, to detect alterations in IL-6, IL-1α, and IL-1β in the prefrontal cortex, striatum, and hippocampal regions of mice to investigate the role of CB2Rs in neuroinflammation induced by alcohol in the brain. The involvement of cannabinoid receptors in alcohol-induced behavior was also evaluated using the non-selective cannabinoid receptor mixed agonist WIN 55,212-2. The results showed that cell-type specific deletion of CB2Rs in dopamine neurons and microglia significantly and differentially altered locomotor activity and rotarod performance activities. The result also revealed that cell-type specific deletion of CB2Rs enhanced alcohol-induced inflammation, and WIN significantly reduced alcohol preference in all genotypes compared to the vehicle controls. These findings suggest that the involvement of CB2Rs in modulating behavioral and neuroinflammatory alterations induced by alcohol may be potential therapeutic targets in the treatment of alcohol use disorder.

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs) of physiological aortic stiffness to investigate the impact of HDAC6 inhibition on the contractile response of angiotensin II stimulated quiescent VSMC function. In this study, we utilise HDAC6 inhibitor BRD 9757 and Tubastatin a HDAC6 inhibitor inducing tubulin hyperacetylation. Results Our data shows that HDAC6 inhibition resulted in increased alpha-tubulin acetylation and decreased VSMC area. Further analysis revealed that although VSMC volume was unaltered, nuclear volume was decreased. Immunofluorescence microscopy revealed that HDAC6 inhibitor treatment resulted in DNA damage accumulation in VSMCs. We hypothesised that altered microtubule stability participated in this phenotype. To test this possibility, we performed a cold-stable microtubule stability assay, which revealed that HDAC6 inhibitor treated VSMCs possessed decreased microtubule stability. To test whether changes in microtubule stability induced DNA damage accumulation, we used the microtubule destabilising agents colchicine and demecholcine, and the microtubule stabilising agent paclitaxel. Importantly, either colchicine or demecholcine treatment increased DNA damage accumulation in VSMCs. In contrast, paclitaxel treatment had no effect on DNA damage levels.

Many patients with sickle cell disease (SCD) suffer from chronic pain, the underlying causes of which are unclear. Recent 16s ribosomal RNA sequencing studies revealed differences in the number and types of bacteria in the gastrointestinal tract of patients and mouse models of SCD relative to controls, but it is unclear if or how these changes contribute to symptomology. In these experiments, we used transgenic SCD mice to determine the extent to which disease related gut dysbiosis contributes to persistent pain. Reflexive pain behaviors were first measured in SCD mice following longitudinal probiotic or antibiotic treatment. Vehicle-treated SCD mice displayed significant mechanical allodynia relative to vehicle-treated wildtype mice, and antibiotic treatment further exacerbated mechanical allodynia in both genotypes. In contrast, probiotic treatment completely reversed persistent touch hypersensitivity in SCD mice. Persistent touch pain was also transiently reversed in SCD mice following fecal material transplant from healthy mice. In complementary experiments, wildtype recipient mice developed cold and touch hypersensitivity that persisted for several weeks after fecal material transplant from SCD donors. Using whole-cell patch clamp recordings, we further determined that these behavioral observations were accompanied by altered intrinsic plasticity in a select class of nodose ganglia sensory neurons, the peripheral terminals of which are well positioned to detect sensory information in the gut. Nodose ganglia neurons isolated from animals that received sickle cell fecal material transplants were hyperexcitable relative to those isolated from animals that received control fecal material transplants. These data are the first to suggest that disease-related gut dysbiosis induces pain through changes in vagal nerve activity. Ongoing studies are examining specific bacterial populations and/or metabolites responsible for these functional changes in order to develop novel therapeutics for chronic SCD pain management. Grant support from National Institutes of Health grants K99HL155791 and R01NS070711.

Aims: the knowledge of the pathogenic mechanisms of severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of Coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. Methods and results: the presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in-situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization (ISH) significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. Conclusions: PD-L1 is overexpressed in the pulmonary tissue of subjects died for COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.