Probes for INS

Bharat Jasani, BSc, PhD, MBChB, FRCPath. Qualifying with honours in Biochemistry (University of Glasgow,1965-1969), PhD in Experimental Pathology and MBChB (honours distinction in Pathology) (University of Birmingham, 1970-1973), Dr Jasani initiated his clinical and research training in Histopathology, at Wales National School of Medicine in 1977, achieving MRCPath (1989), FRCPath (1997), and a Specialist Consultant’s status in Histopathology (Immunohistochemistry) (1993); and Personal Chair in Oncological Pathology (2003). Following his retirement as the Head of Histopathology and Academic Pathology (2003-2011), he continued as Professor of Pathology, Institute of Cancer & Genetics, Cardiff University & Associate Postgraduate Dean for Academic Medicine in Wales (2011-2015). He was then appointed as the Chair of Biomedical Sciences, Nazarbayev University School of Medicine. Astana, Kazakhstan, with Adjunct Professorship in Pathology, University of Pittsburgh School of Medicine, Pittsburg, USA (2015-2016).  IN 2016 HE TOOK UP HIS CURRENT POSITION AS THE DIRECTOR OF PATHOLOGY, TARGOS MOLECULAR PATHOLOGY GMBH, KASSEL, GERMANY, WHICH WAS ACQUIRED BY DISCOVERY LIFE SCIENCES IN 2021. Over the past 40 years Dr Jasani has devoted himself to advancing cancer pathology based on the use of cancer biomarkers. As the Head of Molecular Diagnostic Unit, University Hospital of Wales (1982-2003), he led the development and establishment of diagnostic immunocytochemistry & molecular pathology services in Cardiff and Wales. He also promoted quality assurance and standardization of cancer biomarker analyses as Breast Cancer Module Leader of UKNEQAS of IHC/ISH, and UK’s lead representative on International Working Group on Standardization of Breast Cancer Biomarking, and the United States Sub-Committee on Quality Assurance for Immunocytochemistry. As Principal Investigator and Co-investigator of several translational research projects on development of predictive biomarkers for colorectal and breast cancer, he has also been invited to act as a Consultant and Key Opinion Leader to several leading biotech and pharmaceutical companies. He is an author of a book, over 50 chapters and reviews and more than 200 peer reviewed research publications.

Kallikrein related peptidase 6 (Klk6) is a secreted serine protease highly expressed in oligodendrocytes and implicated in demyelinating conditions. To gain insights into the significance of Klk6 to oligodendrocyte biology, we investigated the impact of global Klk6 gene knockout on CNS developmental myelination using the spinal cord of male and female mice as a model. Results demonstrate that constitutive loss of Klk6 expression accelerates oligodendrocyte differentiation developmentally, including increases in the expression of myelin proteins such as MBP, PLP and CNPase, in the number of CC-1+ mature oligodendrocytes, and myelin thickness by the end of the first postnatal week. Co-ordinate elevations in the pro-myelinating signaling pathways ERK and AKT, expression of fatty acid 2-hydroxylase, and myelin regulatory transcription factor were also observed in the spinal cord of 7d Klk6 knockouts. LC/MS/MS quantification of spinal cord lipids showed sphingosine and sphingomyelins to be elevated in Klk6 knockouts at the peak of myelination. Oligodendrocyte progenitor cells (OPCs)-derived from Klk6 knockouts, or wild type OPCs-treated with a Klk6 inhibitor (DFKZ-251), also showed increased MBP and PLP. Moreover, inhibition of Klk6 in OPC cultures enhanced brain derived neurotrophic factor-driven differentiation. Altogether, these findings suggest that oligodendrocyte-derived Klk6 may operate as an autocrine or paracrine rheostat, or brake, on pro-myelinating signaling serving to regulate myelin homeostasis developmentally and in the adult. These findings document for the first time that inhibition of Klk6 globally, or specifically in oligodendrocyte progenitors, is a strategy to increase early stages of oligodendrocyte differentiation and myelin production in the CNS.

Both preclinical and clinical studies have demonstrated that exposures to acetaminophen (APAP) at levels that cause hepatic injury cause pulmonary injury as well. However, whether exposures that do not result in hepatic injury have acute pulmonary implications is unknown. Thus, we sought to determine how APAP exposures at levels that do not result in significant hepatic injury impact the mature lung. Adult male ICR mice (8-12 wk) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice [280 mg/kg, intraperitoneal (ip)], as well as a lower dose previously reported to not cause hepatic injury (140 mg/kg, ip). We confirm that the lower dose exposures did not result in significant hepatic injury. However, like high dose, lower exposure resulted in increased cellular content of the bronchoalveolar lavage fluid and induced a proinflammatory pulmonary transcriptome. Both the lower and higher dose exposures resulted in measurable changes in lung morphometrics, with the lower dose exposure causing alveolar wall thinning. Using RNAScope, we were able to detect dose-dependent, APAP-induced pulmonary Cyp2e1 expression. Finally, using FLIM we determined that both APAP exposures resulted in acute pulmonary metabolic changes consistent with mitochondrial overload in lower doses and a shift to glycolysis at a high dose. Our findings demonstrate that APAP exposures that do not cause significant hepatic injury result in acute inflammatory, morphometric, and metabolic changes in the mature lung. These previously unreported findings may help explain the potential relationship between APAP exposures and pulmonary-related morbidity.

Emerging insights from numerous laboratories have revealed important roles for non-neoplastic cells in the development and progression of brain tumors. One of these non-neoplastic cellular constituents, glioma-associated microglia (GAM), represents a unique population of brain monocytes within the tumor microenvironment that have been reported to both promote and inhibit glioma proliferation. To elucidate the role of GAM in the setting of low-grade glioma (LGG), we leveraged RNA sequencing meta-analysis, genetically engineered mouse strains, and human biospecimens. Publically available disease-associated microglia (DAM) RNA-seq datasets were used, followed by immunohistochemistry and RNAScope validation. CD11a-deficient mouse microglia were used for in vitro functional studies, while LGG growth in mice was assessed using anti-CD11a neutralizing antibody treatment of Nf1 optic glioma mice in vivo. We identified Itgal/CD11a enrichment in GAM relative to other DAM populations, which was confirmed in several independently generated murine models of Neurofibromatosis type 1 (Nf1) optic glioma. Moreover, ITGAL/CD11A expression was similarly increased in human LGG (pilocytic astrocytoma) specimens from several different datasets, specifically in microglia from these tumors. Using CD11a-knockout mice, CD11a expression was shown to be critical for murine microglia CX3CL1 receptor (Cx3cr1) expression and CX3CL1-directed motility, as well as glioma mitogen (Ccl5) production. Consistent with an instructive role for CD11a + microglia in stromal control of LGG growth, antibody-mediated CD11a inhibition reduced mouse Nf1 LGG growth in vivo. Collectively, these findings establish ITGAL/CD11A as a critical microglia regulator of LGG biology relevant to future stroma-targeted brain tumor treatment strategies.

Amelogenins, the principal proteins in the developing enamel microenvironment, self-assemble into supramolecular structures to govern the remodeling of a proteinaceous organic matrix into longitudinally ordered hydroxyapatite nanocrystal arrays. Extensive in vitro studies using purified native or recombinant proteins have revealed the potential of N-terminal amelogenin on protein self-assembly and its ability to guide the mineral deposition. We have previously identified a 14-aa domain (P2) of N-terminal amelogenin that can self-assemble into amyloid-like fibrils in vitro. Here, we investigated how this domain affects the ability of amelogenin self-assembling and stability of enamel matrix protein scaffolding in an in vivo animal model. Mice harboring mutant amelogenin lacking P2 domain had a hypoplastic, hypomineralized, and aprismatic enamel. In vitro, the mutant recombinant amelogenin without P2 had a reduced tendency to self-assemble and was prone to accelerated hydrolysis by MMP20, the prevailing metalloproteinase in early developing enamel matrix. A reduced amount of amelogenins and a lack of elongated fibrous assemblies in the development enamel matrix of mutant mice were evident compared with that in the wild-type mouse enamel matrix. Our study is the first to demonstrate that a subdomain (P2) at the N-terminus of amelogenin controls amelogenin's assembly into a transient protein scaffold that resists rapid proteolysis during enamel development in an animal model. Understanding the building blocks of fibrous scaffold that guides the longitudinal growth of hydroxyapatites in enamel matrix sheds light on protein-mediated enamel bioengineering.

In females, ovarian estradiol (E2) exerts both negative and positive feedback regulation on the neural circuits governing reproductive hormone secretion, but the cellular and molecular mechanisms underlying this remain poorly understood. In rodents, ERα-expressing kisspeptin neurons in the hypothalamic anteroventral periventricular region (AVPV) are prime candidates to mediate E2 positive feedback induction of preovulatory GnRH and LH surges. E2 stimulates AVPV Kiss1 expression, but the full extent of estrogen effects in these neurons is unknown; whether E2 stimulates or inhibits other genes in AVPV Kiss1 cells has not been determined. Indeed, understanding of the function(s) of AVPV kisspeptin cells is limited, in part, by minimal knowledge of their overall molecular phenotype, as only a few genes are currently known to be co-expressed in AVPV Kiss1 cells. To provide a more detailed profiling of co-expressed genes in AVPV Kiss1 cells, including receptors and other signaling factors, and test how these genes respond to E2, we selectively isolated actively-translated mRNAs from AVPV Kiss1 cells of female mice and performed RNA-Seq. This identified >13,000 mRNAs co-expressed in AVPV Kiss1 cells, including multiple receptor and ligand transcripts positively or negatively regulated by E2. We also performed RNAscope to validate high co-expression of several transcripts identified by RNA-Seq, including Pdyn (prodynorphin), Penk (proenkephalin), Vgf (VGF), and Cartpt (CART), in female AVPV Kiss1 cells. Given the important role of AVPV kisspeptin cells in positive feedback, E2 effects on identified genes may relate to the LH surge mechanism and/or other physiological processes involving these AVPV kisspeptin cells.

Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

We show for the first time that the neuropeptide orexin modulates pupillary light response (PLR), a non-image forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (ipRGCs) (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and non-selective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be due to no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENTThis study reveals the role of the neuropeptide orexin in mouse pupillary light response (PLR), a non-image forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs) by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and non-selective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.

[Histologic lesions and porcine morbillivirus (PoMV) RNA in situ hybridization (ISH, red) of tissue of infected swine. A) Histologic section of cerebrum from fetus A stained by hematoxylin and eosin. Arrowheads indicate neuronal necrosis; arrows indicate mineralization and viral inclusion bodies in a neuron and glial cell. B) Cerebellum of fetus A with extensive detection of PoMV by ISH. C) Cerebrum of fetus A; arrowheads indicate ISH labeling within the cytoplasmic and nuclear compartment of neurons; arrow indicates ISH labelling in an axon. D) Cerebrum of fetus B; arrows indicate multiple viral inclusion bodies in neurons; inset displays satellitosis. E) Cerebrum of fetus B showing extensive PoMV detection by ISH. Arrowheads indicate the border of white and gray matter. F) Detection of PoMV by ISH in the spleen of fetus C. G) Detection of PoMV by ISH in a placenta from litter D; arrowhead indicates allantoic epithelium. H) Detection of PoMV by ISH in a renal vessel of a fetus from litter D; arrows indicate the endothelium and arrowhead indicates the vessel lumen. I) Detection of PoMV by ISH in conducting airways (arrowheads) and alveolar septa in the lung of fetus from litter E. J) Detection of PoMV by ISH in the allantoic connective tissue of the placenta and leukocytes from litter F; arrowhead indicates infiltration of leukocytes.] Figure 2 [/eid/article/27/7/20-3971-f2]. Histologic lesions and porcine morbillivirus (PoMV) RNA in situ hybridization (ISH, red) of tissue of infected swine. A) Histologic section of cerebrum from fetus A stained by hematoxylin and eosin....

The 27th Scientific Conference of the Society on Neuroimmune Pharmacology (SNIP) in New Delhi, India, on March 15-18, 2023 is a historic summit of experts from around the world. The four day conference provides insights into the latest and most advanced science in the intersecting areas of neuroscience, immunology, pharmacology, and its translational aspects, in particular, HIV and drug abuse. Abstracts are ordered in three major groups: (1) Symposium speakers (S1-S64), (2) Investigator Posters (I1-I18), and (3) Trainee Poster (T1-T28).

Tumour evolution is a complex interplay between the acquisition of somatic (epi)genomic changes in tumour cells and the phenotypic consequences they cause, all in the context of a changing microenvironment. Single-cell sequencing offers a window into this dynamic process at the ultimate resolution of individual cells. In this review, we discuss the transformative insight offered by single-cell sequencing technologies for understanding tumour evolution.

There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the muscarinic agonist oxotremorine, we revealed a unique concentration-response curve and a sensitivity to repeated forced swim stress or restraint stress exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia assayed using the sucrose preference test or on stress coping strategy assayed using the forced swim test. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in response to environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.Significance Statement:The basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without impacting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.