Probes for INS

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.

Background Following a dose-escalation study, a dose-expansion study for ISU104 (monotherapy and combination therapy with CET) has been conducted in R/M HNSCC (Ann Oncol, abst #928P, 2020). Here we report updated final safety, clinical efficacy and biomarker analysis results from the dose-expansion study. Methods Eighteen R/M HNSCC pts excluding nasopharyngeal cancer, were enrolled and allocated to Mono (ISU104, 20 mg/kg/day, Q3W; N=6) or Combo groups (ISU104 20 mg/kg, Q3W and CET, initially 400 mg/m2 followed by 250 mg/m2, Q1W; N=12). Tumor response assessments (RECIST 1.1), safety and occurrence of anti-drug antibodies (ADA) were determined. Immunohistochemistry, RNAscope™ Assay-based in situ hybridization (ISH) and next generation sequencing were performed on sections of biopsy samples. Results Most common treatment emergent adverse events (AEs) included decreased appetite (66.7%) and stomatitis (50%) in Mono, and diarrhea (75.0%) and dermatitis acneiform (50%) in Combo. Serious AEs were reported 16.7% in Mono and 58.33% in Combo, but no AEs led to treatment discontinuation. One patient (1/18, 5.56%) developed ADA, which did not have neutralizing activity. Four pts in Combo were responsive to treatment (1 CR and 3 PR out of 11 analyzed pts; 36.36%); one patient remained CR up to now. Duration of responses were 46, 62, 163+ and 449+ days in Combo, and median progression-free survival was 54 and 99 days in Mono and Combo groups, respectively, in median follow-up period of 480 days (as of 30th April 2021). H-scores of potential biomarkers including EGFR-ISH at pre-treatment were correlated with tumor size changes following combination therapy. Potential implication of TP53 mutations and EGFR amplification in patient selection was also noted. Conclusions ISU104 alone or in combination with CET were safe and tolerable in R/M HNSCC pts. Encouraging clinical efficacies and potential biomarkers were demonstrated from combination therapy. A phase II study of ISU104 (Q3W, 20 mg/kg/day) in combination with CET (Q1W) is planned to further strengthen the clinical utility of ISU104.

Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of semaphorins to act both as signaling receptors and ligands yields a multitude of responses. Here, we describe a novel role for Semaphorin-6D (Sema6D) and Plexin-A1 in the positioning and targeting of retinogeniculate axons. In Plexin-A1 or Sema6D mutant mice of either sex, the optic tract courses through rather than at the border of the dorsolateral geniculate nucleus (dLGN); and some retinal axons ectopically arborize adjacent and lateral to the optic tract rather than defasciculating and entering the target region. We find that Sema6D and Plexin-A1 act together and in a dose-dependent manner, as the number of the ectopic retinal projections is altered in proportion to the level of Sema6D or Plexin-A1 expression. Moreover, using retinal in utero electroporation of Sema6D or Plexin-A1 shRNA, we show that Sema6D and Plexin-A1 are both required in retinal ganglion cells (RGCs) for axon positioning and targeting. Strikingly, non-electroporated RGC axons also mistarget in the tract region, indicating that Sema6D and Plexin-A1 can act non-cell autonomously, potentially through axon-axon interactions. These data provide novel evidence for a dose-dependent and non-cell autonomous role for Sema6D and Plexin-A1 in retinal axon organization in the optic tract and dLGN.SIGNIFICANCE STATEMENT:Before innervating their central brain targets, retinal ganglion cell (RGC) axons fasciculate in the optic tract then branch and arborize in their target areas. Upon deletion of the guidance molecules Plexin-A1 or Semaphorin-6D, the optic tract becomes disorganized near and extends within the dorsal lateral geniculate nucleus. In addition, some retinal axons form ectopic aggregates within the defasciculated tract. Sema6D and Plexin-A1 act together as a receptor-ligand pair in a dose-dependent manner, and non-cell autonomously, to produce this developmental aberration. Such a phenotype highlights an under-appreciated role for axon guidance molecules in tract cohesion and appropriate defasciculation near and arborization within targets.