Probes for INS

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.

Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.

Delayed and often impaired wound healing in the elderly presents major medical and socioeconomic challenges. A comprehensive understanding of the cellular/molecular changes that shape complex cell-cell communications in aged skin wounds is lacking. Here, we use single-cell RNA sequencing to define the epithelial, fibroblast, immune cell types, and encompassing heterogeneities in young and aged skin during homeostasis and identify major changes in cell compositions, kinetics, and molecular profiles during wound healing. Our comparative study uncovers a more pronounced inflammatory phenotype in aged skin wounds, featuring neutrophil persistence and higher abundance of an inflammatory/glycolytic Arg1Hi macrophage subset that is more likely to signal to fibroblasts via interleukin (IL)-1 than in young counterparts. We predict systems-level differences in the number, strength, route, and signaling mediators of putative cell-cell communications in young and aged skin wounds. Our study exposes numerous cellular/molecular targets for functional interrogation and provides a hypothesis-generating resource for future wound healing studies.

Pulpitis refers to inflammation of the inner pulp by invading microbes, and tissue repair occurs due to odontogenic differentiation of human dental pulp cells (hDPCs) with multidifferentiation potential. Long noncoding RNAs (lncRNAs) can modulate numerous pathological and biological processes; however, the role of lncRNAs in the inflammation and regeneration of the dentin-pulp complex in pulpitis is unclear. Here, we performed high-throughput sequencing to identify differentially expressed lncRNAs between human normal and inflamed pulp and concluded that lncMEG3 (lncRNA maternally expressed gene 3, MEG3) was significantly upregulated in both inflamed pulp and LPS-treated hDPCs. MEG3 expression in the pulp tissue was detected using the RNAscope™ technique. RNA pulldown assays identified the MEG3-interacting proteins and the potential mechanisms. With MEG3 knockdown, we investigated the role of MEG3 in the secretion of inflammatory cytokines in LPS-treated hDPCs and odontogenic differentiation of hDPCs. MEG3 downregulation inhibited the secretion of TNF-α, IL-1β and IL-6 in LPS-treated hDPCs, and the p38/MAPK signaling pathway may be related to this effect. MEG3 knockdown promoted odontogenic differentiation of hDPCs by regulating the Wnt/β-catenin signaling pathway. Our study suggested that MEG3 has a negative effect on inflammation and regeneration of the dentin-pulp complex in pulpitis.

Adult hippocampal neurogenesis is implicated in antidepressant action in rodents and primates, yet relationships to human major depression (MDD) and antidepressant effects are unclear. In postmortem human hippocampus, we found doublecortin (DCX) protein and mRNA+ cells co-expressing neuronal but not astroglial or microglial markers. We defined neuroblasts as DCX+ cells located in the subgranular zone that co-expressed neuron-specific beta-tubulin (TUJ1) or lacked co-expression with neuronal nuclear marker (NeuN). Untreated MDD, regardless of clinical state, had fewer DCX-positive cells and neuroblasts in the rostral dentate gyrus compared with non-psychiatric controls. High-functioning, but not low-functioning, antidepressant-treated MDD, exhibited more DCX/TUJ1+ neuroblasts than untreated MDD. Groups did not differ in number of immature neurons, defined as DCX/NeuN+ cells in the inner granule cell layer. Deficient neuroblasts may be linked to hippocampal-dependent cognitive deficits in MDD. Similar neuroblast number between controls and higher-functioning antidepressant treated subjects warrants evaluation of neuroblasts as a treatment target.

The maturation and function of osteoblasts (OBs) rely heavily on the reversible phosphorylation of signaling proteins. To date, most of the work in OBs has focused on phosphorylation by tyrosyl kinases, but little has been revealed about dephosphorylation by protein tyrosine phosphatases (PTPases). SHP2 (encoded by PTPN11) is a ubiquitously expressed PTPase. PTPN11 mutations are associated with both bone and cartilage manifestations in patients with Noonan syndrome (NS) and metachondromatosis (MC), although the underlying mechanisms remain elusive. Here, we report that SHP2 deletion in bone gamma-carboxyglutamate protein-expressing (Bglap+) bone cells leads to massive osteopenia in both trabecular and cortical bones due to the failure of bone cell maturation and enhanced osteoclast activity, and its deletion in Bglap+ chondrocytes results in the onset of enchondroma and osteochondroma in aged mice with increased tubular bone length. Mechanistically, SHP2 was found to be required for osteoblastic differentiation by promoting RUNX2/OSTERIX signaling and for the suppression of osteoclastogenesis by inhibiting STAT3-mediated RANKL production by osteoblasts and osteocytes. These findings are likely to explain the compromised skeletal system in NS and MC patients and to inform the development of novel therapeutics to combat skeletal disorders.

Single-cell Multi-Omics (SCM-Omics) and Spatial Multi-Omics (SM-Omics) technologies provide state-of-the-art methods for exploring the composition and function of cell types in tissues/organs. Since its emergence in 2009, single-cell RNA sequencing (scRNA-seq) has yielded many groundbreaking new discoveries. The combination of this method with the emergence and development of SM-Omics techniques has been a pioneering strategy in neuroscience, developmental biology, and cancer research, especially for assessing tumor heterogeneity and T-cell infiltration. In recent years, the application of these methods in the study of metabolic diseases has also increased. The emerging SCM-Omics and SM-Omics approaches allow the molecular and spatial analysis of cells to explore regulatory states and determine cell fate, and thus provide promising tools for unraveling heterogeneous metabolic processes and making them amenable to intervention. Here, we review the evolution of SCM-Omics and SM-Omics technologies, and describe the progress in the application of SCM-Omics and SM-Omics in metabolism-related diseases, including obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). We also conclude that the application of SCM-Omics and SM-Omics approaches can help resolve the molecular mechanisms underlying the pathogenesis of metabolic diseases in the body and facilitate therapeutic measures for metabolism-related diseases. This review concludes with an overview of the current status of this emerging field and the outlook for its future.

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft (PDX) models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a basal-like A subtype-specific transcribed enhancer program (B-STEP) in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histological approach for PDAC patient stratification by performing RNA in situ hybridization analyses for subtype-specific eRNAs on pathological tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single cell level in patient material can be used as a potential tool for treatment stratification.

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Delta-like ligand 3 (DLL3) is highly expressed on SCLC and several other types of neuroendocrine cancers, with limited normal tissue RNA expression in brain, pituitary, and testis, making it a promising CAR T-cell target for SCLC and other solid tumor indications.A large panel of anti-DLL3 scFv-based CARs were characterized for both in vitro and in vivo activity. To understand the potential for pituitary and brain toxicity, subcutaneous or intracranial tumors expressing DLL3 were implanted in mice and treated with mouse cross-reactive DLL3 CAR T cells.A subset of CARs demonstrated high sensitivity for targets with low DLL3 density and long-term killing potential in vitro. Infusion of DLL3 CAR T cells led to robust antitumor efficacy, including complete responses, in subcutaneous and systemic SCLC in vivo models. CAR T-cell infiltration into intermediate and posterior pituitary was detected, but no tissue damage in brain or pituitary was observed, and the hormone-secretion function of the pituitary was not ablated.In summary, the preclinical efficacy and safety data presented here support further evaluation of DLL3 CAR T cells as potential clinical candidates for the treatment of SCLC.

Chronic-pain is a debilitating illness that has become exceedingly widespread with currently limited treatments. Differences in the molecular signature of nociceptors, have been demonstrated between human and the commonly-used mouse model, suggesting functional differences in detection and transmission of noxious-stimuli. Therefore, direct understanding of pain-physiology in humans is required for pain treatment. This could be facilitated by studying humans carrying deleterious genetic mutations affecting pain sensation. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with several body-functions, in particular, noxious-heat detection and inflammatory-pain. Reports of adverse effects in human trials have hinder the clinical development of TRPV1 antagonists as novel pain relievers. Hence, studies on the functional roles of TRPV1, which currently rely mainly on evidences obtained from rodents, should be extended to humans. Here, we examined humans carrying a unique missense mutation in TRPV1, rendering the channel non-functional. The affected individual demonstrated lack of aversion towards capsaicin and elevated heat-pain threshold. Surprisingly, he showed elevated cold-pain threshold and extensive neurogenic inflammatory flare and pain-responses following application of the TRPA1 channel-activator, mustard-oil. Our study provides the first direct evidence for pain-related functional-changes linked to TRPV1 in humans, which is a prime target in the development of novel pain-relievers.