Probes for INS

Olfaction relies on a coordinated partnership between odorant flow and neuronal communication. Disruption in our ability to detect odors, or anosmia, has emerged as a hallmark symptom of infection with SARS-CoV-2, yet the mechanism behind this abrupt sensory deficit remains elusive. Here, using molecular evaluation of human olfactory epithelium (OE) from subjects succumbing to COVID-19 and a hamster model of SARS-CoV-2 infection, we discovered widespread downregulation of olfactory receptors (ORs) as well as key components of their signaling pathway. OR downregulation likely represents a non-cell autonomous effect, since SARS-CoV-2 detection in OSNs is extremely rare both in human and hamster OEs. A likely explanation for the reduction of OR transcription is the striking reorganization of nuclear architecture observed in the OSN lineage, which disrupts multi-chromosomal compartments regulating OR expression in humans and hamsters. Our experiments uncover a novel molecular mechanism by which a virus with a very selective tropism can elicit persistent transcriptional changes in cells that evade it, contributing to the severity of COVID-19.

Genetically engineered T cell therapy can induce remarkable tumor responses in hematologic malignancies. However, it is not known if this type of therapy can be applied effectively to epithelial cancers, which account for 80-90% of human malignancies. We have conducted a first-in-human, phase 1 clinical trial of T cells engineered with a T cell receptor targeting HPV-16 E7 for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). The primary endpoint was maximum tolerated dose. Cell dose was not limited by toxicity with a maximum dose of 1 × 1011 engineered T cells administered. Tumor responses following treatment were evaluated using RECIST (Response Evaluation Criteria in Solid Tumors) guidelines. Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease. Responses included extensive regression of bulky tumors and complete regression of most tumors in some patients. Genomic studies, which included intra-patient tumors with dichotomous treatment responses, revealed resistance mechanisms from defects in critical components of the antigen presentation and interferon response pathways. These findings demonstrate that engineered T cells can mediate regression of common carcinomas, and they reveal immune editing as a constraint on the curative potential of cellular therapy and possibly other immunotherapies in advanced epithelial cancer.

Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

The medial septum (MS) is involved in arousal-based behaviors and modulates general anesthesia response. However, the role of MS in wakefulness control remains unknown. Here, combining double fluorescence in situ hybridization and optrode recording, we showed that MS glutamatergic neurons exhibited higher activities preferentially during wakefulness. Activating these neurons, either optogenetically or chemogenetically, strongly promoted wakefulness, mainly through the transition from non-rapid eye movement (NREM) sleep to wakefulness. In contrast, inactivation of these neurons reduced wakefulness by the transition from wakefulness to NREM sleep. Furthermore, both rabies-mediated monosynaptic retrograde and anterograde tracing showed that MS glutamatergic neurons monosynaptically innervated lateral hypothalamus (LH) glutamatergic neurons, which were also wake-active as well as wake-promoting. Activating MS-derived glutamatergic terminals in LH enhanced wakefulness, whereas silencing MS glutamatergic neurons destabilized the wake-active preference of LH glutamatergic neurons. These results reveal a vital role of MS glutamatergic neurons in wakefulness control and depict a novel septo-hypothalamic circuit for wakefulness.

Zebrafish can regenerate adult cardiac tissue following injuries from ventricular apex amputation, cryoinjury, and cardiomyocyte genetic ablation. Here, we characterize cardiac regeneration from cardiomyocyte chemoptogenetic ablation caused by localized near-infrared excited photosensitizer-mediated Reactive Oxygen Species (ROS) generation. Exposure of transgenic adult zebrafish, Tg(myl7:fapdl5-cerulean), to di-iodinated derivative of the cell- permeable Malachite Green ester fluorogen (MG-2I) and whole-body illumination with 660nm light resulted in cytotoxic damage to about 30% of cardiac tissue. After chemoptogenetic cardiomyocyte ablation, heart function was compromised, and macrophage infiltration was detected, but epicardial and endocardial activation response was much muted when compared to ventricular amputation. The spared cardiomyocytes underwent proliferation and restored the heart structure and function in 45-60 days after ablation. This cardiomyocyte ablation system did not appear to activate the epicardium and endocardium as is noted in other cardiac injury models. This approach represents a useful model to study specifically cardiomyocyte injury, proliferation and regeneration in the absence of whole organ activation. Moreover, this system can be adapted to ablate distinct cell populations in any organ system to study their function in regeneration. This article is protected by

Interaction between adult stem cells and their progeny is critical for tissue homeostasis and regeneration. In multiple organs, mesenchymal stem cells (MSCs) give rise to transit amplifying cells (TACs), which then differentiate into different cell types. However, whether and how MSCs interact with TACs remains unknown. Using the adult mouse incisor as a model, we present in vivo evidence that TACs and MSCs have distinct genetic programs and engage in reciprocal signaling cross talk to maintain tissue homeostasis. Specifically, an IGF-WNT signaling cascade is involved in the feedforward from MSCs to TACs. TACs are regulated by tissue-autonomous canonical WNT signaling and can feedback to MSCs and regulate MSC maintenance via Wnt5a/Ror2-mediated non-canonical WNT signaling. Collectively, these findings highlight the importance of coordinated bidirectional signaling interaction between MSCs and TACs in instructing mesenchymal tissue homeostasis, and the mechanisms identified here have important implications for MSC-TAC interaction in other organs.

Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkwormBombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

Metastatic medullary thyroid cancer (MTC) is a rare, but often, aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC.

Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that causes coronavirus disease, has been shown to infect several species. The role of domestic livestock and associated risks for humans in close contact with food production animals remains unknown for many species. Determining the susceptibility of pigs to SARS-CoV-2 is critical to a One Health approach to manage potential risk for zoonotic transmission. We found that pigs are susceptible to SARS-CoV-2 after oronasal inoculation. Among 16 animals, we detected viral RNA in group oral fluids and in nasal wash from 2 pigs, but live virus was isolated from only 1 pig. Antibodies also were detected in only 2 animals at 11 and 13 days postinoculation but were detected in oral fluid samples at 6 days postinoculation, indicating antibody secretion. These data highlight the need for additional livestock assessment to determine the potential role of domestic animals in the SARS-CoV-2 pandemic.

4521 Background: Rogaratinib (R) is a novel pan-FGFR inhibitor that showed promising efficacy and safety in a Phase I trial in pts with advanced solid tumors, including UC, with FGFR1-3 mRNA overexpression. The Phase Ib/II FORT-2 study (NCT03473756) of R plus atezolizumab (A) in pts with first-line cisplatin-ineligible, FGFR-positive, advanced/metastatic UC previously identified a maximum tolerated dose of R 600 mg twice daily (BID) plus A (1200 mg every 3 weeks) . We report updated safety, efficacy, and the recommended Phase II dose (RP2D) for combination therapy from the Phase Ib study. Methods: Pts with cisplatin-ineligible, locally advanced/metastatic UC with FGFR1/3 mRNA overexpression detected by RNA in situ hybridization of archival tissue (RNAscope) received oral R 600 mg BID plus A 1200 mg on day 1 of a 21-day cycle. Archival tissue was examined for programmed cell-death ligand 1 (PD-L1) protein expression levels, FGFR3-activating mutations via a targeted Illumina NGS panel, and FGFR fusions via an Archer fusion plex assay. Primary objectives were safety, tolerability, and determination of the RP2D. Results: 26 pts (enrolled May 25, 2018 to Nov 25, 2020) were treated; 89% were male, median age was 76 years (range 47-85), 58% had an ECOG performance status of 1, and 77% displayed low or absent (negative or non-detectable) PD-L1 expression (combined positive score < 10%). Common treatment-emergent adverse events (TEAEs) included diarrhea (n = 17, 65%; 1 grade [G] 3), hyperphosphatemia (n = 15, 58%; all G1 or 2), and nausea (n = 11, 42%; 1 G3). The most common G3/4 TEAEs were elevated lipase without pancreatitis (n = 5, 19%), elevated amylase (n = 3, 12%), and rash and syncope (n = 2, 8% each). TEAEs led to interruption/reduction/discontinuation of R in 69%/46%/19% of pts. R-related unique TEAEs were hyperphosphatemia in 15 pts (58%) and retinal pigment epithelium detachment in 1 pt (4%). G5 events occurred in 3 pts (12%), unrelated to treatment. 13 of 24 evaluable pts (54%) had an objective response (OR) per RECIST v1.1. The disease control rate was 83%, including 3 pts (13%) with a complete response (CR), 10 (42%) with a partial response (PR), and 7 (29%) with stable disease. Median duration of response was not reached. OR rate was 56% (2 CRs and 7 PRs) in the 16 pts with tumors having low/negative PD-L1 protein and FGFR3 mRNA overexpression without mutation. The RP2D for R+A was 600 mg BID. Conclusions: First-line treatment with the RP2D of R+A achieved favorable clinical efficacy and tolerability in pts with cisplatin-ineligible, metastatic UC characterized by high FGFR1/3 mRNA expression and generally low/negative PD-L1 expression. Encouraging efficacy was observed regardless of PD-L1 expression or FGFR3 mutation status, warranting future investigation. Clinical trial information: NCT03473756.