Probes for INS

Somatosensory information is signaled by primary sensory neurons located in dorsal root ganglia (DRG) or trigeminal ganglia. Type C-low threshold mechanoreceptors (C-LTMRs) are proposed to sense light touch. The differentiation and maturation of C-LTMRs are regulated by multiple transcript factors, including Zfp521 and Runx1. However, the molecular mechanism of C-LTMR development still remains largely unclear. RNA sequencing (RNA-seq) was performed to detect transcriptional changes in Tlx3cko DRGs compared to controls. In situ hybridization and RNAscope were used to verify RNA-seq data. RNA-seq identified 203 up- and 372 downregulated genes in DRG by loss of Tlx3 function. KEGG and Gene ontology analysis indicated that the biological properties and molecular functions were closely associated with neural signal processing and transmitting somatosensory information. In addition, the expression of marker genes of C-LTMRs was significantly decreased in Tlx3 mutants. However, Tlx3cko mice exhibited normal response to static and dynamic touch. Furthermore, Tlx3 was required to regulate the expression of Zfp521 and Runx1. Tlx3, Runx1 and Zfp521 may form a hierarchical regulation pathway to control C-LTMR development.

Most cells in the body are mononuclear whereas skeletal muscle fibres are uniquely multinuclear. The nuclei of muscle fibres (myonuclei) are usually situated peripherally which complicates the equitable distribution of gene products. Myonuclear abundance can also change under conditions such as hypertrophy and atrophy. Specialised zones in muscle fibres have different functions and thus distinct synthetic demands from myonuclei. The complex structure and regulatory requirements of multinuclear muscle cells understandably led to the hypothesis that myonuclei govern defined 'domains' to maintain homeostasis and facilitate adaptation. The purpose of this review is to provide historical context for the myonuclear domain and evaluate its veracity with respect to mRNA and protein distribution resulting from myonuclear transcription. We synthesise insights from past and current in vitro and in vivo genetically modified models for studying the myonuclear domain under dynamic conditions. We also cover the most contemporary knowledge on mRNA and protein transport in muscle cells. Insights from emerging technologies such as single myonuclear RNA-sequencing further inform our discussion of the myonuclear domain. We broadly conclude: (1) the myonuclear domain can be flexible during muscle fibre growth and atrophy, (2) the mechanisms and role of myonuclear loss and motility deserve further consideration, (3) mRNA in muscle is actively transported via microtubules and locally restricted, but proteins may travel far from a myonucleus of origin and (4) myonuclear transcriptional specialisation extends beyond the classic neuromuscular and myotendinous populations. A deeper understanding of the myonuclear domain in muscle may promote effective therapies for ageing and disease.

The field of precision medicine allows for tailor-made treatments specific to a patient and thereby improve the efficiency and accuracy of disease prevention, diagnosis, and treatment and at the same time would reduce the cost, redundant treatment, and side effects of current treatments. Here, the combination of organ-on-a-chip and bioprinting into engineering high-content in vitro tissue models is envisioned to address some precision medicine challenges. This strategy could be employed to tackle the current coronavirus disease 2019 (COVID-19), which has made a significant impact and paradigm shift in our society. Nevertheless, despite that vaccines against COVID-19 have been successfully developed and vaccination programs are already being deployed worldwide, it will likely require some time before it is available to everyone. Furthermore, there are still some uncertainties and lack of a full understanding of the virus as demonstrated in the high number new mutations arising worldwide and reinfections of already vaccinated individuals. To this end, efficient diagnostic tools and treatments are still urgently needed. In this context, the convergence of bioprinting and organ-on-a-chip technologies, either used alone or in combination, could possibly function as a prominent tool in addressing the current pandemic. This could enable facile advances of important tools, diagnostics, and better physiologically representative in vitro models specific to individuals allowing for faster and more accurate screening of therapeutics evaluating their efficacy and toxicity. This review will cover such technological advances and highlight what is needed for the field to mature for tackling the various needs for current and future pandemics as well as their relevancy towards precision medicine.

Emerging literature suggests that experiences of discrimination negatively influence health and well-being. It is unfortunately common for people living with HIV (PLWH) to be stigmatized and discriminated against because of their HIV status and other marginalized identities (e.g., ethnicity/race, sexual identity and orientation). To date, little research has specifically examined discrimination in PWLH and its associations with pain and other pain-relevant factors such as mood and sleep. The purpose of this ongoing study was to preliminarily analyze associations among daily experiences of discrimination, pain severity and interference, depressive symptoms, and sleep in PLWH. Participants included 24 PLWH recruited from a local HIV treatment center. Participants completed The Everyday Discrimination Scale (TEDS) followed by the Brief Pain Inventory - Short Form (BPI-SF), the Insomnia Severity Index (ISI), and the Center for Epidemiologic Studies - Depression Scale (CES-D). Initial findings tentatively suggest that more frequent daily experiences of discrimination may be significantly associated with greater pain interference on the BPI-SF (p = .030) and greater severity of insomnia symptoms on the ISI (p = .059). However, it appears that daily experiences of discrimination may not be meaningfully associated with pain severity on the BPI-SF (p = .401) or depressive symptoms on the CES-D (p = .235). Our findings highlight the potentially deleterious effects of daily discrimination experiences on pain and sleep in in PLWH. As this ongoing study recruits a larger sample of PLWH, data will need to be reanalyzed to better determine the durability of these preliminary findings. However, there is potential that findings from this study may assist in elucidating causal pathways linking discrimination to pain and pain relevant health behaviors like sleep in PLWH. Grant support from The Impact of Insomnia on Pain, Physical Function, and Inflammation in HIV (3R01HL147603-03S1).

Respiratory chemoreceptor activity encoding arterial PCO2 and PO2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomical evidence suggest that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-depedent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis due to alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests RTN Nmb neurons are dedicated to the respiratory effects of arterial PCO2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.Significance Statement:Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial PCO2 and PO2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomical data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Background: Adoptive T cell therapies have led to remarkable advances in hematologic cancers but with less effect in ST. Actively recruited tumor associated macrophages (TAM) are abundant in the ST microenvironment (TME) and typically display immunosuppressive behavior. Macrophages engineered to be proinflammatory may be an ideal vector for adoptive ST cellular therapy. Engineered CAR-M selectively recognize and phagocytose antigen overexpressing cancer cells, reprogram TME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis in many cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived proinflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies show that CT-0508 induces targeted cancer cell phagocytosis while sparing normal cells, decreases tumor burden and prolongs survival, and was safe and effective in a semi-immunocompetent mouse model of human HER2-overexpressing ovarian cancer. Methods: This FIH Phase 1 study is evaluating safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary efficacy in 18 subjects with locally advanced/unresectable or metastatic ST overexpressing HER2, with progression on available therapies, including anti-HER2 therapies. Filgrastim is used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis prior to CT-0508 CAR macrophage infusion. Group 1 subjects receive CT-0508 on D1, 3, & 5. Group 2 subjects will receive full dose on D1. A Safety Review Committee will review dose limiting toxicities. Pre/post-treatment biopsies and blood samples will be collected for correlative analysis of immunogenicity, trafficking (PCR, RNA scope), CT-0508 persistence in blood and tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Table 1. Her2 Overexpression Across Tumor Types Tumor HER2 Overexpression (%) Bladder 8-70 Salivary duct 30-40 Gastric 7-34 Ovarian 26 Breast 11-25 Salivary mucoepidermoid 17.6 Esophageal 12-14 Gallbladder 9.8-12.8 Cholangiocarcinoma 6.3-9 Colorectal 1.6-5 Cervical 2.8-3.9 Uterine 3 Testicular 2.4 Citation Format: Kim A. Reiss, Yuan Yuan, Naoto T. Ueno, Yara Abdou, Debora Barton, Ramona F. Swaby, Amy Ronczka, Daniel J. Cushing, Sascha Abramson, Thomas Condamine, Michael Klichinsky, E. Claire Dees. A phase 1, first in human (FIH) study of autologous anti-HER2 chimeric antigen receptor macrophages (CAR-M) in HER2-overexpressing solid tumors (ST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT524.

Although RNA plays a vital role in the process of gene expression, it is less used as an in situ biomarker for clinical diagnostics compared to DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules themselves. To tackle this issue, methods that are sensitive and specific are needed. Here we present an RNA single molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity on the RNA molecules, they form V shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We not only successfully applied our method to assess HER2 RNA mRNA expression status in invasive breast cancer tissue, but also to investigate the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicates the great potential of our method to be applied in the diagnosis of disease using RNA biomarkers.

We made 2 Z-based in situ hybridization (ISH) probes for the detection of rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) nucleic acid in formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that had died during an outbreak of RHD in Washington, USA. One probe system was made for detection of negative-sense RNA (i.e., the replicative intermediate RNA for the virus), and the other probe system was constructed for detection of genomic and mRNA of the virus (viral mRNA). Tissue sets were tested separately, and the viral mRNA probe system highlighted much broader tissue distribution than that of the replicative intermediate RNA probe system. The latter was limited to liver, lung, kidney, spleen, myocardium, and occasional endothelial staining, whereas signal for the viral mRNA was seen in many more tissues. The difference in distribution suggests that innate phagocytic activity of various cell types may cause overestimation of viral replication sites when utilizing ISH of single-stranded, positive-sense viruses.

Pulmonary fibrosis is an interstitial lung disease caused by various factors such as exposure to workplace environmental contaminants, drugs, or X-rays. Epithelial cells are among the driving factors of pulmonary fibrosis. Immunoglobulin A (IgA), traditionally thought to be secreted by B cells, is an important immune factor involved in respiratory mucosal immunity. In the current study, we found that lung epithelial cells are involved in IgA secretion, which, in turn, promotes pulmonary fibrosis. Spatial transcriptomics and single-cell sequencing suggest that Igha transcripts were highly expressed in the fibrotic lesion areas of lungs from silica-treated mice. Reconstruction of B-cell receptor (BCR) sequences revealed a new cluster of AT2-like epithelial cells with a shared BCR and high expression of genes related to IgA production. Furthermore, the secretion of IgA by AT2-like cells was trapped by the extracellular matrix and aggravated pulmonary fibrosis by activating fibroblasts. Targeted blockade of IgA secretion by pulmonary epithelial cells may be a potential strategy for treating pulmonary fibrosis.

Precise monitoring of internal temperature is vital for thermal homeostasis in mammals. For decades, warm-sensitive neurons (WSNs) within the preoptic area (POA) were thought to sense internal warmth, using this information as feedback to regulate body temperature (Tcore). However, the cellular and molecular mechanisms by which WSNs measure temperature remain largely undefined. Via a pilot genetic screen, we found that silencing the TRPC4 channel in mice substantially attenuated hypothermia induced by light-mediated heating of the POA. Loss-of-function studies of TRPC4 confirmed its role in warm sensing in GABAergic WSNs, causing additional defects in basal temperature setting, warm defense, and fever responses. Furthermore, TRPC4 antagonists and agonists bidirectionally regulated Tcore. Thus, our data indicate that TRPC4 is essential for sensing internal warmth and that TRPC4-expressing GABAergic WSNs function as a novel cellular sensor for preventing Tcore from exceeding set-point temperatures. TRPC4 may represent a potential therapeutic target for managing Tcore.

The transcriptome of a cell constitutes an essential piece of cellular identity and contributes to the multifaceted complexity and heterogeneity of cell-types within the mammalian brain. Thus, while a wealth of studies have investigated transcriptomic alterations underlying the pathophysiology of diseases of the brain, their use of bulk-tissue homogenates makes it difficult to tease apart whether observed differences are explained by disease state or cellular composition. Cell-type-specific enrichment strategies are, therefore, crucial in the context of gene expression profiling. Laser capture microdissection (LCM) is one such strategy that allows for the capture of specific cell-types, or regions of interest, under microscopic visualization. In this review, we focus on using LCM for cell-type specific gene expression profiling in post-mortem human brain samples. We begin with a discussion of various LCM systems, followed by a walk-through of each step in the LCM to gene expression profiling workflow and a description of some of the limitations associated with LCM. Throughout the review, we highlight important considerations when using LCM with post-mortem human brain samples. Whenever applicable, commercially available kits that have proven successful in the context of LCM with post-mortem human brain samples are described.

Detection and transduction of photic cues by nonvisual photoreceptors, located in the deep brain, is a critical component of timing seasonal reproduction in birds. However, the precise identity of the photoreceptors responsible for detection of salient photic cues remains uncertain and debated. Here I review of the existing evidence for each of the three candidate photoreceptive opsins: Vertebrate Ancient Opsin, Melanopsin, and Neuropsin, including localization, action spectrum, and data from experimental manipulation of opsin expression. These findings are compared to an updated list of key criteria established in the literature as a litmus for classifying an opsin as the "breeding photoreceptor." Integrating evidence for each of the candidate photoreceptors with respect to these criteria reveals support for all three opsins in regulation of seasonal reproduction. Taken together these findings strongly suggest that transduction of seasonal photoperiodic information involves the activity of multiple photoreceptor types and populations functioning in concert. This review also highlights the need to shift attention from simply identifying "the breeding photoreceptor" to a more integrative approach aiming to parse the contribution of specific photoreceptor populations within the brain.