Probes for INS

Satiety and hunger are controlled by a complex and distributed neural network. The ‘standard model’ of energy homeostasis as the net product of orexigenic agouti-related protein and anorexigenic pro-opiomelanocortin neurons within the hypothalamus is the cornerstone of our understanding. It is, however, patently incomplete, and fundamental gaps exist in our understanding of the identity and organisation of cell types forming the appetitive neurocircuitry, their functions and the relevance of those identified and characterised in mice to the equivalent human neurocircuitry. Technological advances in single-cell and spatial transcriptomics, increasingly refined genetic tools for neuronal manipulation in mice, and the development of human hypothalamic cell models provide tools capable of addressing these fundamental questions and offer hope of one day approaching a ‘grand unifying theory’ of energy homeostasis.

Batrachochytrium salamandrivorans is an emerging fungus that is causing salamander declines in Europe. We evaluated whether an invasive frog species (Cuban treefrog, Osteopilus septentrionalis) that is found in international trade could be an asymptomatic carrier when exposed to zoospore doses known to infect salamanders. We discovered that Cuban treefrogs could be infected with B. salamandrivorans and, surprisingly, that chytridiomycosis developed in animals at the two highest zoospore doses. To fulfill Koch's postulates, we isolated B. salamandrivorans from infected frogs, exposed eastern newts (Notophthalmus viridescens) to the isolate, and verified infection and disease by histopathology. This experiment represents the first documentation of B. salamandrivorans chytridiomycosis in a frog species and substantially expands the conservation threat and possible mobilization of this pathogen in trade.

In the 2002 outbreak of severe acute respiratory syndrome (SARS) a number of patients presented abnormalities in the thyroid functioning, neuroendocrine and calcium homeostasis. It was detected in autopsies from SARS Coronavirus (SARS-CoV) patients that the thyroid gland was significantly affected by the disease, with extensive injury and death of follicular and parafollicular cells. In the present SARS-CoV-2 pandemic some studies start to report acute thyroiditis and alterations in the levels of thyroid hormones [(triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH)]. Thyroid cells present high levels of mRNA expression of angiotensin-converting enzyme 2 (ACE2), the host receptor for SARS-CoV-2. It remains poorly studied the thyroid expression of proteins that predispose to SARS-CoV-2 infection and if thyroid cells can be a direct or indirect target of SARS-CoV-2 infection.

Circular RNAs (circRNAs) have been studied extensively in the last few years, uncovering functional roles in a diverse range of cell types and organisms. As shown for a few cases, these functions may be mediated by trans-acting factors, in particular RNA-binding proteins (RBPs). However, the specific interaction partners for most circRNAs remain unknown. This is mainly due to technical difficulties in their identification and in differentiating between interactors of circRNAs and their linear counterparts. Here we review the currently used methodology to systematically study circRNA-protein complexes (circRNPs), focusing either on a specific RNA or protein, both on the gene-specific or global level, and discuss advantages and challenges of the available approaches.

Spatial transcriptional profiling provides gene expression information within the important anatomical context of tissue architecture. This approach is well suited to characterizing solid tumors, which develop within a complex landscape of malignant cells, immune cells, and stroma. In a single assay, spatial transcriptional profiling can interrogate the role of spatial relationships among these cell populations as well as reveal spatial patterns of relevant oncogenic genetic events. The broad utility of this approach is reflected in the array of strategies that have been developed for its implementation as well as in the recent commercial development of several profiling platforms. The flexibility to apply these technologies to both hypothesis-driven and discovery-driven studies allows widespread applicability in research settings. This review discusses available technologies for spatial transcriptional profiling and several applications for their use in cancer research.

Birds display advanced behaviors, including vocal learning and problem-solving, yet lack a layered neocortex, a structure associated with complex behavior in mammals. To determine whether these behavioral similarities result from shared or distinct neural circuits, we used single-cell RNA sequencing to characterize the neuronal repertoire of the songbird song motor pathway. Glutamatergic vocal neurons had considerable transcriptional similarity to neocortical projection neurons; however, they displayed regulatory gene expression patterns more closely related to neurons in the ventral pallium. Moreover, while γ-aminobutyric acid-releasing neurons in this pathway appeared homologous to those in mammals and other amniotes, the most abundant avian class is largely absent in the neocortex. These data suggest that songbird vocal circuits and the mammalian neocortex have distinct developmental origins yet contain transcriptionally similar neurons.

Morphine-induced itch is a very common and debilitating side effect that occurs in laboring women who receive epidural analgesia and in patients who receive spinal morphine for relief of perioperative pain. Although antihistamines are still widely prescribed for the treatment of morphine-induced itch, their use is controversial because the cellular basis for morphine-induced itch remains unclear. Here, we used animal models and show that neuraxial morphine causes itch through neurons and not mast cells. In particular, we found that spinal dynorphin (Pdyn) neurons are both necessary and sufficient for morphine-induced itch in mice. Agonism of the kappa-opioid receptor alleviated morphine-induced itch in mice and nonhuman primates. Thus, our findings not only reveal that morphine causes itch through a mechanism of disinhibition but also challenge the long-standing use of antihistamines, thereby informing the treatment of millions worldwide.

Embryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much underlying microscopic variability is compatible with normal development. Here, we quantify embryo-to-embryo variability in vertebrate development by studying cell number variation in the zebrafish endoderm. We notice that the size of a sub-population of the endoderm, the dorsal forerunner cells (DFCs, which later form the left-right organizer), exhibits significantly more embryo-to-embryo variation than the rest of the endoderm. We find that, with incubation of the embryos at elevated temperature, the frequency of left-right laterality defects is increased drastically in embryos with a low number of DFCs. Furthermore, we observe that these fluctuations have a large stochastic component among fish of the same genetic background. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be associated with maternal effects in the specification of the DFCs.

mRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technology, vaccinology, and nanotechnology have generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual molecules, allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochemical characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clinical trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.

Human papillomavirus (HPV) is associated with numerous malignancies in the head and neck, as well as other body sites. Human papillomavirus-related mulitphenotypic sinonasal carcinoma (HMSC), previously described as HPV-related carcinoma with adenoid cystic-like features, represents a rare carcinoma that morphologically and immunohistochemically resembles a salivary gland tumor.1 A majority of these lesions have been reported in the nasal cavity, but the lesion also occurs in the paranasal sinuses, especially the maxillary and ethmoid sinuses.2 Unlike other malignancies with which HPV types 16 and 18 drive a majority of the malignant diagnoses, HPV type 33 is seen in approximately two-thirds of cases, although other types, including HPV type 16, have been reported.2,3 This HPV association is in contrast to salivary gland tumors of the major glands that show no association with HPV.1

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

Neonatal hydrocephalus presents with various degrees of neuroinflammation and long-term neurological deficits in surgically treated patients, provoking a need for additional medical treatment. We previously reported elevated neuroinflammation and severe periventricular white matter damage in the progressive hydrocephalus (prh) mutant which contains a point mutation in the Ccdc39 gene, causing loss of cilia-mediated unidirectional cerebrospinal fluid (CSF) flow. In this study, we identified cortical neuropil maturation defects such as impaired excitatory synapse maturation and loss of homeostatic microglia, and swimming locomotor defects in early postnatal prh mutant mice. Strikingly, systemic application of the anti-inflammatory small molecule bindarit significantly supports healthy postnatal cerebral cortical development in the prh mutant. While bindarit only mildly reduced the ventricular volume, it significantly improved the edematous appearance and myelination of the corpus callosum. Moreover, the treatment attenuated thinning in cortical layers II-IV, excitatory synapse formation, and interneuron morphogenesis, by supporting the ramified-shaped homeostatic microglia from excessive cell death. Also, the therapeutic effect led to the alleviation of a spastic locomotor phenotype of the mutant. We found that microglia, but not peripheral monocytes, contribute to amoeboid-shaped activated myeloid cells in prh mutants' corpus callosum and the pro-inflammatory cytokines expression. Bindarit blocks NF-kB activation and its downstream pro-inflammatory cytokines, including monocyte chemoattractant protein-1, in the prh mutant. Collectively, we revealed that amelioration of neuroinflammation is crucial for white matter and neuronal maturation in neonatal hydrocephalus. Future studies of bindarit treatment combined with CSF diversion surgery may provide long-term benefits supporting neuronal development in neonatal hydrocephalus.SIGNIFICANCE STATEMENTIn neonatal hydrocephalus, little is known about the signalling cascades of neuroinflammation or the impact of such inflammatory insults on neural cell development within the perinatal cerebral cortex. Here, we report that pro-inflammatory activation of myeloid cells, the majority of which are derived from microglia, impairs periventricular myelination and cortical neuronal maturation using the mouse prh genetic model of neonatal hydrocephalus. Administration of bindarit, an anti-inflammatory small molecule that blocks NF-kB activation, restored the cortical thinning and synaptic maturation defects in the prh mutant brain through suppression of microglial activation. These data indicate the potential therapeutic use of anti-inflammatory reagents targeting neuroinflammation in the treatment of neonatal hydrocephalus.