Probes for INS

Background: Mucus buildup in multiple mucin-producing organs, including the lungs and the intestine, is the hallmark of CF. In the gastrointestinal (GI) tract, aberrant mucus properties may play a critical role in bowel obstruction, inflammation, and bacterial overgrowth, as well as reduced nutrient absorption. Interestingly, all animal models of CF (e.g., mice, rats, ferrets, pigs) experience gastrointestinal complications, with poor growth and intestinal obstruction, as a result of excessive mucus accumulation. However, the precise biochemical change(s) within the CF gut mucus is still undetermined. Disrupted transepithelial Cl− and HCO3 − secretions due to CFTR malfunction affect the viscoelastic properties of airway mucus and may affect gut mucus similarly. Using WT and F508delCftr (CF) mice, we explored how interactions between Cftr and Muc2, the dominant GI mucin, resulted in the intestinal obstructive phenotype observed in CF mice. Methods: CF mice were weaned from laxative for 48 h prior to experiments. Intestinal sections (duodenum, jejunum, ileum, and colon) from WT and F508del-cftr CF mice were analyzed using H&E and AB-PAS staining to examine tissue structure, cell morphology, and mucus accumulation. RNAScope and qRT-PCR were used to determine Muc2 and Cftr mRNA expression along the proximal-to-distal and crypt-villus axes of the murine gut. IHC was used to determine the mucin compositions of gut sections, and fluorescent in-situ hybridization (FISH) was used to observe bacterial penetration of the mucus layer in the mouse intestine. Comparing WT and CF mice, we measured % solids of the luminal content and relative changes in gut mucus concentration via Western blotting on intestinal lavages. Results: Gross pathology revealed that intestinal blockage frequently emerged from the distal ileum. In parallel, we demonstrated that Muc2 expression increased from proximal to distal, while Cftr expression remained uniform throughout the GI tract of WT animals. However, Cftr expression changed gradually along the crypt-villus axis, with a homogenous signal distribution in the duodenum that progressively concentrated to the crypts in the ileum. FISH confirmed that a layer of mucus devoid of bacteria protected the WT murine epithelium from pathogen invasion. Histological and IHC examinations of obstructed ileal regions revealed that distal regions of intestinal plugs were mainly composed of mucus and inflammatory cells, while more proximal regions of the blockages were contaminated by feces and other luminal debris. Luminal content % solids was significantly increased in the CF (32.7%) compared to the WT ileum (22.3%), with concentrations approaching that of the WT colon (37.3%). Western blot analysis of intestinal lavages revealed that highmolecular-weight Muc2 was more concentrated (fold increase ∼2) in the small intestine of CF compared to WT mice. Conclusion: In CF mice, elevated mucus concentrations are caused by the combination of high Muc2 expression and lack of fluid secretion due to defective Cftr protein in the distal ileum, which creates optimal conditions for bowel obstruction in this particular region. Unlike the colon, the ileum is not adapted to high friction and is prone to villi sloughing, further exacerbating the gut phenotype in CF animals.

The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) of the rodent thalamus process various signals and participate in circadian entrainment In both structures, cells exhibiting infra-slow oscillatory activity as well as non-rhythmically firing neurons can be observed Here, we reveal that only one of these two groups of cells responds to anorexigenic (CCK, GLP-1 and OXM) and orexigenic (Ghrl and OXA) peptides. Neuronal responses vary depending on the time of day (day vs. night) and on the diet (standard vs. high-fat diet) Additionally, we visualized receptors to the tested peptides in the IGL/VLG using in situ hybridisation Our results suggest that two electrophysiologically different subpopulations of IGL/VLG neurons are involved in two separate functions: the one related to body's energy homeostasis and the one associated with the subcortical visual system ABSTRACT: The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) are subcortical structures involved in entrainment of the brain's circadian system to photic and non-photic (e.g. metabolic and arousal) cues. Both receive information about environmental light from photoreceptors, exhibit an infra-slow oscillations (ISO) in vivo, and connect to the master circadian clock. Although current evidence demonstrates that the IGL/VLG communicate metabolic information and are crucial for entrainment of circadian rhythms to time-restricted feeding, their sensitivity to food-intake-related peptides has not been investigated yet. We examined the effect of metabolically relevant peptides on the spontaneous activity of IGL/VLG neurons. Using ex vivo and in vivo electrophysiological recordings as well as in situ hybridisation, we tested potential sensitivity of the IGL/VLG to anorexigenic and orexigenic peptides, such as cholecystokinin, glucagon-like peptide 1, oxyntomodulin, peptide YY, orexin A, and ghrelin. We explored neuronal responses to these drugs during day and night, and in standard vs. high-fat diet conditions. We found that IGL/VLG neurons response to all the substances tested, except peptide YY. Moreover, more neurons responded to anorexigenic drugs at night, while a high-fat diet affected the IGL/VLG sensitivity to orexigenic peptides. Interestingly, ISO neurons responded to light and orexin A, but did not respond to the other food-intake-related peptides.. In contrast, non-ISO cells were activated by metabolic peptides, with only some being responsive to light. Our results show for the first time that peptides involved in the body's energy homeostasis stimulate the thalamus and suggest functional separation of the IGL/VLG cells. Abstract figure legend (1) Sprague Dawley rats were fed ad libitum with control (CD) or a high-fat (HFD) chow in a 12:12 h light-dark cycle. (2) The subject of the study was a thalamic intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (VLG) showing spontaneous infra-slow oscillatory (ISO) or nonâ¿¿oscillatory (nonâ¿¿ISO) activity. (3) Neuronal activity of the IGL and VLG was recorded using ex vivo and in vivo electrophysiological techniques. (4) Anorexigenic (in green) and orexigenic (in red) peptides such as cholecystokinin (CCK), glucagon-like peptide 1 (GLPâ¿¿1), oxyntomodulin (OXM), peptide YY (pYY), orexin A (OXA) and ghrelin (Ghrl) were administered during IGL/VLG recordings. (5) We found that non-ISO neurons of the IGL/VLG responded with an increase in firing rate to all the substances tested, except peptide YY. The amplitude (marked with arrows) and frequency of responses (marked with <, >) varied depending on the diet and the phase of the day. (6) In situ hybridization was performed on IGL/VLGâ¿¿containing brain sections to visualise receptors to the tested peptides. This article is protected by

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Xuming Zhu is first author on ‘ FZD2 regulates limb development by mediating β-catenin-dependent and -independent Wnt signaling pathways’, published in DMM. Xuming is an instructor in the lab of Sarah E. Millar at Icahn School of Medicine at Mount Sinai, New York, NY, USA, investigating the molecular mechanisms that underlie the development of appendages, epithelial homeostasis and diseases.

Mike May, is a freelance writer and editor with more than 30 years of experience. He earned an MS in biological engineering from the University of Connecticut and a PhD in neurobiology and behavior from Cornell University. He worked as an associate editor at American Scientist, and he is the author of more than 1,000 articles for clients that include GEN, Nature, Science, Scientific American and many others. In addition, he served as the editorial director of many publications, including several Nature Outlooks and Scientific American Worldview.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). 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, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. 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, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. 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.

Neuropathic pain is amongst the most common non-communicable disorders and the poor effectiveness of current treatment is an unmet need. Although pain is a universal experience, there are significant inter-individual phenotypic differences. Developing models that can accurately recapitulate the clinical pain features is crucial to better understand underlying pathophysiological mechanisms and find innovative treatments. Current data from heterologous expression systems that investigate properties of specific molecules involved in pain signaling, and from animal models, show limited success with their translation into the development of novel treatments for pain. This is in part because they do not recapitulate the native environment in which a particular molecule functions, and due to species-specific differences in the properties of several key molecules that are involved in pain signaling. The limited availability of post-mortem tissue, in particular dorsal root ganglia (DRG), has hampered research using human cells in pre-clinical studies. Human induced-pluripotent stem cells (iPSCs) have emerged as an exciting alternative platform to study patient-specific diseases. Sensory neurons that are derived from iPSCs (iPSC-SNs) have provided new avenues towards elucidating peripheral pathophysiological mechanisms, the potential for development of personalized treatments, and as a cell-based system for high-throughput screening for discovering novel analgesics. Nevertheless, reprogramming and differentiation protocols to obtain nociceptors have mostly yielded immature homogenous cell populations that do not recapitulate the heterogeneity of native sensory neurons. To close the gap between native human tissue and iPSCs, alternative strategies have been developed. We will review here recent developments in differentiating iPSC-SNs and their use in pre-clinical translational studies. Direct conversion of stem cells into the cells of interest has provided a more cost- and time-saving method to improve reproducibility and diversity of sensory cell types. Furthermore, multi-cellular strategies that mimic in vivo microenvironments for cell maturation, by improving cell contact and communication (co-cultures), reproducing the organ complexity and architecture (three-dimensional organoid), and providing iPSCs with the full spatiotemporal context and nutrients needed for acquiring a mature phenotype (xenotransplantation), have led to functional sensory neuron-like systems. Finally, this review touches on novel prospective strategies, including fluorescent-tracking to select the differentiated neurons of relevance, and dynamic clamp, an electrophysiological method that allows direct manipulation of ionic conductances that are missing in iPSC-SNs.

The milestone achievement of reprogramming a human somatic cell into a pluripotent stem cell by Yamanaka and Takahashi in 2007 has changed the stem cell research landscape tremendously. Their discovery opened the unprecedented opportunity to work with human-induced pluripotent stem cells and the differentiated progeny thereof, without major ethical restrictions. Additionally, the new method offers the possibility to generate pluripotent stem cells from patients with various genetic diseases which is of great importance (a) to understand the basic mechanisms of a specific disease in a human cellular context and (b) to help find suitable therapies for the persons concerned. In individual cases, this can even help to develop personalized treatment options. Chronic pain is a disease that affects roughly one in five people worldwide, but its onset is rarely based upon genetic alterations. Nevertheless, the work with sensory-like neurons derived from human pluripotent stem cells has become a more widely used tool also in the field of pain research, as during the past years several differentiation procedures have been published that describe the generation of different types of sensory-like neurons and their useful contribution to studying mechanisms of sensitization. Especially also to complement and verify cellular and molecular mechanisms identified in rodent model systems, the model of choice for decades. Although a sole cellular system is not able to mimic a disease as complex as pain, it is a valid tool to understand basic mechanisms of sensitization in specific subsets of human neurons that might be at the onset of the disease. In addition, the creativity of basic researchers and the more and more advanced available technologies will most likely find ways to implement the derived human cells in more complex networks. In this chapter, I want to introduce a selection of published differentiation strategies that result in the generation of human sensory-like neurons. Additionally, I will point out some studies whose results helped to further understand pain-related mechanisms and which were conducted using the aforementioned differentiation procedures.

Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown that this activity is greatly influenced by local GABAergic signaling primarily due to somatostatin-expressing GABAergic neurons (SST). To further understand the network dynamics associated with gastric motility control in the DVC, we focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of these neurons on gastric motility remains unknown. Here we investigate the anatomical and functional characteristics of the NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both sexes. We sought to determine if NPY neurons influence the activity of gastric projecting neurons, synaptically interact with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in vivo show that NPY neurons: are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from the gastric antrum; are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum projecting neurons; are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a potent inhibitory effect on the action potential firing of the NPY neurons; and affect gastric tone and motility as reflected by their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the network that controls vagal transmission to the stomach.Significance StatementThe brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach via tone and motility. Two distinct non-overlapping populations of predominantly excitatory neuropeptide Y (NPY) neurons and predominantly inhibitory somatostatin (SST) neurons form reciprocal connections with each other in the nucleus of the tractus solitarius (NTS) and with premotor neurons in the dorsal motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS. NPY neurons increased and decreased gastric motility, respectively, while both activation and inhibition of NTS SST neurons enhanced gastric motility.

The lateral hypothalamic area (LHA) is a highly conserved brain region critical for maintaining physiological homeostasis and goal-directed behavior. LHA neurons that express melanin-concentrating hormone (MCH) are key regulators of arousal, energy balance and motivated behavior. However, cellular and functional diversity among LHAMCH neurons is not well understood. Previous anatomical and molecular data suggest that LHAMCH neurons may be parsed into at least two distinct subpopulations, one of which is enriched in neurokinin-3 receptor (NK3R), the receptor for neurokinin B (NKB), encoded by the Tac2 gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory and stress in other brain regions, but NKB interactions with LHAMCH neurons is poorly understood. We first identified how LHAMCH subpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHAMCH neurons, we used Cre-dependent retrograde and anterograde viral tracing in male Tac2-Cre mice and identified Tac2/EYFP+ neurons in the bed nucleus of the stria terminalis (BNST) and central nucleus of the amygdala (CeA), the central extended amygdala, as major sources of NKB input onto LHAMCH neurons. In addition to innervating the LHA, these limbic forebrain NKB neurons also project to midbrain and brainstem targets. Finally, using a dual virus approach, we found that optogenetic activation of these inputs in slices evokes GABA release onto a subset of LHAMCH neurons, but lacked specificity for the NK3R+ subpopulation. Overall, these data define parallel tachykininergic/GABAergic limbic forebrain projections that are positioned to modulate multiple nodes of homeostatic and behavioral control.SIGNIFICANCE STATEMENTThe lateral hypothalamic area (LHA) orchestrates fundamental behavioral states in the mammalian hypothalamus, including arousal, energy balance, memory, stress and motivated behavior. The neuropeptide melanin-concentrating hormone (MCH) defines one prominent population of LHA neurons, with multiple roles in the regulation of homeostatic behavior. Outstanding questions remain concerning the upstream inputs that control MCH neurons. We sought to define neurochemically distinct pathways in the mouse brain that may communicate with specific MCH neuron subpopulations using viral-based retrograde and anterograde neural pathway tracing and optogenetics in brain slices. Here we identify a specific neuropeptide-defined forebrain circuit that makes functional synaptic connections with MCH neuron subpopulations. This work lays the foundation for further manipulating molecularly distinct neural circuits that modulate innate behavioral states.

Background: Pseudomonas aeruginosa, the predominant pathogen in chronic lung infection of adults with cystic fibrosis (CF), possesses a number of mechanisms that contribute to antimicrobial resistance (AMR). Even aggressive antibiotic treatment is unable to effectively clear chronic P. aeruginosa, which may partially be due to the rapid diversification displayed by P. aeruginosa during long-term CF infection, but our knowledge of the role of population heterogeneity on AMR is limited, because prior studies have undersampled P. aeruginosa isolates in CF lungs. Specifically, the role of evolutionary trade-offs on AMR in these patients has been overlooked. We propose that P. aeruginosa trades off between AMR, surface attachment, and growth rate to sustain diverse populations in the CF lung. Methods: We sampled 75 P. aeruginosa isolates from expectorated sputum samples of 4 adults with CF chronically infected with P. aeruginosa (n = 300) and tested each for growth rate in lysogeny broth and synthetic CF sputum media, susceptibility profiles to 6 antibiotics commonly prescribed to CF patients, and surface attachment to assess the role of population heterogeneity on AMR. Results: We found significant within-patient heterogeneity in AMR across all patients and antibiotics. The majority of isolates were well within the range of susceptibility for the tested antibiotics, despite ineffective clearing of P. aeruginosa infection for each of these patients. One patient harbored isolates that grew better in the presence of tobramycin. This patient showed evidence of trade-offs between surface attachment and AMR, whereas the other 3 did not. There was some evidence of within-patient trade-offs between AMR and growth rate, but these relationships were not found to be consistent across patients. Conclusion: Overall, our results demonstrate that in vitro susceptibility testing is not representative of in situ AMR levels; further work is needed to address this. Furthermore, we found weak evidence of evolutionary tradeoffs as a driver of heterogeneity in AMR in diverse P. aeruginosa populations sourced from the CF lung, although this may mean that these trade-offs exist at below detectable levels.

Background: Culture-independent microbiota analysis has permitted comprehensive investigation of bacterial diversity in cystic fibrosis (CF) lung infections and is being increasingly used to examine fungal communities. The prevalence and clinical impact of fungi in CF is relatively poorly understood, with studies largely focused on adults. We investigated fungal diversity in children with CF aged 1 to 18 years using bronchoalveolar lavage (BAL) and induced-sputum (IS) samples to capture multiple niches within the lung. Methods: Sequencing and analysis of the fungal ITS2 region was performed on 22 matched sets of BAL-IS samples collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). Each set comprised 4 samples: BAL1 (right middle lobe), BAL2 (left lingular lobe), BAL3 (pooled right and left lower and upper lobes), and IS. Bioinformatic analysis was performed in QIIME2, with downstream analysis using R statistical software (R packages phyloseq and vegan). Fungal community diversity and composition were evaluated at the genus level for each individual and the different sampling types. Results: All 88 samples (22 individuals) had evidence of fungi, and 370 fungal genera were identified across the dataset. The fungal diversity (Shannon index) captured in BAL was not significantly different from that captured in IS, and all 4 sampling types overlapped in mycobiome composition. A core group of 29 genera were identified across all BAL and IS samples, with Candida, Aspergillus, Dipodascus, Simplicillium, and Lecanicillium being the most prevalent and abundant. Candida was found at a higher average relative abundance in IS samples (30%) than in BAL samples (10%). Co-occurrence network analysis showed variable interactions between fungal genera, with positive and negative interactions identified irrespective of sample type. At the individual patient level, there was evidence of both concordance and dissimilarity between the fungal community profiles captured by BAL1, BAL2, BAL3, and IS, indicating that compartmentalization of the lung mycobiome can occur. Although this cross-sectional dataset was limited, there were also trends for greater Candida, Aspergillus, and Exophiala relative abundance and decreasing fungal diversity with increasing age. Conclusion: This study has shown that the mycobiome in pediatric CF samples is diverse and complex. There was overlap between the fungal communities identified in BAL and IS samples, suggesting that IS can capture fungal genera associated with the lower airway. The data show that IS is suitable for large-scale studies to relate clinical outcome to individual mycobiome heterogeneity

Peters plus syndrome, characterized by defects in eye and skeletal development with isolated cases of ventriculomegaly/hydrocephalus, is caused by mutations in the β3-glucosyltransferase (B3GLCT) gene. In the endoplasmic reticulum, B3GLCT adds glucose to O-linked fucose on properly folded Thrombospondin Type 1 Repeats (TSRs). The resulting glucose-fucose disaccharide is proposed to stabilize the TSR fold and promote secretion of B3GLCT substrates, with some substrates more sensitive than others to loss of glucose. Mouse B3glct mutants develop hydrocephalus at high frequency. In this study, we demonstrated that B3glct mutant ependymal cells had fewer cilia basal bodies and altered translational polarity compared to controls. Localization of mRNA encoding A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 20 (ADAMTS20) and ADAMTS9, suggested that reduced function of these B3GLCT substrates contributed to ependymal cell abnormalities. In addition, we showed that multiple B3GLCT substrates (Adamts3, Adamts9, and Adamts20) are expressed by the subcommissural organ, that subcommissural organ-spondin (SSPO) TSRs were modified with O-linked glucose-fucose, and that loss of B3GLCT reduced secretion of SSPO in cultured cells. In the B3glct mutant subcommissural organ intracellular SSPO levels were reduced and BiP levels increased, suggesting a folding defect. Secreted SSPO colocalized with BiP, raising the possibility that abnormal extracellular assembly of SSPO into Reissner's fiber also contributed to impaired CSF flow in mutants. Combined, these studies underscore the complexity of the B3glct mutant hydrocephalus phenotype and demonstrate that impaired cerebrospinal fluid (CSF) flow likely stems from the collective effects of the mutation on multiple processes.