Odorant Receptor Choice Mechanism Revealed by Analysis of a Highly Represented Odorant Receptor Transgene

SSRN Electronic Journal

2022 May 28

Makhlouf, M;D'Hulst, C;Omura, M;Rosa, A;Mina, R;Bernal-Garcia, S;Lempert, E;Saraiva, L;Feinstein, P;
| DOI: 10.2139/ssrn.4119003

In the mouse, more than 1,100 odorant receptors (ORs) are expressed in a monogenic and monoallelic fashion, referred to as singular gene expression. Using a 21bp singular-choice enhancer (x21), we radically increase representation of olfactory sensory neurons (OSNs) choosing a 5x21 enhanced OR transgene, but not overexpression of its mRNA on a per cell basis. RNA-sequencing and differential expression analysis identified 425 differentially expressed genes (DEGs). ORs make up 86% of all DEGs, of which 325 have decreased representation and 40 have increased representation. Underrepresented ORs include Class I, Class II and TAAR genes and within each of their respective olfactory bulb domains: DI, DII, and DIII (TAAR) we committedly observe multiple homogeneous glomeruli with an OR1A1-identity. The underrepresentation of endogenous, class-specific ORs across evolutionarily distinct cell types in favor of the expression of the 5x21-OR1A1 transgene argues that a common mechanism of singular gene choice is present for all OR-expressing OSNs.

Autonomous sensing of the insulin peptide by an olfactory G protein-coupled receptor modulates glucose metabolism

Cell metabolism

2022 Feb 01

Cheng, J;Yang, Z;Ge, XY;Gao, MX;Meng, R;Xu, X;Zhang, YQ;Li, RZ;Lin, JY;Tian, ZM;Wang, J;Ning, SL;Xu, YF;Yang, F;Gu, JK;Sun, JP;Yu, X;
PMID: 35108512 | DOI: 10.1016/j.cmet.2021.12.022

Along with functionally intact insulin, diabetes-associated insulin peptides are secreted by β cells. By screening the expression and functional characterization of olfactory receptors (ORs) in pancreatic islets, we identified Olfr109 as the receptor that detects insulin peptides. The engagement of one insulin peptide, insB:9-23, with Olfr109 diminished insulin secretion through Gi-cAMP signaling and promoted islet-resident macrophage proliferation through a β cell-macrophage circuit and a β-arrestin-1-mediated CCL2 pathway, as evidenced by β-arrestin-1-/- mouse models. Systemic Olfr109 deficiency or deficiency induced by Pdx1-Cre+/-Olfr109fl/fl specifically alleviated intra-islet inflammatory responses and improved glucose homeostasis in Akita- and high-fat diet (HFD)-fed mice. We further determined the binding mode between insB:9-23 and Olfr109. A pepducin-based Olfr109 antagonist improved glucose homeostasis in diabetic and obese mouse models. Collectively, we found that pancreatic β cells use Olfr109 to autonomously detect self-secreted insulin peptides, and this detection arrests insulin secretion and crosstalks with macrophages to increase intra-islet inflammation.

Innate Type 2 Immunity Controls Hair Follicle Commensalism by Demodex Mites

SSRN Electronic Journal

2022 Jan 22

Ricardo-Gonzalez, R;Kotas, M;O'Leary, C;Tenvooren, I;Marquez, D;Singh, K;Damsky, W;Schroeder, A;Cohen, J;Fassett, M;Lee, J;Daniel, S;Bittinger, K;Díaz, R;Fraser, J;Ansel, M;Spitzer, M;Liang, H;Locksley, R;
| DOI: 10.2139/ssrn.4013912

_Demodex_ mites are obligate commensal parasites of hair follicles (HF) in mammals. Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction and aging, but mechanisms restricting _Demodex_ outgrowth and pathogenesis are not defined. Here, we show that control over mite HF colonization of mice requires ILC2s, IL-13, and its receptor IL-4Ra, but not IL-4 or the adaptive immune system. Epithelial HF-associated ILC2s elaborate IL-13 that attenuates HF and epithelial cell proliferation at anagen onset; in their absence, _Demodex_ colonization leads to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammatory programs leading to loss of barrier function and premature HF exhaustion over time. Humans with rhinophymatous acne rosacea, a nasal inflammatory condition associated with a high burden of _Demodex_, had increased HF inflammatory cells with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a critical role for skin ILC2s and IL-13, which comprise an immune checkpoint necessary to sustain cutaneous integrity and restrict pathologic infestation by colonizing HF mites.

Hemophilia A Gene Therapy: Current and Next-Generation Approaches

Expert opinion on biological therapy

2021 Nov 16

Pipe, SW;Gonen-Yaacovi, G;Segurado, OG;
PMID: 34781798 | DOI: 10.1080/14712598.2022.2002842

: Hemophilia comprises a group of X-linked hemorrhagic disorders that result from a deficiency of coagulation factors. The disorder affects mainly males and leads to chronic pain, joint deformity, reduced mobility, and increased mortality. Current therapies require frequent administration of replacement clotting factors, but the emergence of alloantibodies (inhibitors) diminishes their efficacy. New therapies are being developed to produce the deficient clotting factors and prevent the emergence of inhibitors.: This article provides an update on the characteristics and disease pathophysiology of hemophilia A, as well as current treatments, with a special focus on ongoing clinical trials related to gene replacement therapies.: Gene replacement therapies provide safe, durable, and stable transgene expression while avoiding the challenges of clotting factor replacement therapies in patients with hemophilia. Improving the specificity of the viral construct and decreasing the therapeutic dose are critical toward minimizing cellular stress, induction of the unfolded protein response, and the resulting loss of protein production in liver cells. Next-generation gene therapies incorporating chimeric DNA sequences in the transgene can increase clotting factor synthesis and secretion, and advance the efficacy, safety, and durability of gene replacement therapy for hemophilia A as well as other blood clotting disorders.

Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons

Nature neuroscience

2021 Dec 20

Yang, NJ;Isensee, J;Neel, DV;Quadros, AU;Zhang, HB;Lauzadis, J;Liu, SM;Shiers, S;Belu, A;Palan, S;Marlin, S;Maignel, J;Kennedy-Curran, A;Tong, VS;Moayeri, M;Röderer, P;Nitzsche, A;Lu, M;Pentelute, BL;Brüstle, O;Tripathi, V;Foster, KA;Price, TJ;Collier, RJ;Leppla, SH;Puopolo, M;Bean, BP;Cunha, TM;Hucho, T;Chiu, IM;
PMID: 34931070 | DOI: 10.1038/s41593-021-00973-8

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions

Neuron

2021 Dec 15

Liu, S;Ye, M;Pao, GM;Song, SM;Jhang, J;Jiang, H;Kim, JH;Kang, SJ;Kim, DI;Han, S;
PMID: 34921781 | DOI: 10.1016/j.neuron.2021.11.029

Breathing can be heavily influenced by pain or internal emotional states, but the neural circuitry underlying this tight coordination is unknown. Here we report that Oprm1 (μ-opioid receptor)-expressing neurons in the lateral parabrachial nucleus (PBL) are crucial for coordinating breathing with affective pain in mice. Individual PBLOprm1 neuronal activity synchronizes with breathing rhythm and responds to noxious stimuli. Manipulating PBLOprm1 activity directly changes breathing rate, affective pain perception, and anxiety. Furthermore, PBLOprm1 neurons constitute two distinct subpopulations in a "core-shell" configuration that divergently projects to the forebrain and hindbrain. Through non-overlapping projections to the central amygdala and pre-Bötzinger complex, these two subpopulations differentially regulate breathing, affective pain, and negative emotions. Moreover, these subsets form recurrent excitatory networks through reciprocal glutamatergic projections. Together, our data define the divergent parabrachial opioidergic circuits as a common neural substrate that coordinates breathing with various sensations and behaviors such as pain and emotional processing.

Reagents and models for detecting endogenous GLP1R and GIPR

EBioMedicine

2021 Dec 01

Ast, J;Broichhagen, J;Hodson, DJ;
PMID: 34911028 | DOI: 10.1016/j.ebiom.2021.103739

Glucagon-like peptide-1 receptor (GLP1R) agonists target the GLP1R, whereas dual GLP1R/ gastric inhibitory polypeptide receptor (GIPR) agonists target both the GLP1R and GIPR. Despite the importance of these drug classes for the treatment of diabetes and obesity, still very little is known about the localization of GLP1R and GIPR themselves. Complicating matters is the low abundance of GLP1R and GIPR mRNA/protein, as well as a lack of specific and validated reagents for their detection. Without knowing where GLP1R and GIPR are located, it is difficult to propose mechanisms of action in the various target organs, and whether this is indirect or direct. In the current review, we will explain the steps needed to properly validate reagents for endogenous GLP1R/GIPR detection, describe the available approaches to visualize GLP1R/GIPR, and provide an update on the state-of-art. The overall aim is to provide a reference resource for researchers interested in GLP1R and GIPR signaling.

Baiting out a full length sequence from unmapped RNA-seq data

BMC genomics

2021 Nov 27

Li, D;Huang, Q;Huang, L;Wen, J;Luo, J;Li, Q;Peng, Y;Zhang, Y;
PMID: 34837950 | DOI: 10.1186/s12864-021-08146-4

As a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored.We develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure.We believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.

A Multi-Omics Atlas of the Human Retina at Single-Cell Resolution

SSRN Electronic Journal

2021 Dec 22

Liang, Q;Cheng, X;Wang, J;Owen, L;Shakoor, A;Lillvis, J;Zhang, C;Farkas, M;Kim, I;Li, Y;DeAngelis, M;Chen, R;
| DOI: 10.2139/ssrn.3991078

Cell types in the human retina are highly heterogeneous with their abundance varying by several orders of magnitude. Here, we generated a multi-omics single-cell atlas of the adult human retina, including over 250K and 150K nuclei for single-nuclei RNA- and ATAC-seq, respectively. This atlas is highly comprehensive, with over 60 distinct cell types identified, achieving a sensitivity of 0.01%. Cross species comparison of the retina atlas among human, monkey, and mice revealed increasing divergences in transcriptomic profiles and cell types toward the downstream layer of the neural circuitry. Interestingly, the overall cell heterogeneity in primate retina decreases compared to that of rodent retina. Furthermore, integrative analysis identified 70k distal cis-element-gene pairs with a large portion being cell type specific. Finally, when combined with eQTLs from bulk retina profiling, the multi-omics cell atlas enables systematic identification of candidate causal variants for a targeted gene along with cell type context information. Taken together, we present a comprehensive single-cell multi-omics atlas for the human retina that enables systematic in-depth molecular characterization of individual cell subtypes.

HPV-Positive and HPV-Negative Vulvar Squamous Cell Carcinoma Are Biologically, but Not Clinically, Distinct

Journal of Investigative Dermatology

2021 Oct 01

Kolitz, E;Lucas, E;Hosler, G;Kim, J;Hammer, S;Lewis, C;Xu, L;Day, A;Mauskar, M;Lea, J;Wang, R;
| DOI: 10.1016/j.jid.2021.10.009

Vulvar squamous cell carcinoma (VSCC) pathogenesis is traditionally defined by the presence or absence of human papillomavirus (HPV), but the definition of these groups and their molecular characteristics remains ambiguous across studies. Here, we present a retrospective cohort analysis of 36 patients with invasive VSCC where HPV status was determined using RNA in situ hybridization (ISH) and polymerase chain reaction (PCR). Clinical annotation, p16 immunohistochemistry (IHC), programmed death ligand-1 (PD-L1) IHC, HPV16 circular E7 RNA (circE7) detection, and RNA-sequencing (RNA-seq) of the cases was performed. A combination of ISH and PCR identified 20 cases (55.6%) as HPV-positive. HPV-status did not impact overall survival (HR: 1.36, 95% CI: 0.307 to 6.037, p=0.6857) or progression-free survival (HR: 1.12, 95% CI: 0.388 to 3.22, p=0.8367), and no significant clinical differences were found between the groups. PD-L1 expression did not correlate with HPV status, but increased expression of PD-L1 correlated with worse overall survival. Transcriptomic analyses (n=23) revealed distinct groups, defined by HPV status, with multiple differentially expressed genes previously implicated in HPV-induced cancers. HPV-positive tumors showed higher global expression of endogenous circular RNAs (circRNAs), including several circRNAs that have previously been implicated in the pathogenesis of other cancers.

Structural evidence for visual arrestin priming via complexation of phosphoinositols

Structure (London, England : 1993)

2021 Oct 21

Sander, CL;Luu, J;Kim, K;Furkert, D;Jang, K;Reichenwallner, J;Kang, M;Lee, HJ;Eger, BT;Choe, HW;Fiedler, D;Ernst, OP;Kim, YJ;Palczewski, K;Kiser, PD;
PMID: 34678158 | DOI: 10.1016/j.str.2021.10.002

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail.

Microglia and BDNF at the crossroads of stressor related disorders: Towards a unique trophic phenotype

Neuroscience and biobehavioral reviews

2021 Sep 16

Prowse, N;Hayley, S;
PMID: 34537262 | DOI: 10.1016/j.neubiorev.2021.09.018

Stressors ranging from psychogenic/social to neurogenic/injury to systemic/microbial can impact microglial inflammatory processes, but less is known regarding their effects on trophic properties of microglia. Recent studies do suggest that microglia can modulate neuronal plasticity, possibly through brain derived neurotrophic factor (BDNF). This is particularly important given the link between BDNF and neuropsychiatric and neurodegenerative pathology. We posit that certain activated states of microglia play a role in maintaining the delicate balance of BDNF release onto neuronal synapses. This focused review will address how different "activators" influence the expression and release of microglial BDNF and address the question of tropomyosin receptor kinase B (TrkB) expression on microglia. We will then assess sex-based differences in microglial function and BDNF expression, and how microglia are involved in the stress response and related disorders such as depression. Drawing on research from a variety of other disorders, we will highlight challenges and opportunities for modulators that can shift microglia to a "trophic" phenotype with a view to potential therapeutics relevant for stressor-related disorders.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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