mRNA distribution in skeletal muscle is associated with mRNA size

Journal of cell science

2021 Jun 24

Pinheiro, H;Pimentel, MR;Sequeira, C;Oliveira, LM;Pezzarossa, A;Roman, W;Gomes, ER;
PMID: 34164679 | DOI: 10.1242/jcs.256388

Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs is enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins - giant mRNAs - are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.

IL-1R1-dependent signaling coordinates epithelial regeneration in response to intestinal damage

Science immunology

2021 May 07

Cox, CB;Storm, EE;Kapoor, VN;Chavarria-Smith, J;Lin, DL;Wang, L;Li, Y;Kljavin, N;Ota, N;Bainbridge, TW;Anderson, K;Roose-Girma, M;Warming, S;Arron, JR;Turley, SJ;de Sauvage, FJ;van Lookeren Campagne, M;
PMID: 33963061 | DOI: 10.1126/sciimmunol.abe8856

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1+ mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1+ mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function.

Single-cell dissection of cellular components and interactions shaping the tumor immune phenotypes in ovarian cancer

Cancer cell

2021 Apr 27

Hornburg, M;Desbois, M;Lu, S;Guan, Y;Lo, AA;Kaufman, S;Elrod, A;Lotstein, A;DesRochers, TM;Munoz-Rodriguez, JL;Wang, X;Giltnane, J;Mayba, O;Turley, SJ;Bourgon, R;Daemen, A;Wang, Y;
PMID: 33961783 | DOI: 10.1016/j.ccell.2021.04.004

Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.

The role of EBV in hematolymphoid proliferations: Emerging concepts relevant to diagnosis and treatment

Histopathology

2021 Apr 07

Ababneh, E;Saad, AM;Crane, GM;
PMID: 33829526 | DOI: 10.1111/his.14379

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus with >90% of the adult population worldwide harboring latent infection. A small subset of those infected develop EBV-associated neoplasms including a range of lymphoproliferative disorders (LPD). The diagnostic distinction of these entities appears increasingly relevant as our understanding of EBV-host interactions and mechanisms of EBV-driven lymphomagenesis improves. EBV may lower the mutational threshold for malignant transformation, create potential vulnerabilities related to viral alteration of cell metabolism and allow for improved immune targeting. However, these tumors may escape immune surveillance by affecting their immune microenvironment, limiting viral gene expression or potential loss of the viral episome. Methods to manipulate the latency state of the virus to enhance immunogenicity are emerging as well as the potential to detect so-called "hit and run" cases where EBV has been lost. Finally, measurement of EBV DNA remains an important biomarker for screening and monitoring of LPD. Methods to distinguish EBV DNA derived from virions during lytic activation from latent, methylated EBV DNA present in EBV-associated neoplasms may broaden the utility of this testing, particularly in patients with compromised immune function. We will highlight some of these emerging areas relevant to the diagnosis and treatment of EBV-associated LPD with potential applicability to other EBV-associated neoplasms. This article is protected by

Top-Down Control of Sweet and Bitter Taste in the Mammalian Brain

Cell

2021 Jan 07

Jin, H;Fishman, ZH;Ye, M;Wang, L;Zuker, CS;
PMID: 33417862 | DOI: 10.1016/j.cell.2020.12.014

Hardwired circuits encoding innate responses have emerged as an essential feature of the mammalian brain. Sweet and bitter evoke opposing predetermined behaviors. Sweet drives appetitive responses and consumption of energy-rich food sources, whereas bitter prevents ingestion of toxic chemicals. Here we identified and characterized the neurons in the brainstem that transmit sweet and bitter signals from the tongue to the cortex. Next we examined how the brain modulates this hardwired circuit to control taste behaviors. We dissect the basis for bitter-evoked suppression of sweet taste and show that the taste cortex and amygdala exert strong positive and negative feedback onto incoming bitter and sweet signals in the brainstem. Finally we demonstrate that blocking the feedback markedly alters responses to ethologically relevant taste stimuli. These results illustrate how hardwired circuits can be finely regulated by top-down control and reveal the neural basis of an indispensable behavioral response for all animals.

Quantitative Analysis of Inflammatory Uterine Lesions of Pregnant Gilts with Digital Image Analysis Following Experimental PRRSV-1 Infection

Animals : an open access journal from MDPI

2023 Feb 24

Horváth, DG;Abonyi-Tóth, Z;Papp, M;Szász, AM;Rümenapf, T;Knecht, C;Kreutzmann, H;Ladinig, A;Balka, G;
PMID: 36899686 | DOI: 10.3390/ani13050830

Reproductive disorders caused by porcine reproductive and respiratory syndrome virus-1 are not yet fully characterized. We report QuPath-based digital image analysis to count inflammatory cells in 141 routinely, and 35 CD163 immunohistochemically stained endometrial slides of vaccinated or unvaccinated pregnant gilts inoculated with a high or low virulent PRRSV-1 strain. To illustrate the superior statistical feasibility of the numerical data determined by digital cell counting, we defined the association between the number of these cells and endometrial, placental, and fetal features. There was strong concordance between the two manual scorers. Distributions of total cell counts and endometrial and placental qPCR results differed significantly between examiner1's endometritis grades. Total counts' distribution differed significantly between groups, except for the two unvaccinated. Higher vasculitis scores were associated with higher endometritis scores, and higher total cell counts were expected with high vasculitis/endometritis scores. Cell number thresholds of endometritis grades were determined. A significant correlation between fetal weights and total counts was shown in unvaccinated groups, and a significant positive correlation was found between these counts and endometrial qPCR results. We revealed significant negative correlations between CD163+ counts and qPCR results of the unvaccinated group infected with the highly virulent strain. Digital image analysis was efficiently applied to assess endometrial inflammation objectively.

Evaluation of the Antiseizure Activity of Endemic Plant Halfordia kendack Guillaumin and Its Main Constituent, Halfordin, on a Zebrafish Pentylenetetrazole (PTZ)-Induced Seizure Model

International journal of molecular sciences

2023 Jan 30

Skiba, A;Kozioł, E;Luca, SV;Budzyńska, B;Podlasz, P;Van Der Ent, W;Shojaeinia, E;Esguerra, CV;Nour, M;Marcourt, L;Wolfender, JL;Skalicka-Woźniak, K;
PMID: 36768918 | DOI: 10.3390/ijms24032598

Epilepsy is a neurological disease that burdens over 50 million people worldwide. Despite the considerable number of available antiseizure medications, it is estimated that around 30% of patients still do not respond to available treatment. Herbal medicines represent a promising source of new antiseizure drugs. This study aimed to identify new drug lead candidates with antiseizure activity from endemic plants of New Caledonia. The crude methanolic leaf extract of Halfordia kendack Guillaumin (Rutaceae) significantly decreased (75 μg/mL and 100 μg/mL) seizure-like behaviour compared to sodium valproate in a zebrafish pentylenetetrazole (PTZ)-induced acute seizure model. The main coumarin compound, halfordin, was subsequently isolated by liquid-liquid chromatography and subjected to locomotor, local field potential (LFP), and gene expression assays. Halfordin (20 μM) significantly decreased convulsive-like behaviour in the locomotor and LFP analysis (by 41.4% and 60%, respectively) and significantly modulated galn, and penka gene expression.

Nucleic Acid Pharmaceutical Agents

Haschek and Rousseaux's Handbook of Toxicologic Pathology, Volume 2 : Safety Assessment Environmental Toxicologic Pathology

2023 Feb 27

Kohnken, R;Harbison, C;Klein, S;Engelhardt, J;
| DOI: 10.1016/B978-0-12-821047-5.00017-8

Nucleic acid pharmaceutical (NAP) agents are a relatively recent class of therapeutics that are uniquely capable of inhibiting protein translation through direct interaction with RNA. These classes of pharmaceuticals have demonstrated clinical benefit for diseases previously considered untreatable by small molecules and biologics by their theoretical ability to target any cellular RNA associated with disease. This chapter provides an overview of the major types of NAPs and the types of indications for which they are being developed, with examples of therapeutics on the market and in clinical development. The reader is also provided with a review of the most common delivery systems and chemical modifications that enable the biodistribution and efficacy of these drugs. A brief discussion of the nonclinical safety package is provided, and finally the most common toxicities that have been observed in preclinical species are discussed.

Reagents for Mass Cytometry

Chemical reviews

2023 Jan 25

Arnett, LP;Rana, R;Chung, WW;Li, X;Abtahi, M;Majonis, D;Bassan, J;Nitz, M;Winnik, MA;
PMID: 36696538 | DOI: 10.1021/acs.chemrev.2c00350

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

Immunity to the microbiota promotes sensory neuron regeneration

Cell

2023 Jan 06

Enamorado, M;Kulalert, W;Han, SJ;Rao, I;Delaleu, J;Link, VM;Yong, D;Smelkinson, M;Gil, L;Nakajima, S;Linehan, JL;Bouladoux, N;Wlaschin, J;Kabat, J;Kamenyeva, O;Deng, L;Gribonika, I;Chesler, AT;Chiu, IM;Le Pichon, CE;Belkaid, Y;
PMID: 36640762 | DOI: 10.1016/j.cell.2022.12.037

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

Targeting Peripheral μ-opioid Receptors or μ-opioid Receptor-Expressing Neurons Does not Prevent Morphine-induced Mechanical Allodynia and Anti-allodynic Tolerance

Neuroscience bulletin

2023 Jan 09

Du, F;Yin, G;Han, L;Liu, X;Dong, D;Duan, K;Huo, J;Sun, Y;Cheng, L;
PMID: 36622575 | DOI: 10.1007/s12264-022-01009-2

The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity (OIH) and analgesic tolerance. Among the different forms of OIH and tolerance, the opioid receptors and cell types mediating opioid-induced mechanical allodynia and anti-allodynic tolerance remain unresolved. Here we demonstrated that the loss of peripheral μ-opioid receptors (MORs) or MOR-expressing neurons attenuated thermal tolerance, but did not affect the expression and maintenance of morphine-induced mechanical allodynia and anti-allodynic tolerance. To confirm this result, we made dorsal root ganglia-dorsal roots-sagittal spinal cord slice preparations and recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs in superficial dorsal horn neurons. Consistent with the behavioral results, peripheral MOR loss did not prevent the opening of Aβ mechanical allodynia pathways in the spinal dorsal horn. Therefore, the peripheral MOR signaling pathway may not be an optimal target for preventing mechanical OIH and analgesic tolerance. Future studies should focus more on central mechanisms.

Incorporating Molecular Classification When Stratifying the Survival Risk of Patients with High-Grade Endometrial Carcinomas

Journal of clinical medicine

2023 Jan 09

Zong, L;Mo, S;Sun, Z;Lu, Z;Chen, J;Yu, S;Xiang, Y;
PMID: 36675462 | DOI: 10.3390/jcm12020530

Assessing survival risk in patients with high-grade endometrial carcinomas has remained challenging. We aimed to investigate the distribution of molecular subtypes and assess their prognostic role in a large cohort of 355 patients with high-grade endometrial carcinoma. Molecular classification was determined using DNA polymerase epsilon (POLE) sequencing as well as immunohistochemical staining for p53 and mismatch repair (MMR) proteins. Endometrial carcinomas were stratified into four subtypes: POLE ultramutated, MMR-deficient, non-specific molecular profile (NSMP), and p53-mutant. This study included 177 and 178 patients with endometrioid and non-endometrioid carcinomas, respectively. Forty-two patients (11.8%) were categorized as POLE ultramutated, 106 (29.9%) as MMR-deficient, 128 (36.1%) as p53-mutant, and 79 (22.2%) as NSMP. Patients of different molecular subtypes had distinct survival times; molecular classification, but not histotype, was significantly associated with survival outcomes. When incorporating molecular classification into the stratification model, 52 patients (15.5%) switched risk groups, with 40 (11.9%) shifting to a lower risk for having a POLE mutation and 12 (3.6%) shifting to a higher risk owing to p53-mutant status. Molecular classification may provide more accurate prognostic information among patients with high-grade endometrial carcinomas and improve their stratification for purposes of clinical management.

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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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