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Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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Imaging mass cytometry: High-dimensional and single-cell perspectives on the microenvironment of solid tumours

Progress in biophysics and molecular biology

2022 Nov 01

Liu, Z;Xun, J;Liu, S;Wang, B;Zhang, A;Zhang, L;Wang, X;Zhang, Q;
PMID: 36252872 | DOI: 10.1016/j.pbiomolbio.2022.10.003

Imaging mass cytometry (IMC) is a new technology integrating mass spectrometry, high-resolution laser ablation and immunohistochemistry/cytochemistry. A unique high-dimensional perspective comprehensively and accurately depicts the complex interaction of phenotype, signalling pathway and tumour microenvironment and is widely used in solid tumours. However, the application scenarios of IMC in basic medicine and clinical research in solid tumours lack systematic introduction and classification. This paper reviews the application of IMC in depicting the panorama of the tumour microenvironment, revealing tumour spatial heterogeneity, clarifying tumour pharmacological mechanisms, assisting in new drug development, and dynamically evaluating the efficacy of immunotherapy in solid tumours.
Spatially resolved transcriptomics and the kidney: many opportunities

Kidney international

2022 Jul 02

Dixon, EE;Wu, H;Sulvarán-Guel, E;Guo, J;Humphreys, BD;
PMID: 35788360 | DOI: 10.1016/j.kint.2022.06.011

Defining changes in gene expression during health and disease is critical for the understanding of human physiology. In recent years, single-cell/nuclei RNA sequencing (sc/snRNAseq) has revolutionized the definition and discovery of cell types and states as well as the interpretation of organ- and cell-type-specific signaling pathways. However, these advances require tissue dissociation to the level of the single cell or single nuclei level. Spatially resolved transcriptomics (SrT) now provides a platform to overcome this barrier in understanding the physiological contexts of gene expression and cellular microenvironment changes in development and disease. Some of these transcriptomic tools allow for high-resolution mapping of hundreds of genes simultaneously in cellular and subcellular compartments. Other tools offer genome depth mapping but at lower resolution. We review advances in SrT, considerations for using SrT in your own research, and applications for kidney biology.
What is happening to immunohistochemistry?

BioEssays : news and reviews in molecular, cellular and developmental biology

2023 Jun 01

Gautron, L;
PMID: 37264690 | DOI: 10.1002/bies.202300056

Immunohistochemistry is a commonly used technique in research and pathology laboratories worldwide. However, in recent years, there has been a significant decrease in the number of Pubmed entries using the term immunohistochemistry. This decline can be attributed to two factors: increased awareness of the issue of unreliable research antibodies and the availability of novel RNA in situ hybridization techniques. Using the example of immunohistochemistry, this text discusses the factors that can affect good laboratory and publishing practices, or their lack thereof.
High-Plex Spatial Profiling of RNA and Protein Using Digital Spatial Profiler

Methods in molecular biology (Clifton, N.J.)

2023 May 16

Wang, N;Li, X;Ding, Z;
PMID: 37191791 | DOI: 10.1007/978-1-0716-3163-8_6

The rapid emergence of spatial multi-omics technologies in recent years has revolutionized biomedical research. Among these, the Digital Spatial Profiler (DSP, commercialized by nanoString) has become one of the dominant technologies in spatial transcriptomics and proteomics and has assisted in deconvoluting complex biological questions. Based on our practical experience in the past 3 years with DSP, we share here a detailed hands-on protocol and key handling notes that will allow the broader community to optimize their work procedure.
Cannabinoid CB2 receptors modulate alcohol induced behavior, and neuro-immune dysregulation in mice

Behavioural brain research

2023 Apr 14

Kibret, BG;Roberts, A;Kneebone, A;Embaby, S;Fernandez, J;Liu, QR;Onaivi, ES;
PMID: 37061199 | DOI: 10.1016/j.bbr.2023.114439

The identification of additional lipid mediators, enzymes, and receptors revealed an expanded endocannabinoid system (ECS) called the endocannabinoidome (eCBome). Furthermore, eCBome research using wild type and genetically modified mice indicate the involvement of this system in modulating alcohol induced neuroinflammatory alterations associated with behavioral impairments and the release of proinflammatory cytokines. We investigated the role of cannabinoid type 2 receptors (CB2Rs) in modulating behavioral and neuro-immune changes induced by alcohol using conditional knockout (cKO) mice with selective deletion of CB2Rs in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2) cKO mice. We used a battery of behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests to evaluate behavioral changes induced by alcohol. ELISA assay was used, to detect alterations in IL-6, IL-1α, and IL-1β in the prefrontal cortex, striatum, and hippocampal regions of mice to investigate the role of CB2Rs in neuroinflammation induced by alcohol in the brain. The involvement of cannabinoid receptors in alcohol-induced behavior was also evaluated using the non-selective cannabinoid receptor mixed agonist WIN 55,212-2. The results showed that cell-type specific deletion of CB2Rs in dopamine neurons and microglia significantly and differentially altered locomotor activity and rotarod performance activities. The result also revealed that cell-type specific deletion of CB2Rs enhanced alcohol-induced inflammation, and WIN significantly reduced alcohol preference in all genotypes compared to the vehicle controls. These findings suggest that the involvement of CB2Rs in modulating behavioral and neuroinflammatory alterations induced by alcohol may be potential therapeutic targets in the treatment of alcohol use disorder.
BS19 Histone deacetylase 6 inhibition induces dna damage accumulation in aortic smooth muscle cells

Basic science

2022 Jun 01

Solanki, R;Warren, D;Johnson, R;
| DOI: 10.1136/heartjnl-2022-bcs.199

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs) of physiological aortic stiffness to investigate the impact of HDAC6 inhibition on the contractile response of angiotensin II stimulated quiescent VSMC function. In this study, we utilise HDAC6 inhibitor BRD 9757 and Tubastatin a HDAC6 inhibitor inducing tubulin hyperacetylation. Results Our data shows that HDAC6 inhibition resulted in increased alpha-tubulin acetylation and decreased VSMC area. Further analysis revealed that although VSMC volume was unaltered, nuclear volume was decreased. Immunofluorescence microscopy revealed that HDAC6 inhibitor treatment resulted in DNA damage accumulation in VSMCs. We hypothesised that altered microtubule stability participated in this phenotype. To test this possibility, we performed a cold-stable microtubule stability assay, which revealed that HDAC6 inhibitor treated VSMCs possessed decreased microtubule stability. To test whether changes in microtubule stability induced DNA damage accumulation, we used the microtubule destabilising agents colchicine and demecholcine, and the microtubule stabilising agent paclitaxel. Importantly, either colchicine or demecholcine treatment increased DNA damage accumulation in VSMCs. In contrast, paclitaxel treatment had no effect on DNA damage levels.
Sickle Cell Disease Associated Changes in the Gut Microbiome Contribute to Persistent Pain

The Journal of Pain

2022 May 01

Sadler, K;Ehlers, V;Brandow, A;Stucky, C;
| DOI: 10.1016/j.jpain.2022.03.027

Many patients with sickle cell disease (SCD) suffer from chronic pain, the underlying causes of which are unclear. Recent 16s ribosomal RNA sequencing studies revealed differences in the number and types of bacteria in the gastrointestinal tract of patients and mouse models of SCD relative to controls, but it is unclear if or how these changes contribute to symptomology. In these experiments, we used transgenic SCD mice to determine the extent to which disease related gut dysbiosis contributes to persistent pain. Reflexive pain behaviors were first measured in SCD mice following longitudinal probiotic or antibiotic treatment. Vehicle-treated SCD mice displayed significant mechanical allodynia relative to vehicle-treated wildtype mice, and antibiotic treatment further exacerbated mechanical allodynia in both genotypes. In contrast, probiotic treatment completely reversed persistent touch hypersensitivity in SCD mice. Persistent touch pain was also transiently reversed in SCD mice following fecal material transplant from healthy mice. In complementary experiments, wildtype recipient mice developed cold and touch hypersensitivity that persisted for several weeks after fecal material transplant from SCD donors. Using whole-cell patch clamp recordings, we further determined that these behavioral observations were accompanied by altered intrinsic plasticity in a select class of nodose ganglia sensory neurons, the peripheral terminals of which are well positioned to detect sensory information in the gut. Nodose ganglia neurons isolated from animals that received sickle cell fecal material transplants were hyperexcitable relative to those isolated from animals that received control fecal material transplants. These data are the first to suggest that disease-related gut dysbiosis induces pain through changes in vagal nerve activity. Ongoing studies are examining specific bacterial populations and/or metabolites responsible for these functional changes in order to develop novel therapeutics for chronic SCD pain management. Grant support from National Institutes of Health grants K99HL155791 and R01NS070711.
Activation of α 6-containing GABA A receptors induces antinociception under physiological and pathological conditions

Pain

2023 May 01

Rodríguez-Palma, EJ;De la Luz-Cuellar, YE;Islas-Espinoza, AM;Félix-Leyva, AE;Shiers, SI;García, G;Torres-López, JE;Delgado-Lezama, R;Murbartián, J;Price, TJ;Granados-Soto, V;
PMID: 36001074 | DOI: 10.1097/j.pain.0000000000002763

The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.
Induction of Anxiety-Like Phenotypes by Knockdown of Cannabinoid Type-1 Receptors in the Amygdala of Marmosets

Neuroscience bulletin

2023 Jun 27

Zhu, L;Zheng, D;Li, R;Shen, CJ;Cai, R;Lyu, C;Tang, B;Sun, H;Wang, X;Ding, Y;Xu, B;Jia, G;Li, X;Gao, L;Li, XM;
PMID: 37368194 | DOI: 10.1007/s12264-023-01081-2

The amygdala is an important hub for regulating emotions and is involved in the pathophysiology of many mental diseases, such as depression and anxiety. Meanwhile, the endocannabinoid system plays a crucial role in regulating emotions and mainly functions through the cannabinoid type-1 receptor (CB1R), which is strongly expressed in the amygdala of non-human primates (NHPs). However, it remains largely unknown how the CB1Rs in the amygdala of NHPs regulate mental diseases. Here, we investigated the role of CB1R by knocking down the cannabinoid receptor 1 (CNR1) gene encoding CB1R in the amygdala of adult marmosets through regional delivery of AAV-SaCas9-gRNA. We found that CB1R knockdown in the amygdala induced anxiety-like behaviors, including disrupted night sleep, agitated psychomotor activity in new environments, and reduced social desire. Moreover, marmosets with CB1R-knockdown had up-regulated plasma cortisol levels. These results indicate that the knockdown of CB1Rs in the amygdala induces anxiety-like behaviors in marmosets, and this may be the mechanism underlying the regulation of anxiety by CB1Rs in the amygdala of NHPs.
Osteocytes: New Kids on the Block for Cancer in Bone Therapy

Cancers

2023 May 07

Anloague, A;Delgado-Calle, J;
PMID: 37174109 | DOI: 10.3390/cancers15092645

The tumor microenvironment plays a central role in the onset and progression of cancer in the bone. Cancer cells, either from tumors originating in the bone or from metastatic cancer cells from other body systems, are located in specialized niches where they interact with different cells of the bone marrow. These interactions transform the bone into an ideal niche for cancer cell migration, proliferation, and survival and cause an imbalance in bone homeostasis that severely affects the integrity of the skeleton. During the last decade, preclinical studies have identified new cellular mechanisms responsible for the dependency between cancer cells and bone cells. In this review, we focus on osteocytes, long-lived cells residing in the mineral matrix that have recently been identified as key players in the spread of cancer in bone. We highlight the most recent discoveries on how osteocytes support tumor growth and promote bone disease. Additionally, we discuss how the reciprocal crosstalk between osteocytes and cancer cells provides the opportunity to develop new therapeutic strategies to treat cancer in the bone.
A Spatial Atlas of Wnt Receptors in Adult Mouse Liver

The American journal of pathology

2023 Feb 10

Gayden, J;Hu, S;Joseph, PN;Delgado, E;Liu, S;Bell, A;Puig, S;Monga, SP;Freyberg, Z;
PMID: 36773785 | DOI: 10.1016/j.ajpath.2023.01.011

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, we quantitatively mapped the spatial expression of Fzd receptors in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors are expressed within a metabolic unit, Fzd receptors 1, 4, and 6 are the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors is not zonated. In contrast, Fzd receptor 6 is preferentially expressed in zone 1. We also verified that Wnt2 and Wnt9b expression is highly zonated and primarily found in zone 3. Therefore, our results suggest that zonated Wnt/β-catenin signaling at baseline is mostly due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, we showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, our establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.
Vascular smooth muscle- and myeloid cell-derived integrin α9β1 does not directly mediate the development of atherosclerosis in mice

Atherosclerosis

2022 Nov 01

Jung, IH;Elenbaas, JS;Burks, KH;Amrute, JM;Xiangyu, Z;Alisio, A;Stitziel, NO;
PMID: 36215801 | DOI: 10.1016/j.atherosclerosis.2022.09.015

Sushi, von Willebrand factor type A, EGF pentraxin domain-containing 1 (SVEP1), an extracellular matrix protein, is a human coronary artery disease locus that promotes atherosclerosis. We previously demonstrated that SVEP1 induces vascular smooth muscle cell (VSMC) proliferation and an inflammatory phenotype in the arterial wall to enhance the development of atherosclerotic plaque. The only receptor known to interact with SVEP1 is integrin α9β1, a cell surface receptor that is expressed by VSMCs and myeloid lineage-derived monocytes and macrophages. Our previous in vitro studies suggested that integrin α9β1 was necessary for SVEP1-induced VSMC proliferation and inflammation; however, the underlying mechanisms mediated by integrin α9β1 in these cell types during the development of atherosclerosis remain poorly understood.Here, using cell-specific gene targeting, we investigated the effects of the integrin α9β1 receptor on VSMCs and myeloid cells in mouse models of atherosclerosis. Interestingly, we found that depleting integrin α9β1 in either VSMCs or myeloid cells did not affect the formation or complexity of atherosclerotic plaque in vessels after either 8 or 16 weeks of high fat diet feeding.Our results indicate that integrin α9β1 in these two cell types does not mediate the in vivo effect of SVEP1 in the development of atherosclerosis. Instead, our results suggest either the presence of other potential receptor(s) or alternative integrin α9β1-expressing cell types responsible for SVEP1 induced signaling in the development of atherosclerosis.

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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
EnEmProbe targets exons n and m
En-EmProbe 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

Enabling research, drug development (CDx) and diagnostics

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