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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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Molecular identification of spatially distinct anabolic responses to mechanical loading in murine cortical bone

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

2022 Aug 24

Chlebek, C;Moore, JA;Ross, FP;van der Meulen, MCH;
PMID: 36054133 | DOI: 10.1002/jbmr.4686

Osteoporosis affects over 200 million women worldwide, one third of whom are predicted to suffer from an osteoporotic fracture in their lifetime. The most promising anabolic drugs involve administration of expensive antibodies. Because mechanical loading stimulates bone formation, our current data, using a mouse model, replicates the anabolic effects of loading in humans and may identify novel pathways amenable to oral treatment. Murine tibial compression produces axially-varying deformations along the cortical bone, inducing highest strains at the mid-diaphysis and lowest at the metaphyseal shell. To test the hypothesis that load-induced transcriptomic responses at different axial locations of cortical bone would vary as a function of strain magnitude, we loaded the left tibiae of 10wk female C57Bl/6 mice in vivo in compression, with contralateral limbs as controls. Animals were euthanized at 1, 3 or 24 h post-loading or loaded for 1 wk (n=4-5/group). Bone marrow and cancellous bone were removed, cortical bone was segmented into the metaphyseal shell, proximal diaphysis and mid-diaphysis, and load-induced differential gene expression and enriched biological processes were examined for the three segments. At each time point, the mid-diaphysis (highest strain) had the greatest transcriptomic response. Similarly, biological processes regulating bone formation and turnover increased earlier and to the greatest extent at the mid-diaphysis. Higher strain induced greater levels of osteoblast and osteocyte genes, whereas expression was lower in osteoclasts. Among the top differentially-expressed genes at 24-hours post-loading, seventeen had known functions in bone biology, of which twelve were present only in osteoblasts, three exclusively in osteoclasts, and two were present in both cell types. Based on these results, we conclude that murine tibial loading induces spatially-unique transcriptomic responses correlating with strain magnitude in cortical bone. This article is protected by
Chemogenetic activation of noradrenergic A5 neurons increases blood pressure and visceral sympathetic activity in adult rats

American journal of physiology. Regulatory, integrative and comparative physiology

2022 Aug 22

Souza, GMPR;Stornetta, DS;Vitali, AJ;Wildner, H;Zeilhofer, HU;Campbell, JN;Abbott, SBG;
PMID: 35993562 | DOI: 10.1152/ajpregu.00119.2022

In mammals, the pontine noradrenergic system influences nearly every aspect of central nervous system function. A subpopulation of pontine noradrenergic neurons, called A5, are thought to be important in the cardiovascular response to physical stressors, yet their function is poorly defined. We hypothesized that activation of A5 neurons stimulates a sympathetically-mediated increase in BP. To test this hypothesis, we conducted a comprehensive assessment of the cardiovascular effects of chemogenetic stimulation of A5 neurons in male and female adult rats using intersectional genetic and anatomical targeting approaches. Chemogenetic stimulation of A5 neurons in freely behaving rats elevated BP by 15 mmHg and increased cardiac baroreflex sensitivity with a negligible effect on resting HR. Importantly, A5 stimulation had no detectable effect on locomotor activity, metabolic rate or respiration. Under anesthesia, stimulation of A5 neurons produced a marked elevation in visceral sympathetic nerve activity (SNA) and no change in skeletal muscle SNA, showing that A5 neurons preferentially stimulate visceral SNA. Interestingly, projection mapping indicates that A5 neurons target sympathetic preganglionic neurons throughout the spinal cord and parasympathetic preganglionic neurons throughout in the brainstem, as well as the nucleus of the solitary tract, and ventrolateral medulla. Moreover, in situ hybridization and immunohistochemistry indicate that a sub-population of A5 neurons co-release glutamate and monoamines. Collectively, this study suggests A5 neurons are a central modulator of autonomic function with a potentially important role in sympathetically-driven redistribution of blood flow from the visceral circulation to critical organs and skeletal muscle.
Microbial DNA enrichment promotes liver steatosis and fibrosis in the course of non-alcoholic steatohepatitis

Acta physiologica (Oxford, England)

2022 May 02

Luo, Z;Ji, Y;Zhang, D;Gao, H;Jin, Z;Yang, M;Ying, W;
PMID: 35500155 | DOI: 10.1111/apha.13827

Low-grade inflammation is the hallmark of non-alcoholic fatty liver diseases (NAFLD) and non-alcoholic steatohepatitis (NASH). The leakage of microbiota-derived products can contribute to liver inflammation during NAFLD/NASH development. Here, we assessed the roles of gut microbial DNA-containing extracellular vesicles (mEVs) in regulating liver cellular abnormalities in the course of NAFLD/NASH.We performed studies with Vsig4-/- , C3-/- , cGAS-/- , and their wild-type littermate mice. Vsig4+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut mEVs were adoptively transferred into Vsig4-/- , C3-/- , cGAS-/- , or littermate WT mice, and hepatocyte inflammation and HSC fibrogenic activation were measured in these mice.Non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis development was concomitant with a diminished liver Vsig4+ macrophage population and a marked bacterial DNA enrichment in both hepatocytes and HSCs. In the absence of Vsig4+ macrophages, gut mEVs translocation led to microbial DNA accumulation in hepatocytes and HSCs, resulting elevated hepatocyte inflammation and HSC fibrogenic activation. In contrast, in lean WT mice, Vsig4+ macrophages remove gut mEVs from bloodstream through a C3-dependent opsonization mechanism and prevent the infiltration of gut mEVs into hepatic cells. Additionally, Vsig4-/- mice more quickly developed significant liver steatosis and fibrosis than WT mice after Western diet feeding. In vitro treatment with NASH mEVs triggered hepatocyte inflammation and HSC fibrogenic activation. Microbial DNAs are key cargo for the effects of gut mEVs by activating cGAS/STING.Accumulation of microbial DNAs fuels the development of NAFLD/NASH-associated liver abnormalities.
Global Analyses of mRNA Expression in Human Sensory Neurons Reveals eIF5a as a Conserved Target for Inflammation-associated Pain

The Journal of Pain

2022 May 01

Chase, R;de la Peña, J;Smith, P;Lawson, J;Lou, T;Stanowick, A;Black, B;Campbell, Z;
| DOI: 10.1016/j.jpain.2022.03.144

Sensory neurons derived from human induced pluripotent stem cells (hiPSCs) are a promising model. One limitation posed by the use of monocultures is the loss of cellular heterogeneity found in tissues. Here we make use of high-throughput RNA sequencing to quantify gene expression in hiPSC-derived mono-cultured and co-cultured sensory neurons. The following groups were compared: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. We find that co-culture of sensory neurons and astrocytes enhances expression of transcripts enriched in native DRG tissues. Numerous well-established genes linked to pain and most translation factors are consistently expressed in both hiPSC and mouse samples. Marker genes for various neuronal subtypes are also present in the hiPSC cultures. As a proof of concept for the potential use of the dataset for hypothesis generation, we validated the expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissue and hiPSC samples. eIF5A is a unique translation factor in that it requires a post-translational hypusine modification to be active. We show that inhibition of hypusine synthesis prevents hyperalgesic priming by inflammatory mediators in vivo and diminishes hiPSC neuronal firing in vitro. In total, we present the transcriptomes of hiPSC sensory neuron models and evaluate the requirement of a functional translation factor. This work was supported by NIH grants R01NS100788 (ZTC), R01NS114018 (ZTC), and 1UG3TR003149 (BJB).
Single-cell high-dimensional imaging mass cytometry: one step beyond in oncology

Seminars in immunopathology

2023 Jan 04

Glasson, Y;Chépeaux, LA;Dumé, AS;Lafont, V;Faget, J;Bonnefoy, N;Michaud, HA;
PMID: 36598557 | DOI: 10.1007/s00281-022-00978-w

Solid tumors have a dynamic ecosystem in which malignant and non-malignant (endothelial, stromal, and immune) cell types constantly interact. Importantly, the abundance, localization, and functional orientation of each cell component within the tumor microenvironment vary significantly over time and in response to treatment. Such intratumoral heterogeneity influences the tumor course and its sensitivity to treatments. Recently, high-dimensional imaging mass cytometry (IMC) has been developed to explore the tumor ecosystem at the single-cell level. In the last years, several studies demonstrated that IMC is a powerful tool to decipher the tumor complexity. In this review, we summarize the potential of this technology and how it may be useful for cancer research (from preclinical to clinical studies).
Patient Selection Approaches in FGFR Inhibitor Trials-Many Paths to the Same End?

Cells

2022 Oct 10

Ellinghaus, P;Neureiter, D;Nogai, H;Stintzing, S;Ocker, M;
PMID: 36231142 | DOI: 10.3390/cells11193180

Inhibitors of fibroblast growth factor receptor (FGFR) signaling have been investigated in various human cancer diseases. Recently, the first compounds received FDA approval in biomarker-selected patient populations. Different approaches and technologies have been applied in clinical trials, ranging from protein (immunohistochemistry) to mRNA expression (e.g., RNA in situ hybridization) and to detection of various DNA alterations (e.g., copy number variations, mutations, gene fusions). We review, here, the advantages and limitations of the different technologies and discuss the importance of tissue and disease context in identifying the best predictive biomarker for FGFR targeting therapies.
Analysis of SNHG14: A Long Non-Coding RNA Hosting SNORD116, Whose Loss Contributes to Prader-Willi Syndrome Etiology

Genes

2022 Dec 29

Ariyanfar, S;Good, D;
| DOI: 10.3390/genes14010097

The Small Nucleolar Host Gene 14 (SNHG14) is a host gene for small non-coding RNAs, including the SNORD116 small nucleolar C/D box RNA encoding locus. Large deletions of the SNHG14 locus, as well as microdeletions of the SNORD116 locus, lead to the neurodevelopmental genetic disorder Prader-Willi syndrome. This review will focus on the SNHG14 gene, its expression patterns, its role in human cancer, and the possibility that single nucleotide variants within the locus contribute to human phenotypes in the general population. This review will also include new in silico data analyses of the SNHG14 locus and new in situ RNA expression patterns of the Snhg14 RNA in mouse midbrain and hindbrain regions.
Emerging approaches for decoding neuropeptide transmission

Trends in neurosciences

2022 Oct 15

Girven, KS;Mangieri, L;Bruchas, MR;
PMID: 36257845 | DOI: 10.1016/j.tins.2022.09.005

Neuropeptides produce robust effects on behavior across species, and recent research has benefited from advances in high-resolution techniques to investigate peptidergic transmission and expression throughout the brain in model systems. Neuropeptides exhibit distinct characteristics which includes their post-translational processing, release from dense core vesicles, and ability to activate G-protein-coupled receptors (GPCRs). These complex properties have driven the need for development of specialized tools that can sense neuropeptide expression, cell activity, and release. Current research has focused on isolating when and how neuropeptide transmission occurs, as well as the conditions in which neuropeptides directly mediate physiological and adaptive behavioral states. Here we describe the current technological landscape in which the field is operating to decode key questions regarding these dynamic neuromodulators.
Contemporary Approaches to the Study of Pain

Neuromethods

2022 May 26

Ferreira, DW;Arokiaraj, CM;Seal, RP;
| DOI: 10.1007/978-1-0716-2039-7#page=50

This volume contains experimental approaches that are currently revolutionizing our understanding of the neurobiology of pain. The chapters cover many cutting-edge methods including the identification of gene expression profiles, transcriptomes or translatomes, from individual cells or defined groups of cells in rodents and primates;  the electrophysiological investigation of human tissues, such as human dorsal root ganglion neurons; ways to assess modality response profiles of neurons using calcium imaging in vitro and in vivo; and somatosensory behaviors in rodents using high-speed videography and machine learning.  In the _Neuromethods_ series style, the chapters include detailed advice from specialists to obtain successful results in your laboratory.
Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling

ChemRxiv Analytical Chemistry

2023 Apr 28

Robles-Remacho*, A;Sanchez-Martin, R;Diaz-Mochon*, J;
| DOI: 10.26434/chemrxiv-2023-n20f0

Spatial transcriptomics technologies are providing new insights to study gene expression, allowing researchers to investigate the spatial organization of transcriptomes in cells and tissues. This approach enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution, utilizing diverse technological approaches. In this perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques, as well as provide information on data analysis. In addition, this perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.
Considerations for the Use of Viral Vectors in Nonhuman Primates

Vectorology for Optogenetics and Chemogenetics

2023 Feb 07

Bohlen, M;Tremblay, S;
| DOI: 10.1007/978-1-0716-2918-5_16

The development of new genetic tools has revolutionized our ability to study the functional role of specific neuronal populations and circuits generating behavior. Although this revolution has already taken place in small animal models such as mice, adoption of these techniques has been relatively slow for animals more closely related to humans, such as nonhuman primates. Current challenges include effective delivery to much larger structural targets in the primate brain, cell-type specific transduction, and immunological responses. In this chapter, we will review some of the challenges and considerations that are specific to using these viral technologies in the nonhuman primate brain. Ultimately, these challenges can be met with new advances in surgical technique and gene therapy that will spin out new viral vectors with enhanced features able to compensate for the limitations of current vectors. As the existing challenges are circumvented, this will lead to a revolution in primate neuroscientific research and a greater understanding of the functional role circuits play in complex behaviors relevant to human neurological and psychiatric diseases.
Developing Vaccines to Improve Preparedness for Filovirus Outbreaks: The Perspective of the USA Biomedical Advanced Research and Development Authority (BARDA)

Vaccines

2023 Jun 19

Parish, LA;Stavale, EJ;Houchens, CR;Wolfe, DN;
PMID: 37376509 | DOI: 10.3390/vaccines11061120

Outbreaks of viral hemorrhagic fever caused by filoviruses have become more prevalent in recent years, with outbreaks of Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV) all occurring in 2022 and 2023. While licensed vaccines are now available for EBOV, vaccine candidates for SUDV and MARV are all in preclinical or early clinical development phases. During the recent outbreak of SUDV virus disease, the Biomedical Advanced Research and Development Authority (BARDA), as part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, implemented key actions with our existing partners to advance preparedness and enable rapid response to the outbreak, while also aligning with global partners involved in the implementation of clinical trials in an outbreak setting. Beyond pre-existing plans prior to the outbreak, BARDA worked with product sponsors to expedite manufacturing of vaccine doses that could be utilized in clinical trials. While the SUDV outbreak has since ended, a new outbreak of MARV disease has emerged. It remains critical that we continue to advance a portfolio of vaccines against SUDV and MARV while also expediting manufacturing activities ahead of, or in parallel if needed, outbreaks.

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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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