Methods in molecular biology (Clifton, N.J.)
Oliver, A;Hagen, J;Yang, S;Kalyuzhny, AE;
PMID: 36513934 | DOI: 10.1007/978-1-0716-2811-9_14
Detection of phosphorylated proteins in tissue sections using immunohistochemistry (IHC) is a challenging task. The absence of tissue staining may be caused by either a lack of protein expression or a lack of protein activation via its phosphorylation. To address this problem, we employed Integrated Co-detection Workflow (ICW) protocol to analyze lung cancer tissue sections by combining in situ hybridization (ISH) with IHC. The target protein of interest was epidermal growth factor receptor (EGFR, also known as ErbB1 and HER1) which is the founding member of the ErbB family of receptor tyrosine kinases. Using phospho-specific antibodies specific for a phosphorylated site Y1173 of EGFR molecule allowed us to analyze IHC and ISH staining at a single cell level in lung cancer tissue. We have observed both a co-localization of IHC with ISH signals and ISH-positive cells lacking IHC labeling for phosphorylated EGFR. ICW appears to be a very powerful spatial biology technique for accurate localization of cancer cells with phosphorylated/activated and non-phosphorylated/nonactivated proteins.
Kinchen J, Chen HH, Parikh K, Antanaviciute A, Jagielowicz M, Fawkner-Corbett D, Ashley N, Cubitt L, Mellado-Gomez E, Attar M, Sharma E, Wills Q, Bowden R, Richter FC, Ahern D, Puri KD, Henault J, Gervais F, Koohy H, Simmons A.
PMID: - | DOI: 10.1016/j.cell.2018.08.067
Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cellfunction. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
Molecular therapy : the journal of the American Society of Gene Therapy
O'Reilly, D;Belgrad, J;Ferguson, C;Summers, A;Sapp, E;McHugh, C;Mathews, E;Boudi, A;Buchwald, J;Ly, S;Moreno, D;Furgal, R;Luu, E;Kennedy, Z;Hariharan, V;Monopoli, K;Yang, XW;Carroll, J;DiFiglia, M;Aronin, N;Khvorova, A;
PMID: 37177784 | DOI: 10.1016/j.ymthe.2023.05.006
Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.
Lund, J;Breum, AW;Gil, C;Falk, S;Sass, F;Isidor, MS;Dmytriyeva, O;Ranea-Robles, P;Mathiesen, CV;Basse, AL;Johansen, OS;Fadahunsi, N;Lund, C;Nicolaisen, TS;Klein, AB;Ma, T;Emanuelli, B;Kleinert, M;Sørensen, CM;Gerhart-Hines, Z;Clemmensen, C;
PMID: 37055619 | DOI: 10.1038/s42255-023-00780-4
Lactate is a circulating metabolite and a signalling molecule with pleiotropic physiological effects. Studies suggest that lactate modulates energy balance by lowering food intake, inducing adipose browning and increasing whole-body thermogenesis. Yet, like many other metabolites, lactate is often commercially produced as a counterion-bound salt and typically administered in vivo through hypertonic aqueous solutions of sodium L-lactate. Most studies have not controlled for injection osmolarity and the co-injected sodium ions. Here, we show that the anorectic and thermogenic effects of exogenous sodium L-lactate in male mice are confounded by the hypertonicity of the injected solutions. Our data reveal that this is in contrast to the antiobesity effect of orally administered disodium succinate, which is uncoupled from these confounders. Further, our studies with other counterions indicate that counterions can have confounding effects beyond lactate pharmacology. Together, these findings underscore the importance of controlling for osmotic load and counterions in metabolite research.
Chemical communications (Cambridge, England)
Sahota, A;Monteza Cabrejos, A;Kwan, Z;Paulose Nadappuram, B;Ivanov, AP;Edel, JB;
PMID: 37039236 | DOI: 10.1039/d3cc00573a
Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.
Staedtler, E;Iadarola, M;Sapio, M;Maric, D;Ghetti, A;Mannes, A;
| DOI: 10.1016/j.jpain.2023.02.059
Nociceptive input to the spinal cord is transmitted by primary afferent neurons in the dorsal root ganglia (DRG). A subset of DRG neurons has the ability to attenuate nociceptive transmission through expression of the µ-opioid receptor. The relationship between algesic and analgesic properties of individual DRG neurons has not yet been evaluated in the human. By using a combination of six different 4-Plex in-situ hybridization experiments, we were able to identify three different main nociceptive populations. First, we detected a population poly-responsive, poly-modulated small-diameter nociceptors that co-express TRPV1, OPRM1, and SCN11A, and in most cases additionally SCN10A and P2RX3 and in subpopulations additionally TAC1, TRPA1, TRPM8, OPRD1, PIEZO2, and/or OPRL1. Second, we detected a medium-sized set of multimodal putative nociceptors with a very similar molecular profile, apart from a lack of expression of TRPA1. Both nociceptor types do express the µ-opioid receptor (encoded by OPRM1) and their activity and input into the spinal cord likely can be attenuated by clinically used µ-receptor agonists. The third population consists of small-diameter neurons that have a molecular expression profile characteristic for nociceptive neurons, such that they express TRPV1 together with SCN11A, SCN10A, P2RX3, PIEZO2, OPRD1, and partially TRPA1. The most distinguishable characteristic of this population is the lack of expression of the µ-opioid receptor and thus are non-susceptible to clinically used opioids. Further molecular characterization of these nociceptive populations might reveal attractive molecular candidates for targeting different types of pain indications.
Journal of the peripheral nervous system : JPNS
White, D;Abdulla, M;Park, SB;Goldstein, D;Moalem-Taylor, G;Lees, JG;
PMID: 36995049 | DOI: 10.1111/jns.12544
The expanding use of chemotherapy in curative cancer treatment has simultaneously resulted in a substantial and growing cohort of cancer survivors with prolonged disability from chemotherapy-induced peripheral neuropathy (CIPN). CIPN is associated with several commonly prescribed chemotherapeutics, including taxanes, platinum-based drugs, vinca alkaloids, bortezomib and thalidomide. These distinct classes of chemotherapeutics, with their varied neurotoxic mechanisms, often cause patients to suffer from a broad profile of neuropathic symptoms including chronic numbness, paraesthesia, loss of proprioception or vibration sensation and neuropathic pain. Decades of investigation by numerous research groups have provided substantial insights describing this disease. Despite these advances, there is currently no effective curative or preventative treatment option for CIPN and only the dual serotonin-norepinephrine reuptake inhibitor Duloxetine is recommended by clinical guidelines for the symptomatic treatment of painful CIPN.In this review, we examine current preclinical models, with our analysis focused on translational relevance and value.Animal models have been pivotal in achieving a better understanding of the pathogenesis of CIPN. However, it has been challenging for researchers to develop appropriate preclinical models that are effective vehicles for the discovery of translatable treatment options.Further development of preclinical models targeting translational relevance will promote value for preclinical outcomes in CIPN studies.
Venkatesan, M;Zhang, N;Marteau, B;Yajima, Y;De Zarate Garcia, NO;Fang, Z;Hu, T;Cai, S;Ford, A;Olszewski, H;Borst, A;Coskun, AF;
PMID: 37005468 | DOI: 10.1038/s41598-023-32474-y
Organelles play important roles in human health and disease, such as maintaining homeostasis, regulating growth and aging, and generating energy. Organelle diversity in cells not only exists between cell types but also between individual cells. Therefore, studying the distribution of organelles at the single-cell level is important to understand cellular function. Mesenchymal stem cells are multipotent cells that have been explored as a therapeutic method for treating a variety of diseases. Studying how organelles are structured in these cells can answer questions about their characteristics and potential. Herein, rapid multiplexed immunofluorescence (RapMIF) was performed to understand the spatial organization of 10 organelle proteins and the interactions between them in the bone marrow (BM) and umbilical cord (UC) mesenchymal stem cells (MSCs). Spatial correlations, colocalization, clustering, statistical tests, texture, and morphological analyses were conducted at the single cell level, shedding light onto the interrelations between the organelles and comparisons of the two MSC subtypes. Such analytics toolsets indicated that UC MSCs exhibited higher organelle expression and spatially spread distribution of mitochondria accompanied by several other organelles compared to BM MSCs. This data-driven single-cell approach provided by rapid subcellular proteomic imaging enables personalized stem cell therapeutics.
Cross, AR;Gartner, L;Hester, J;Issa, F;
PMID: 36944604 | DOI: 10.1097/TP.0000000000004587
The last 5 y have seen the development and widespread adoption of high-plex spatial transcriptomic technology. This technique detects and quantifies mRNA transcripts in situ, meaning that transcriptomic signatures can be sampled from specific cells, structures, lesions, or anatomical regions while conserving the physical relationships that exist within complex tissues. These methods now frequently implement next-generation sequencing, enabling the simultaneous measurement of many targets, up to and including the whole mRNA transcriptome. To date, spatial transcriptomics has been foremost used in the fields of neuroscience and oncology, but there is potential for its use in transplantation sciences. Transplantation has a clear dependence on biopsies for diagnosis, monitoring, and research. Transplant patients represent a unique cohort with multiple organs of interest, clinical courses, demographics, and immunosuppressive regimens. Obtaining high complexity data on the disease processes underlying rejection, tolerance, infection, malignancy, and injury could identify new opportunities for therapeutic intervention and biomarker identification. In this review, we discuss currently available spatial transcriptomic technologies and how they can be applied to transplantation.
Choe, K;Pak, U;Pang, Y;Hao, W;Yang, X;
PMID: 36671541 | DOI: 10.3390/biom13010156
Development from single cells to multicellular tissues and organs involves more than just the exact replication of cells, which is known as differentiation. The primary focus of research into the mechanism of differentiation has been differences in gene expression profiles between individual cells. However, it has predominantly been conducted at low throughput and bulk levels, challenging the efforts to understand molecular mechanisms of differentiation during the developmental process in animals and humans. During the last decades, rapid methodological advancements in genomics facilitated the ability to study developmental processes at a genome-wide level and finer resolution. Particularly, sequencing transcriptomes at single-cell resolution, enabled by single-cell RNA-sequencing (scRNA-seq), was a breath-taking innovation, allowing scientists to gain a better understanding of differentiation and cell lineage during the developmental process. However, single-cell isolation during scRNA-seq results in the loss of the spatial information of individual cells and consequently limits our understanding of the specific functions of the cells performed by different spatial regions of tissues or organs. This greatly encourages the emergence of the spatial transcriptomic discipline and tools. Here, we summarize the recent application of scRNA-seq and spatial transcriptomic tools for developmental biology. We also discuss the limitations of current spatial transcriptomic tools and approaches, as well as possible solutions and future prospects.
Brain, Behavior, and Immunity
Nemeth, D;Liu, X;Kocak, N;Niu, H;Smirnova, M;McGovern, S;Herd, A;DiSabato, D;Floyd, T;Atluri, R;Nusstein, A;Oliver, B;Witcher, K;McKim, D;Gajewski-Kurdziel, P;Godbout, J;Zhang, Q;Blakely, R;Sheridan, J;Quan, N;
| DOI: 10.1016/j.bbi.2022.07.065
Methods: Global and neuronal specific IL-1R1 reporter mice, RNA sequencing analysis, and double-immunofluorescent labeling were used to map and validate nIL-1R1 expression. NF-κB/IL-1R1 co-reporter mice were utilized to detect IL-1R1 and NF-κB expression following intracerebroventricular (i.c.v.) IL-1 injections. Basescope in situ hybridization was utilized to detect splice variants of IL-1R Accessory Protein (IL-1AcP). Unpredictable foot shock (6x shocks over 1hr for 6d) was employed as a chronic stress paradigm. Results: IL-1R1 is expressed in subsets of glutamatergic or serotonergic neurons, with highest expression in the dentate gyrus (DG) and dorsal raphe nucleus (DRN). I.c.v. IL-1β injection reveals nIL-1R1 does not signal through the canonical NF-κB pathway, whereas endothelia and ventricular IL-1R1s do. We identified that neurons of the DG and DRN express the alternatively spliced IL-1RAcP Isoform B (IL-1RAcPb). Additional results suggest that nIL-1R1 may become reactive to IL-1 when neuronal expression of IL-1RAcPb shifts to the canonical IL-1RAcP following stress. Consequently, nIL-1R1 mediates activation of microglia near nIL-1R1 neurons. Conclusions: These data suggest that regional specific nIL-1R1 may require a culmination of stress and inflammatory signals to unlock nIL-1R1 signaling. Overall, these data provide a map of nIL-1R1 and its corresponding accessory protein in the brain along with a potential output of nIL-1R1 signaling.
Gala, DS;Titlow, JS;Teodoro, RO;Davis, I;
PMID: 36442969 | DOI: 10.1261/rna.079422.122
Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses requiring mRNA transport and localized translation to regulate synaptic plasticity efficiently. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, this topic has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial sub-types, containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing for example synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed using cutting-edge tools already available for neurons.
