SSRN Electronic Journal

2022 May 28

Cui, Y;Bondarenko, E;Thörn Perez, C;Chiu, D;Feldman, J;
| DOI: 10.2139/ssrn.4117921

We elucidated neural mechanisms underlying sighing. Photostimulation of parafacial (pF) neuromedin B ( NMB) or gastrin releasing peptide (GRP) or preBötC NMBR or GRPR neurons elicited ectopic sighs with latency inversely related to time from the preceding endogenous sigh. Of particular note, ectopic sighs could be produced without involvement of these peptides or their receptors in preBötC. Moreover, chemogenetic or optogenetic activation of preBötC SST neurons induced sighing, even in the presence of NMBR or GRPR antagonists. We propose that an increase in the excitability of preBötC NMBR or GRPR neurons not requiring activation of their peptide receptors activates partially overlapping pathways to generate sighs, and that preBötC SST neurons are a downstream element in the sigh generation circuit that converts normal breaths into sighs.

Methods and applications for single-cell and spatial multi-omics

Nature reviews. Genetics

2023 Mar 02

Vandereyken, K;Sifrim, A;Thienpont, B;Voet, T;
PMID: 36864178 | DOI: 10.1038/s41576-023-00580-2

The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.

Single-cell proteomics enabled by next-generation sequencing or mass spectrometry

Nature methods

2023 Mar 01

Bennett, HM;Stephenson, W;Rose, CM;Darmanis, S;
PMID: 36864196 | DOI: 10.1038/s41592-023-01791-5

In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.

Spatial profiling technologies illuminate the tumor microenvironment

Cancer cell

2023 Feb 13

Elhanani, O;Ben-Uri, R;Keren, L;
PMID: 36800999 | DOI: 10.1016/j.ccell.2023.01.010

The tumor microenvironment (TME) is composed of many different cellular and acellular components that together drive tumor growth, invasion, metastasis, and response to therapies. Increasing realization of the significance of the TME in cancer biology has shifted cancer research from a cancer-centric model to one that considers the TME as a whole. Recent technological advancements in spatial profiling methodologies provide a systematic view and illuminate the physical localization of the components of the TME. In this review, we provide an overview of major spatial profiling technologies. We present the types of information that can be extracted from these data and describe their applications, findings and challenges in cancer research. Finally, we provide a future perspective of how spatial profiling could be integrated into cancer research to improve patient diagnosis, prognosis, stratification to treatment and development of novel therapeutics.

Single-cell atlases: shared and tissue-specific cell types across human organs

Nature reviews. Genetics

2022 Feb 25

Elmentaite, R;Domínguez Conde, C;Yang, L;Teichmann, SA;
PMID: 35217821 | DOI: 10.1038/s41576-022-00449-w

The development of single-cell and spatial transcriptomics methods was instrumental in the conception of the Human Cell Atlas initiative, which aims to generate an integrated map of all cells across the human body. These technology advances are bringing increasing depth and resolution to maps of human organs and tissues, as well as our understanding of individual human cell types. Commonalities as well as tissue-specific features of primary and supportive cell types across human organs are beginning to emerge from these human tissue maps. In this Review, we highlight key biological insights obtained from cross-tissue studies into epithelial, fibroblast, vascular and immune cells based on single-cell gene expression data in humans and contrast it with mechanisms reported in mice.

Baseline skin cytokine profiles determined by RNA in situ hybridization correlate with response to dupilumab in patients with eczematous dermatitis

Journal of the American Academy of Dermatology

2023 Feb 11

Singh, K;Valido, K;Swallow, M;Okifo, KO;Wang, A;Cohen, JM;Damsky, W;
PMID: 36780951 | DOI: 10.1016/j.jaad.2022.12.052

Dupilumab has revolutionized the treatment of atopic dermatitis. However, not all patients respond optimally, and this may relate to underlying molecular heterogeneity. Nevertheless, clinically useful and accessible methods to assess such heterogeneity have not been developed.We assessed whether cytokine staining and/or histologic features correlate with clinical response to dupilumab in patients with eczematous dermatitis.We retrospectively analyzed biopsies from 61 patients with eczematous dermatitis treated with dupilumab (90.2% met Hanifin-Rajka criteria for atopic dermatitis). RNA in situ hybridization was used to measure markers of type 2 (interleukin [IL]4, IL13), type 1 (interferon gamma) and type 3 (IL17A, IL17F, IL22) inflammation. Histologic features were also assessed. Patterns were compared among complete (n = 16), partial (n = 37), and nonresponders (n = 8) to dupilumab.We found that increased IL13 expression was associated with optimal response to dupilumab. In contrast, nonresponders tended to express less IL13 and relatively greater levels of type 1 and 3 cytokines. In addition, certain histologic features tended to correlate with improved response to dupilumab.Retrospective approach and small size of the nonresponder group.Cytokine RNA in situ hybridization may aid in treatment selection for eczematous disorders. Moreover, personalization of treatment selection for inflammatory skin diseases may be possible.

Identification of a protective microglial state mediated by miR-155 and interferon-γ signaling in a mouse model of Alzheimer's disease

Nature neuroscience

2023 Jun 08

Yin, Z;Herron, S;Silveira, S;Kleemann, K;Gauthier, C;Mallah, D;Cheng, Y;Margeta, MA;Pitts, KM;Barry, JL;Subramanian, A;Shorey, H;Brandao, W;Durao, A;Delpech, JC;Madore, C;Jedrychowski, M;Ajay, AK;Murugaiyan, G;Hersh, SW;Ikezu, S;Ikezu, T;Butovsky, O;
PMID: 37291336 | DOI: 10.1038/s41593-023-01355-y

Microglia play a critical role in brain homeostasis and disease progression. In neurodegenerative conditions, microglia acquire the neurodegenerative phenotype (MGnD), whose function is poorly understood. MicroRNA-155 (miR-155), enriched in immune cells, critically regulates MGnD. However, its role in Alzheimer's disease (AD) pathogenesis remains unclear. Here, we report that microglial deletion of miR-155 induces a pre-MGnD activation state via interferon-γ (IFN-γ) signaling, and blocking IFN-γ signaling attenuates MGnD induction and microglial phagocytosis. Single-cell RNA-sequencing analysis of microglia from an AD mouse model identifies Stat1 and Clec2d as pre-MGnD markers. This phenotypic transition enhances amyloid plaque compaction, reduces dystrophic neurites, attenuates plaque-associated synaptic degradation and improves cognition. Our study demonstrates a miR-155-mediated regulatory mechanism of MGnD and the beneficial role of IFN-γ-responsive pre-MGnD in restricting neurodegenerative pathology and preserving cognitive function in an AD mouse model, highlighting miR-155 and IFN-γ as potential therapeutic targets for AD.

The long and short: Non-coding RNAs in the mammalian inner ear

Hearing research

2022 Dec 16

Koffler-Brill, T;Noy, Y;Avraham, KB;
PMID: 36566643 | DOI: 10.1016/j.heares.2022.108666

Non-coding RNAs (ncRNAs) play a critical role in the entire body, and their mis-regulation is often associated with disease. In parallel with the advances in high-throughput sequencing technologies, there is a great deal of focus on this broad class of RNAs. Although these molecules are not translated into proteins, they are now well established as significant regulatory components in many biological pathways and pathological conditions. ncRNAs can be roughly divided into two main sub-groups based on the length of the transcript, with both the small and long non-coding RNAs having diverse regulatory functions. The smaller length group includes ribosomal RNAs (rRNA), transfer RNAs (tRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), microRNAs (miRNA), small interfering RNAs (siRNA), and PIWI-associated RNAs (piRNA). The longer length group includes linear long non-coding RNAs (lncRNA) and circular RNAs (circRNA). This review is designed to present the different classes of small and long ncRNA molecules and describe some of their known roles in physiological and pathological conditions, as well as methods used to assess the validity and function of miRNAs and lncRNAs, with a focus on their role and functions in the inner ear, hearing and deafness.

Durable response in a patient with recurrent respiratory papillomatosis treated with immune checkpoint blockade

Head & neck

2022 Jul 11

Bai, K;Norberg, SM;Sievers, C;Meyer, T;Friedman, J;Hinrichs, C;Allen, CT;
PMID: 35815785 | DOI: 10.1002/hed.27144

Immune checkpoint blockade can provide clinical benefit for patients with advanced cancer. Here, we report durable disease control over many years following PD-L1 blockade through induction of a viral antigen-specific T cell response in an adult patient with recurrent respiratory papillomatosis.Antigen-specific T cell response assays, single cell RNA-sequencing, and RNA-scope was used to study clinical tissues.An HPV6 E2-specific T cell clone restricted to HLA-B*55, present at low frequency in the pre-treatment papilloma, significantly expanded after six doses of PD-L1 blockade and remained present and functional at the site of initial response in the larynx as a tissue resident memory T cell for 4 years. An associated reduction in E2 target gene was observed following treatment.Although demonstrated in a single exceptional responder, these results highlight that immune checkpoint blockade may induce durable, viral antigen-specific immunity of sufficient magnitude to control disease in patients with nonmalignant disorders.

Understanding spatiotemporal coupling of gene expression using single molecule RNA imaging technologies

Transcription

2023 Apr 12

Gerber, A;van Otterdijk, S;Bruggeman, FJ;Tutucci, E;
PMID: 37050882 | DOI: 10.1080/21541264.2023.2199669

Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field.Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA.

Towards multiomic analysis of oral mucosal pathologies

Seminars in immunopathology

2023 Jan 01

Einhaus, J;Han, X;Feyaerts, D;Sunwoo, J;Gaudilliere, B;Ahmad, SH;Aghaeepour, N;Bruckman, K;Ojcius, D;Schürch, CM;Gaudilliere, DK;
PMID: 36790488 | DOI: 10.1007/s00281-022-00982-0

Oral mucosal pathologies comprise an array of diseases with worldwide prevalence and medical relevance. Affecting a confined space with crucial physiological and social functions, oral pathologies can be mutilating and drastically reduce quality of life. Despite their relevance, treatment for these diseases is often far from curative and remains vastly understudied. While multiple factors are involved in the pathogenesis of oral mucosal pathologies, the host's immune system plays a major role in the development, maintenance, and resolution of these diseases. Consequently, a precise understanding of immunological mechanisms implicated in oral mucosal pathologies is critical (1) to identify accurate, mechanistic biomarkers of clinical outcomes; (2) to develop targeted immunotherapeutic strategies; and (3) to individualize prevention and treatment approaches. Here, we review key elements of the immune system's role in oral mucosal pathologies that hold promise to overcome limitations in current diagnostic and therapeutic approaches. We emphasize recent and ongoing multiomic and single-cell approaches that enable an integrative view of these pathophysiological processes and thereby provide unifying and clinically relevant biological signatures.

Single-cell/nucleus transcriptomic and muscle pathologies

Transcriptome Profiling

2022 Oct 14

Chemello, F;Sales, G;Cagnin, S;
| DOI: 10.1016/b978-0-323-91810-7.00011-x

Recent years have seen a dramatic improvement in RNA and DNA sequencing technologies allowing the analysis of gene expression and chromatin conformation at the single-cell or nuclei level. This permitted to evidence that cells of the human brain may have different genomes, the different cell types living in a tumor or during its development, and many other biological features, promising significant future biomedical and clinical impacts. In this chapter, we will develop the concept of single-cell or nucleus RNA sequencing discussing methods and applications in the field of muscle pathologies. We will focus on all the three types of muscles: skeletal muscle is particularly important to sustain the body and regulate the metabolism, cardiac muscle is fundamental for blood movement within vessels and oxygen and nutrient distribution, and smooth muscle is involved in the maintenance of blood pressure and in the movement of the bolus within the intestine.

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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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