Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain

Nature communications

2021 Mar 08

Kupari, J;Usoskin, D;Parisien, M;Lou, D;Hu, Y;Fatt, M;Lönnerberg, P;Spångberg, M;Eriksson, B;Barkas, N;Kharchenko, PV;Loré, K;Khoury, S;Diatchenko, L;Ernfors, P;
PMID: 33686078 | DOI: 10.1038/s41467-021-21725-z

Distinct types of dorsal root ganglion sensory neurons may have unique contributions to chronic pain. Identification of primate sensory neuron types is critical for understanding the cellular origin and heritability of chronic pain. However, molecular insights into the primate sensory neurons are missing. Here we classify non-human primate dorsal root ganglion sensory neurons based on their transcriptome and map human pain heritability to neuronal types. First, we identified cell correlates between two major datasets for mouse sensory neuron types. Machine learning exposes an overall cross-species conservation of somatosensory neurons between primate and mouse, although with differences at individual gene level, highlighting the importance of primate data for clinical translation. We map genomic loci associated with chronic pain in human onto primate sensory neuron types to identify the cellular origin of chronic pain. Genome-wide associations for chronic pain converge on two different neuronal types distributed between pain disorders that display different genetic susceptibilities, suggesting both unique and shared mechanisms between different pain conditions.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche

Nature

2021 Mar 03

Wu, N;Sun, H;Zhao, X;Zhang, Y;Tan, J;Qi, Y;Wang, Q;Ng, M;Liu, Z;He, L;Niu, X;Chen, L;Liu, Z;Li, HB;Zeng, YA;Roulis, M;Liu, D;Cheng, J;Zhou, B;Ng, LG;Zou, D;Ye, Y;Flavell, RA;Ginhoux, F;Su, B;
PMID: 33658717 | DOI: 10.1038/s41586-021-03283-y

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

Tumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell

Science advances

2021 Feb 01

Kildisiute, G;Kholosy, WM;Young, MD;Roberts, K;Elmentaite, R;van Hooff, SR;Pacyna, CN;Khabirova, E;Piapi, A;Thevanesan, C;Bugallo-Blanco, E;Burke, C;Mamanova, L;Keller, KM;Langenberg-Ververgaert, KPS;Lijnzaad, P;Margaritis, T;Holstege, FCP;Tas, ML;Wijnen, MHWA;van Noesel, MM;Del Valle, I;Barone, G;van der Linden, R;Duncan, C;Anderson, J;Achermann, JC;Haniffa, M;Teichmann, SA;Rampling, D;Sebire, NJ;He, X;de Krijger, RR;Barker, RA;Meyer, KB;Bayraktar, O;Straathof, K;Molenaar, JJ;Behjati, S;
PMID: 33547074 | DOI: 10.1126/sciadv.abd3311

Neuroblastoma is a childhood cancer that resembles developmental stages of the neural crest. It is not established what developmental processes neuroblastoma cancer cells represent. Here, we sought to reveal the phenotype of neuroblastoma cancer cells by comparing cancer (n = 19,723) with normal fetal adrenal single-cell transcriptomes (n = 57,972). Our principal finding was that the neuroblastoma cancer cell resembled fetal sympathoblasts, but no other fetal adrenal cell type. The sympathoblastic state was a universal feature of neuroblastoma cells, transcending cell cluster diversity, individual patients, and clinical phenotypes. We substantiated our findings in 650 neuroblastoma bulk transcriptomes and by integrating canonical features of the neuroblastoma genome with transcriptional signals. Overall, our observations indicate that a pan-neuroblastoma cancer cell state exists, which may be attractive for novel immunotherapeutic and targeted avenues.

CRISPR Systems for COVID-19 Diagnosis

ACS sensors

2021 Jan 27

Rahimi, H;Salehiabar, M;Barsbay, M;Ghaffarlou, M;Kavetskyy, T;Sharafi, A;Davaran, S;Chauhan, SC;Danafar, H;Kaboli, S;Nosrati, H;Yallapu, MM;Conde, J;
PMID: 33502175 | DOI: 10.1021/acssensors.0c02312

The emergence of the new coronavirus 2019 (COVID-19) was first seen in December 2019, which has spread rapidly and become a global pandemic. The number of cases of COVID-19 and its associated mortality have raised serious concerns worldwide. Early diagnosis of viral infection undoubtedly allows rapid intervention, disease management, and substantial control of the rapid spread of the disease. Currently, the standard approach for COVID-19 diagnosis globally is the RT-qPCR test; however, the limited access to kits and associated reagents, the need for specialized lab equipment, and the need for highly skilled personnel has led to a detection slowdown. Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems has reshaped molecular diagnosis. The benefits of the CRISPR system such as speed, precision, specificity, strength, efficiency, and versatility have inspired researchers to develop CRISPR-based diagnostic and therapeutic methods. With the global COVID-19 outbreak, different groups have begun to design and develop diagnostic and therapeutic programs based on the efficient CRISPR system. CRISPR-based COVID-19 diagnostic systems have advantages such as a high detection speed (i.e., 30 min from raw sample to reach a result), high sensitivity and precision, portability, and no need for specialized laboratory equipment. Here, we review contemporary studies on the detection of COVID-19 based on the CRISPR system.

Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis

Nature communications

2021 Jan 13

Wu, CL;Dicks, A;Steward, N;Tang, R;Katz, DB;Choi, YR;Guilak, F;
PMID: 33441552 | DOI: 10.1038/s41467-020-20598-y

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Clinicopathological and prognostic significance of immunophenotypic characterization of endocervical adenocarcinoma using CLDN18, CDH17, and PAX8 in association with HPV status

Virchows Archiv : an international journal of pathology

2021 Sep 28

Asaka, S;Nakajima, T;Ida, K;Asaka, R;Kobayashi, C;Ito, M;Miyamoto, T;Uehara, T;Ota, H;
PMID: 34581850 | DOI: 10.1007/s00428-021-03207-1

In 2020, the WHO published a new system for classifying invasive endocervical adenocarcinoma based on histological features and high-risk human papillomavirus (HPV) infection. However, immunophenotypes of each histological subtype require further investigation. We immunohistochemically analyzed 66 invasive endocervical adenocarcinomas using three cell-lineage-specific markers: claudin 18 (CLDN18) for gastric, cadherin 17 (CDH17) for intestinal, and PAX8 for Müllerian epithelial cells. We identified five immunophenotypes of endocervical adenocarcinoma: gastric (21%); intestinal (14%); gastrointestinal (11%); Müllerian (35%); and not otherwise specified (NOS) (20%). Adenocarcinomas with gastric immunophenotype, characterized by aging (p = 0.0050), infrequent HPV infection (p < 0.0001), concurrent lobular endocervical glandular hyperplasia (p = 0.0060), lymphovascular invasion (p = 0.0073), advanced clinical stage (p = 0.0001), and the poorest progression-free (p < 0.0001) and overall (p = 0.0023) survivals, were morphologically compatible with gastric-type adenocarcinoma of the WHO 2020 classification. Conversely, most adenocarcinomas with Müllerian (91%) and intestinal (89%) immunophenotypes were HPV associated and morphologically compatible with usual- or intestinal-type adenocarcinomas of the WHO 2020 classification. The morphology of adenocarcinomas with gastrointestinal immunophenotype was intermediate or mixed between those of gastric and intestinal immunophenotypes; 57% were HPV associated. Adenocarcinomas with NOS immunophenotype were mainly HPV associated (85%) and histologically poorly differentiated. Multivariate analysis revealed that gastric (p = 0.008), intestinal + gastrointestinal (p = 0.0103), and NOS (p = 0.009) immunophenotypes were independent predictors of progression-free survival. Immunophenotypes characterized by CLDN18, CDH17, and PAX8 exhibited clinicopathological relevance and may improve the diagnostic accuracy and prognostic value of conventional histological classification.

Human papillomavirus status and prognosis of oropharyngeal high-grade neuroendocrine carcinoma

Oral oncology

2023 Jan 24

de Sousa, LG;Lazar Neto, F;Dal Lago, EA;Sikora, A;Hanna, E;Moreno, A;Phan, J;Glisson, BS;Bell, D;Ferrarotto, R;
PMID: 36702015 | DOI: 10.1016/j.oraloncology.2023.106311

The prognostic impact of human papillomavirus (HPV) infection or smoking on oropharyngeal high-grade neuroendocrine carcinoma (HG-NEC) is not established.Retrospective study with patients with oropharyngeal HG-NEC seen at MD Anderson Cancer Center from 1997 to 2020, and previously reported patients with oropharyngeal HG-NEC and known p16 and HPV status from the literature review. Survival was estimated with the Kaplan-Meier method, and survival differences assessed with the log-rank test and Cox proportional hazards models.Thirty patients were included; most had a heavy (≥10 pack-years) smoking history (52%), locoregional disease (stage III-IVB; 77%), and p16-positive tumor (92%). HPV was positive in 65% of tested samples (15/23). Of 24 patients treated with curative intent, the objective response rates was 90% (9/10) and 81% (17/21), respectively, for induction chemotherapy and definitive radiotherapy. During follow-up, 54% (13/24) recurred, mostly (11/13) at distant sites. Median overall survival (OS) was 46 months (95% CI, 14.3 - NA). OS was not associated with HPV status (HR 0.73, P = 0.6) or smoking (HR 1.16, P = 0.8). Among 63 patients with known HPV status after the literature review (19 HPV- and 44 HPV + ), HPV status remained unassociated with OS (P = 0.92).This is the largest retrospective cohort of oropharyngeal HG-NEC. Distant recurrence rate after curative treatment was high, suggesting that multimodality treatment including systemic therapy may benefit patients with locally advanced disease. HPV infection did not affect survival outcomes, therefore should not lead to therapy de-intensification for this histology.

Le virus Zika se réplique dans le rein fœtal

Néphrologie & Thérapeutique

2022 Sep 01

Dejucq-Rainsford, N;Robinet, G;Satie, A;Aubry, F;Rioux-Leclercq, N;Lavoué, V;Vigneau, C;Mazaud-Guittot, S;
| DOI: 10.1016/j.nephro.2022.07.200

Introduction Le virus ZIKA (ZIKV) est un virus transmis par les moustiques et par le sperme, avec un fort potentiel d’émergence. Lors d’une infection pendant la grossesse, ce virus peut entraîner des anomalies fœtales cérébrales mais aussi uro-génitales, comme révélé lors de l’épidémie de 2015-2016 dans les Amériques. Description L’objectif de notre étude est de déterminer la permissivité du rein fœtal au ZIKV et les conséquences de cette infection. Méthodes Pour cela nous avons infecté ex vivo avec ZIKV des cultures organotypiques de reins fœtaux disséqués à partir de produits d’IVG obtenus entre 11 et 14 semaines d’aménorrhée. Résultats Nos résultats montrent que le ZIKV se réplique efficacement dans le rein fœtal, comme attesté par l’augmentation de l’ARN viral dans les cultures au cours du temps et par la détection in situ en RNAscope de l’ARN brin négatif produit lors de la réplication du virus. L’ARN réplicatif du ZIKV a été retrouvé dans le tissu interstitiel ainsi que dans des tubules et des glomérules en formation. Les cellules cibles du virus ont été identifiées par immunohistochimie à l’aide d’anticorps contre la protéine virale non structurale NS2b et contre des marqueurs cellulaires. Le virus est retrouvé au niveau du compartiment interstitiel dans des macrophages CD68+ et des fibroblastes SMA+ et au niveau des cellules épithéliales tubulaires CK18+. La localisation dans des cellules glomérulaires WT1+ reste à déterminer. L’infection virale n’a pas d’effet délétère majeur sur la morphologie, la viabilité et la prolifération cellulaire du rein à 6 jours post-infection. Conclusion En conclusion, ces résultats révèlent pour la première fois que le rein fœtal est permissif au virus Zika. Il serait nécessaire d’évaluer l’effet à plus long terme de l’infection sur le rein en développement. Notre modèle ex vivo pourrait permettre de tester l’efficacité d’antiviraux visant à empêcher la réplication du ZIKV dans le rein foetal.

Low Grade Papillary Sinonasal (Schneiderian) Carcinoma: A Series of Five Cases of a Unique Malignant Neoplasm with Comparison to Inverted Papilloma and Conventional Nonkeratinizing Squamous Cell Carcinoma

Head and neck pathology

2021 May 26

Saab-Chalhoub, MW;Guo, X;Shi, Q;Chernock, RD;Lewis, JS;
PMID: 34041710 | DOI: 10.1007/s12105-021-01335-3

There have been a few case reports and one small series of low grade papillary sinonasal (Schneiderian) carcinomas (LGPSC) which mimic papillomas but have overtly invasive growth and which occasionally metastasize. We describe the morphologic, clinical, immunohistochemical, and molecular features of five patients with LGPSC compared with eight cases each of inverted papilloma (IP) and conventional nonkeratinizing squamous cell carcinoma (SCC) with papillary growth. All LGPSC were nested with predominantly pushing invasion, no stromal reaction, and frequent surface papillary growth. All consisted of one cell type only, with polygonal cells with round nuclei, no (or limited) cytologic atypia, low mitotic activity, and prominent neutrophilic infiltrate. One patient had slightly more infiltrative bone invasion, another lymphovascular, perineural, and skeletal muscle invasion, and a third nodal metastasis after 17 years. By comparison, IPs had bland cytology, neutrophilic microabscesses, mixed immature squamous, goblet cell, and respiratory epithelium, and extremely low mitotic activity. Nonkeratinizing SCCs had basaloid-appearing cells with nuclear pleomorphism, brisk mitotic activity, and apoptosis. All LGPSC were p63 positive. Mitotic activity and Ki67 indices were significantly higher for LGPSCs than IPs and significantly lower than NKSCCs, while p53 immunohistochemistry in LGPSC was identical to nonkeratinizing SCC and higher than for IP. Sequencing showed all five tumors to harbor a MUC6 mutation, one tumor to harbor CDKN2A and PIK3R1 mutations, and one tumor to harbor a NOTCH1 mutation. All LGPSC lacked EGFR and KRAS mutations and lacked copy number variations of any main cancer genes. At a median follow up of 12 months, two LGPSC recurred locally, and one patient died after massive local recurrences and nodal metastases. LGPSC is a distinct, de novo sinonasal carcinoma that can be differentiated from papillomas by morphology and selected immunohistochemistry.

Timing, number, and type of sexual partners associated with risk of oropharyngeal cancer

Cancer

2021 Jan 11

Drake, VE;Fakhry, C;Windon, MJ;Stewart, CM;Akst, L;Hillel, A;Chien, W;Ha, P;Miles, B;Gourin, CG;Mandal, R;Mydlarz, WK;Rooper, L;Troy, T;Yavvari, S;Waterboer, T;Brenner, N;Eisele, DW;D'Souza, G;
PMID: 33426652 | DOI: 10.1002/cncr.33346

Case-control studies from the early 2000s demonstrated that human papillomavirus-related oropharyngeal cancer (HPV-OPC) is a distinct entity associated with number of oral sex partners. Using contemporary data, we investigated novel risk factors (sexual debut behaviors, exposure intensity, and relationship dynamics) and serological markers on odds of HPV-OPC. HPV-OPC patients and frequency-matched controls were enrolled in a multicenter study from 2013 to 2018. Participants completed a behavioral survey. Characteristics were compared using a chi-square test for categorical variables and a t test for continuous variables. Adjusted odds ratios (aOR) were calculated using logistic regression. A total of 163 HPV-OPC patients and 345 controls were included. Lifetime number of oral sex partners was associated with significantly increased odds of HPV-OPC (>10 partners: odds ratio [OR], 4.3 [95% CI, 2.8-6.7]). After adjustment for number of oral sex partners and smoking, younger age at first oral sex (<18 vs >20 years: aOR, 1.8 [95% CI, 1.1-3.2]) and oral sex intensity (>5 sex-years: aOR, 2.8 [95% CI, 1.1-7.5]) remained associated with significantly increased odds of HPV-OPC. Type of sexual partner such as older partners when a case was younger (OR, 1.7 [95% CI, 1.1-2.6]) or having a partner who had extramarital sex (OR, 1.6 [95% CI, 1.1-2.4]) was associated with HPV-OPC. Seropositivity for antibodies to HPV16 E6 (OR, 286 [95% CI, 122-670]) and any HPV16 E protein (E1, E2, E6, E7; OR, 163 [95% CI, 70-378]) was associated with increased odds of HPV-OPC. Number of oral sex partners remains a strong risk factor for HPV-OPC; however, timing and intensity of oral sex are novel independent risk factors. These behaviors suggest additional nuances of how and why some individuals develop HPV-OPC.

Patch‐to‐Seq Reveals Unique Transcriptomic Profiles of Chemosensitive Serotonergic Neurons

The FASEB Journal

2021 Jan 01

Mouradian, G;Duffy, E;Liu, P;Gomez‐Vargas, J;
| DOI: 10.1096/fasebj.2021.35.S1.04448

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Prior data indicate that a sub-population of brainstem serotonin (5-HT) neurons are likely central respiratory chemoreceptors. However, it remains unclear which 5-HT neurons develop chemosensitivity and what molecular markers may identify this unique sub-population of 5-HT neurons. Here we employed the “patch-to-seq” technique to measure action potential firing rate responses of 5-HT neuron to hypercapnic acidosis using cell-attached patch-clamp electrophysiology followed by isolation of that neurons’ intracellular RNA content for single cell RNA Sequencing. Acute brainstem slices (200 µm) from young (P18-23) transgenic rats expressing eGFP in all 5-HT neurons (SSeGFP) were recorded while superfused with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D-AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). The Chemosensitivity Index (C.I.) determined cell phenotypes. pH-sensitive eGFP+ neurons (n=48) had an average C.I. of 169.8± 8.25 SEM whereas pH-insensitive eGFP+ neurons (n=44) had an average C.I. of 100.3 ± 1.4 (P < 0.0001; t-test). Cell extracts from 11 chemosensitive and 10 insensitive recorded cells were subjected to single cell RNA Sequencing (scRNA-Seq), from which an average of 48.3M reads were generated with a quality score of ~32.7 and ~75% mapping rate per sample with an average of ~8,000 genes detected. Purity of the isolated samples was confirmed by significantly more expression (Log2(FPKM+1)) of 6 serotonergic vs 7 neuronal and 8 glial gene markers, and more expression of 7 neuronal vs 8 known glial gene markers. There were 166differentially expressed genes (q < 0.05) between pH-sensitive and -insensitive 5-HT neuron populations none of which have a known contribution to pH regulation or sensing. Predicted upstream regulators of 352 DEGs (p

Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse

Proceedings of the National Academy of Sciences of the United States of America

2022 Nov 29

Sanders, TR;Kelley, MW;
PMID: 36409884 | DOI: 10.1073/pnas.2203935119

The afferent innervation of the cochlea is comprised of spiral ganglion neurons (SGNs), which are characterized into four subtypes (Type 1A, B, and C and Type 2). However, little is known about the factors and/or processes that determine each subtype. Here, we present a transcriptional analysis of approximately 5,500 single murine SGNs collected across four developmental time points. All four subtypes are transcriptionally identifiable prior to the onset of coordinated spontaneous activity, indicating that the initial specification process is under genetic control. Trajectory analysis indicates that SGNs initially split into two precursor types (Type 1A/2 and Type 1B/C), followed by subsequent splits to give rise to four transcriptionally distinct subtypes. Differential gene expression, pseudotime, and regulon analyses were used to identify candidate transcription factors which may regulate the subtypes specification process. These results provide insights into SGN development and comprise a transcriptional atlas of SGN maturation across the prenatal period.

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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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