Inhibition of Proliferation, Migration, and Invasion of Keloid Fibroblasts By miR-183-5p Through Downregulating EGR1 / Inhibición de la Proliferación, Migración e Invasión de Fibroblastos Queloides por miR-183-5p Mediante la Regulación Negativa de EGR1

SUMMARY: Keloid scar is a unique benign fibroproliferative tumor of the human skin. Previously, it was reported that early growth response 1 (EGR1), a transcription factor, promotes keloid fibrosis; however, the mechanism by which EGR1 modulates keloid formation was not elaborated. In this research, the specific function and the microRNA (miRNA) regulatory network of EGR1 in keloids was examined. Keloid fibroblasts (KFs) were transfected with EGR1-small interfering RNA (siEGR1), EGR1-overexpression plasmid (pcDNA3.1-EGR1), and microRNA (miR-183-5p)-mimics to regulate the expression of EGR1 and miR-183-5p. The study employed dual-luciferase reporter assays to explore the targeting regulation of miR-183-5p on EGR1. Additionally, Western blotting, flow cytometry, qRT-PCR, cell count kit-8 (CCK-8), transwell, and wound healing assays, and RNA sequencing were conducted. EGR1 was upregulated in KFs, and EGR1 silencing diminished proliferation, fibrosis, migration, invasion, and apoptosis of cells. In KFs, the expression of miR- 183-5p was reduced, leading to the inhibition of cell proliferation, migration, and invasion. Conversely, it enhanced apoptosis. By targeting EGR1, miR-183-5p partially counteracted the impact of EGR1 on migration, invasion, and fibrosis in KFs. The findings imply that miR-183-5p suppresses keloid formation by targeting EGR1. As a result, EGR1 holds promise as a potential therapeutic target for preventing and treating keloids.

La cicatriz queloide es un tumor fibroproliferativo benigno único de la piel humana. Anteriormente, se informó que la respuesta de crecimiento temprano 1 (EGR1), un factor de transcripción, promueve la fibrosis queloide; sin embargo, no se explicó el mecanismo por el cual EGR1 modula la formación de queloides. En esta investigación, se examinó la función específica y la red reguladora de microARN (miARN) de EGR1 en queloides. Se transfectaron fibroblastos queloides (KF) con ARN de interferencia pequeño de EGR1 (siEGR1), plásmido de sobreexpresión de EGR1 (pcDNA3.1-EGR1) y miméticos de microARN (miR-183-5p) para regular la expresión de EGR1 y miR-183. -5p. El estudio empleó ensayos de indicador de luciferasa dual para explorar la regulación dirigida de miR-183-5p en EGR1. Además, se realizaron pruebas de transferencia Western, citometría de flujo, qRT-PCR, kit de recuento celular-8 (CCK-8), transwell y curación de heridas, y secuenciación de ARN. EGR1 estaba regulado positivamente en KF, y el silenciamiento de EGR1 disminuyó la proliferación, fibrosis, migración, invasión y apoptosis de las células. En KF, la expresión de miR- 183-5p se redujo, lo que llevó a la inhibición de la proliferación, migración e invasión celular. Por el contrario, mejoró la apoptosis. Al apuntar a EGR1, miR-183-5p contrarrestó parcialmente el impacto de EGR1 en la migración, invasión y fibrosis en KF. Los hallazgos implican que miR-183-5p suprime la formación de queloides al apuntar a EGR1. Como resultado, EGR1 es prometedor como objetivo terapéutico potencial para prevenir y tratar los queloides.

Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury / 中华医学杂志(英文版)

BACKGROUND@#Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys.@*METHODS@#In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining.@*RESULTS@#15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI.@*CONCLUSION@#Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.

FARSB stratifies prognosis and cold tumor microenvironment across different cancer types: an integrated single cell and bulk RNA sequencing analysis / 南方医科大学学报

OBJECTIVE@#Immunotherapy has brought significant clinical benefits to a subset of patients, but has thus far been disappointing in the treatment of immunologically "cold" tumors. Existing biomarkers that can precisely identify these populations are insufficient. In this context, a potential cold tumor microenvironment (TME) marker FARSB was investigated to reveal its impact on TME and patients' response to immunotherapy across pan-cancer.@*METHODS@#The expression levels and mutational landscape of FARSB in pan-cancer were investigated. Kaplan-Meier and univariate Cox regression analyses were applied to analyze the prognostic significance of FARSB. Pathways affected by FARSB were investigated by gene set enrichment and variation analysis. The relationship between FARSB expression and immune infiltration was examined using the TIMER2 and R packages. Single-cell RNA sequencing (scRNA-seq) data of several cancer types from GSE72056, GSE131907, GSE132465, GSE125449 and PMID32561858 were analyzed to validate the impact of FARSB on the TME. The predictive effect of FARSB on immunotherapy efficacy was explored in 3 immune checkpoint inhibitors (ICIs)- treated cohorts (PMID32472114, GSE176307, and Riaz2017).@*RESULTS@#FARSB expression was significantly higher in 25 tumor tissues than in normal tissues and was associated with poor prognosis in almost all tumor types. FARSB expression exhibited a strong association with several DNA damage repair pathways and was significantly associated with TP53 mutation in lung adenocarcinoma (P < 0.0001, OR=2.25). FARSB characterized a typical immune desert TME and correlated with impaired expression of chemokines and chemokines receptors. Large-scale scRNA-seq analysis confirmed the immunosuppressive role of FARSB and revealed that FARSB potentially shapes the cold TME by impeding intercellular interactions. In 3 ICI-treated cohorts, FARSB demonstrated predictive value for immunotherapy.@*CONCLUSION@#This study provides a pan-cancer landscape of the FARSB gene by integrated single-cell and bulk DNA sequencing analysis and elucidates its biological function to promote DNA damage repair and construct the immune desert TME, suggesting the potential value of FARSB as a novel marker for stratifying patients with poor immunotherapeutic benefits and "cold" TME.

Acute Developmental Toxicity of Panax notoginseng in Zebrafish Larvae / 中国结合医学杂志

OBJECTIVE@#To evaluate toxicity of raw extract of Panax notoginseng (rPN) and decocted extract of PN (dPN) by a toxicological assay using zebrafish larvae, and explore the mechanism by RNA sequencing assay.@*METHODS@#Zebrafish larvae was used to evaluate acute toxicity of PN in two forms: rPN and dPN. Three doses (0.5, 1.5, and 5.0 µ g/mL) of dPN were used to treat zebrafishes for evaluating the developmental toxicity. Behavior abnormalities, body weight, body length and number of vertebral roots were used as specific phenotypic endpoints. RNA sequencing (RNA-seq) assay was applied to clarify the mechanism of acute toxicity, followed by real time PCR (qPCR) for verification. High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb.@*RESULTS@#The acute toxicity result showed that rPN exerted higher acute toxicity than dPN in inducing death of larval zebrafishes (P<0.01). After daily oral intake for 21 days, dPN at doses of 0.5, 1.5 and 5.0 µ g/mL decreased the body weight, body length, and vertebral number of larval zebrafishes, indicating developmental toxicity of dPN. No other adverse outcome was observed during the experimental period. RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes, of which carboxypeptidase A1 (cpa1) and opioid growth factor receptor-like 2 (ogfrl2) were identified as functional genes in regulating body development of zebrafishes. qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2 (both P<0.01), verifying cpa1 and ogfrl2 as target genes for dPN.@*CONCLUSION@#This report uncovers the developmental toxicity of dPN, suggesting potential risk of its clinical application in children.

Study of the unique cellular molecular characteristics of moderately intrauterine adhesion based on single-cell RNA sequencing / 中华医学遗传学杂志

OBJECTIVE@#To depict the cell landscape and molecular biological characteristics of human intrauterine adhesion (IUA) so as to better understand its immune microenvironment and provide new inspirations for clinical treatment.@*METHODS@#Four patients with IUA who underwent hysteroscopic treatment at Dongguan Maternal and Child Health Care Hospital from February 2022 to April 2022 were selected as the study subjects. Hysteroscopy was used to collect the tissues of IUA, which were graded based on the patient's medical history, menstrual history and status of IUA. Library construction, sequencing, single cell data comparison and gene expression matrix construction were carried out in strict accordance with the single cell RNA sequencing process. Thereafter, the UMAP dimension reduction analysis of cell population and genetic analysis were carried out based on the cell types.@*RESULTS@#A total of 27 511 cell transcripts were obtained from four moderately graded IUA tissue samples and assigned to six cell lineages including T cells, mononuclear phagocytes, epithelial cells, fibroblasts, endothelial cells and erythrocytes. Compared with normal uterine tissue cells, the four samples showed different cell distribution, and the proportions of mononuclear phagocytes and T cells in sample IUA0202204 were significantly increased, suggesting a strong cellular immune response.@*CONCLUSION@#The cell diversity and heterogeneity of moderate IUA tissues have been described. Each cell subgroup has unique molecular characteristics, which may provide new clues for further study of the pathogenesis of IUA and heterogeneity among the patients.

Alterações sistêmicas na assinatura do ciclo celular de pacientes com COVID-19 / Systemic changes in the cell cycle signature of patients with COVID-19

Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Secuenciación de próxima generación para la detección de patógenos en cirugía de cadera: experiencia y viabilidad diagnóstica en un centro de atención terciaria de la Argentina / Next generation sequencing for the detection of pathogens in hip surgery. Experience and diagnostic feasibility in a tertiary care center in Argentina

Introducción: El diagnóstico rápido y definitivo con identificación del patógeno es fundamental cuando hay una infección periprotésica. La secuenciación de próxima generación permite identificar el ADN en un germen determinado en poco tiempo. Hasta donde sabemos, no hay reportes sobre su empleo para el manejo de la infección periprotésica en Sudamérica. Nuestro objetivo fue demostrar la viabilidad diagnóstica de las muestras obtenidas de una serie de pacientes operados en Buenos Aires, Argentina, y analizadas con la técnica de secuenciación de próxima generación. materiales y métodos: Se analizó a una serie prospectiva de 20 pacientes sometidos a cirugía de revisión séptica y aséptica de cadera desde diciembre de 2019 hasta marzo de 2020. Se obtuvieron muestras intraoperatorias de líquido sinovial, tejido profundo y canal endomedular, que fueron enviadas para su análisis al laboratorio NexGen Microgen. Resultados: Se seleccionaron 17 pacientes, porque tenían una muestra apta para analizar. Los resultados se recibieron dentro de las 72 h de la cirugía. En un caso, el resultado de la secuenciación de próxima generación informó un germen distinto del identificado en los cultivos posoperatorios de partes blandas, esto permitió corregir la antibioticoterapia. En otro, esta técnica identificó Parabacteroides gordonii en una revisión aséptica, en otro, Morganella morganii, a partir de cultivos negativos en una revisión en un tiempo. Conclusión: Se demostró la viabilidad diagnóstica con la secuenciación de próxima generación, se pueden obtener resultados de microorganismos patógenos dentro de las 72 h posteriores a la cirugía en pacientes con infección periprotésica y cultivos negativos. Nivel de Evidencia: IV

Introduction: Early diagnosis of a periprosthetic joint infection (PJI) and identification of the pathogen are paramount. Next-generation sequencing (NGS) can identify the nucleic acids in a given germ in a short period. To our knowledge, there are no reports of its use in the management of PJI in South America. Our objective was to demonstrate the diagnostic feasibility of the NGS technique on the samples obtained from a series of patients operated on in Buenos Aires, Argentina. Materials and methods: A prospective series of 20 patients undergoing septic and aseptic hip revision surgery from December 2019 to March 2020 was analyzed. Intraoperative samples of synovial fluid, deep tissue, and intramedullary canal were obtained and sent to the NexGen Microgen laboratory (Texas, USA) for analysis. Results: Seventeen patients were finally eligible to present a sample suitable for analysis. In 100% of the samples, NGS results were obtained within 72 hours of surgery. In one case, the NGS result reported a germ different from the one identified in the postoperative soft tissue cultures, allowing antibiotic therapy to be corrected. In another case, NGS identified Parabacteroides gordonii in aseptic revision surgery. In another patient, the NGS identified Morganella morganii, in which conventional postoperative cultures were negative in single-stage revision surgery. Conclusion: In this study, we demonstrated the diagnostic feasibility of NGS, obtaining results within 72 hours immediately after surgery for pathogenic organisms in patients with PJI and negative cultures. Level of Evidence: IV

Application of CRISPR-Cas9 to interrogate novel gene functions in cutaneous melanoma

Melanoma accounts for 3% of skin neoplasms and is the leading cause of death from skin disorders worldwide. The high mortality rate associated with this disease stems from the high capacity of melanoma patients to develop metastases and treatment relapse with inhibitors of the MAPK signaling pathway (such as BRAF inhibitors), commonly used in melanoma therapy. Thus, the investigation of genes involved in the mechanisms of melanoma development is essential for new and more effective therapeutic strategies. Hence, we describe in this thesis two projects involving the genes SIN3B and IRF4 as possible biomarkers for cutaneous melanoma. Initially, through bioinformatics analyses performed by our group, an upregulation of SIN3B was found in metastatic melanomas. This result together with the understanding of SIN3B role in regulating gene expression and oncogenic transformation, prompted us to describe in this thesis some mechanisms by which SIN3B may influence melanoma development. We then sought to characterize the gene function using SIN3B-deleted cells, generated by the CRISPR-Cas9 methodology. Initially, we observed increased SIN3B expression in BRAF-mutant metastatic melanomas, where we noted that the long splicing variant of the gene (NM_001297595.1) was effectively prevalent in melanomas. Subsequently, we designed gRNAs between the exons 2 and 3 of the human SIN3B gene and engineered three knockout clones and three control clones (containing empty lentiCRISPRv2 plasmid) from different melanoma cell lines (SKMEL28, A2058, and A375). Through functional analyses, it was observed that the absence of the gene did not interfere in the proliferation of tumor cells; however, it led to a decrease in invasive properties. These results were verified by Boyden chamber assays and transcriptome analysis (total RNA sequencing of deleted cells), where a decrease in migration and motility pathways was observed. Additionally, a screening of synthetically lethal genes with SIN3B was performed with a genome wide CRISPR library. These results showed that USP7 and STK11 genes, which belong to the FoxO signaling pathway, were essential in SIN3B-depleted melanoma cells. Finally, through a collaborative project with the Wellcome Trust Sanger Institute, previous large-scale sequencing analyses demonstrated that deletion of the IRF4 gene was lethal for melanoma cells. Accordingly, we performed IRF4 silencing in vitro and noticed that the lack of IRF4 promotes cell death and apoptosis, independently of MYC and MITF, known in the literature to be downstream targets of this gene. Therefore, these data suggest that IRF4 plays a vital role in melanoma cell survival. Taken together, both works herein described in this thesis demonstrate how CRISPR-Cas9 can be applied to study the functions and mechanisms of genes involved in melanoma progression, collectively helping in the development of more effective therapeutic strategies for this tumor

O melanoma representa 3% dos tipos de neoplasias cutâneas e é a maior causa das mortes por distúrbios de pele no mundo. A alta taxa de mortalidade associada à essa doença advém da alta capacidade de pacientes com melanoma desenvolverem metástases, e apresentarem recidiva após tratamento com inibidores da via de sinalização MAPK (como da proteína BRAF), comumente utilizados no tratamento de pacientes metastáticos. Assim, a investigação de genes envolvidos nos mecanismos de desenvolvimento do melanoma é primordial para novas estratégias terapêuticas mais efetivas. Dessa forma, descrevemos no presente trabalho dois projetos envolvendo os genes SIN3B e IRF4 como possíveis biomarcadores para melanoma cutâneo. Em análises prévias de bioinformática realizados pelo nosso grupo, SIN3B foi identificado tendo maior expressão em melanomas metastáticos. Além disso, diversos estudos mostraram que o gene está envolvido na regulação da expressão gênica e transformação oncogênica. Dessa forma, descrevemos nessa tese alguns mecanismos pelos quais SIN3B pode influenciar no desenvolvimento do melanoma, através da caracterização funcional de células SIN3B-deletadas pela metodologia CRISPR-Cas9. Inicialmente, observamos aumento na expressão de SIN3B em melanomas metastáticos BRAF-mutados, onde notamos que a variante de splicing longa do gene (NM_001297595.1), era efetivamente prevalente em melanomas. Assim, desenhamos sequências de RNA guias entre os éxons 2 e 3 do gene SIN3B humano e, obtivemos três clones knockout e outros três clones controle (contendo plasmídeo vazio) em diferentes linhagens de melanoma (SKMEL28, A2058 e A375), para caracterização funcional. Observou-se que a ausência do gene não interferiu na proliferação das células tumorais, contudo, acarretou na diminuição de processos invasivos. Esses resultados foram averiguados através de ensaios em câmara de Boyden e análises de transcriptoma (sequenciamento de RNA total das células deletadas), onde notou-se diminuição das vias de migração e motilidade. Adicionalmente, um rastreamento de genes sinteticamente letais com SIN3B foi realizado com uma biblioteca de CRISPR capaz de silenciar todo o genoma. Esses resultados mostraram que os genes USP7 e STK11, ambos pertencentes à via de sinalização de FoxO, são essenciais nas células SIN3B deletadas. Por fim, através de um projeto colaborativo com o Wellcome Trust Sanger Institute, análises prévias de sequenciamento de larga escala demonstraram que a deleção do gene IRF4 era letal para células de melanoma. Dessa forma, realizamos o silenciamento de IRF4 in vitro e notamos que a ausência do gene promove morte celular e apoptose, independentemente de MYC e MITF, conhecidos na literatura por serem alvos downstream do gene. Portanto, esses dados sugerem que IRF4 tem um papel importante na sobrevivência de células de melanoma. Em conjunto, ambos trabalhos descritos nessa tese, demonstram como a metodologia CRISPR-Cas9 pode auxiliar no entendimento de processos importantes para a malignidade do melanoma e contribuir para estratégias terapêuticas mais efetivas para esse tumor

Cancer biology deciphered by single-cell transcriptomic sequencing

Tumors are complex ecosystems in which heterogeneous cancer cells interact with their microenvironment composed of diverse immune, endothelial, and stromal cells. Cancer biology had been studied using bulk genomic and gene expression profiling, which however mask the cellular diversity and average the variability among individual molecular programs. Recent advances in single-cell transcriptomic sequencing have enabled a detailed dissection of tumor ecosystems and promoted our understanding of tumorigenesis at single-cell resolution. In the present review, we discuss the main topics of recent cancer studies that have implemented single-cell RNA sequencing (scRNA-seq). To study cancer cells, scRNA-seq has provided novel insights into the cancer stem-cell model, treatment resistance, and cancer metastasis. To study the tumor microenvironment, scRNA-seq has portrayed the diverse cell types and complex cellular states of both immune and non-immune cells interacting with cancer cells, with the promise to discover novel targets for future immunotherapy.

Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Application of single-cell transcriptome sequencing in mechanistic toxicology / 中华预防医学杂志

Traditional bulk RNA sequencing assesses the average expression level of genes in tissues rather than the differences in cellular responses. Accordingly, it is hard to differentiate sensitive responding cells, leading to inaccurate identification of toxicity pathways. Single-cell RNA sequencing (scRNA-seq) isolated single cells from tissue and subjected them to cell subtypes-specific transcriptome analysis. This technique in toxicological studies realizes the heterogeneous cellular responses in the tissue microenvironment upon chemical exposure. Thus it helps to identify sensitive responding cells and key molecular events, providing a powerful tool and a new perspective for exploring the mechanisms of toxicity and the modes of action. This review summarizes the development, principle, method, application and limitations of scRNA-seq in mechanistic toxicological researches, and discusses the prospect of multi-directional applications.

Investigation on the growth factor regulatory network of dermal fibroblasts in mouse full-thickness skin defect wounds based on single-cell RNA sequencing / 中华烧伤杂志

Objective: To explore the heterogeneity and growth factor regulatory network of dermal fibroblasts (dFbs) in mouse full-thickness skin defect wounds based on single-cell RNA sequencing. Methods: The experimental research methods were adopted. The normal skin tissue from 5 healthy 8-week-old male C57BL/6 mice (the same mouse age, sex, and strain below) was harvested, and the wound tissue of another 5 mice with full-thickness skin defect on the back was harvested on post injury day (PID) 7. The cell suspension was obtained by digesting the tissue with collagenase D and DNase Ⅰ, sequencing library was constructed using 10x Genomics platform, and single-cell RNA sequencing was performed by Illumina Novaseq6000 sequencer. The gene expression matrices of cells in the two kinds of tissue were obtained by analysis of Seurat 3.0 program of software R4.1.1, and two-dimensional tSNE plots classified by cell group, cell source, and gene labeling of major cells in skin were used for visual display. According to the existing literature and the CellMarker database searching, the expression of marker genes in the gene expression matrices of cells in the two kinds of tissue was analyzed, and each cell group was numbered and defined. The gene expression matrices and cell clustering information were introduced into CellChat 1.1.3 program of software R4.1.1 to analyze the intercellular communication in the two kinds of tissue and the intercellular communication involving vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) signal pathways in the wound tissue, the relative contribution of each pair of FGF subtypes and FGF receptor (FGFR) subtypes (hereinafter referred to as FGF ligand receptor pairs) to FGF signal network in the two kinds of tissue, and the intercellular communication in the signal pathway of FGF ligand receptor pairs with the top 2 relative contributions in the two kinds of tissue. The normal skin tissue from one healthy mouse was harvested, and the wound tissue of one mouse with full-thickness skin defect on the back was harvested on PID 7. The multiple immunofluorescence staining was performed to detect the expression and distribution of FGF7 protein and its co-localized expression with dipeptidyl peptidase 4 (DPP4), stem cell antigen 1 (SCA1), smooth muscle actin (SMA), and PDGF receptor α (PDGFRα) protein. Results: Both the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7 contained 25 cell groups, but the numbers of cells in each cell group between the two kinds of tissue were different. Genes PDGFRα, platelet endothelial cell adhesion molecule 1, lymphatic endothelial hyaluronic acid receptor 1, receptor protein tyrosine phosphatase C, keratin 10, and keratin 79 all had distinct distributions on two-dimensional tSNE plots, indicating specific cell groups respectively. The 25 cell groups were numbered by C0-C24 and divided into 9 dFb subgroups and 16 non-dFb groups. dFb subgroups included C0 as interstitial progenitor cells, C5 as adipose precursor cells, and C13 as contractile muscle cells related fibroblasts, etc. Non-dFb group included C3 as neutrophils, C8 as T cells, and C18 as erythrocytes, etc. Compared with that of the normal skin tissue of healthy mice, the intercellular communication in the wound tissue of full-thickness skin defected mice on PID 7 was more and denser, and the top 3 cell groups in intercellular communication intensity were dFb subgroups C0, C1, and C2, of which all communicated with other cell groups in the wound tissue. In the wound tissue of full-thickness skin defected mice on PID 7, VEGF signals were mainly sent by the dFb subgroup C0 and received by vascular related cell groups C19 and C21, PDGF signals were mainly sent by peripheral cells C14 and received by multiple dFb subgroups, EGF signals were mainly sent by keratinocyte subgroups C9 and C11 and received by the dFb subgroup C0, and the main sender and receiver of FGF signals were the dFb subgroup C6. In the relative contribution rank of FGF ligand receptor pairs to FGF signal network in the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7, FGF7-FGFR1 was the top 1, and FGF7-FGFR2 or FGF10-FGFR1 was in the second place, respectively; compared with those in the normal skin tissue, there was more intercellular communication in FGF7-FGFR1 signal pathway, while the intercellular communication in FGF7-FGFR2 and FGF10-FGFR1 signal pathways decreased slightly or did not change significantly in the wound tissue; the intercellular communication in FGF7-FGFR1 signal pathway in the wound tissue was stronger than that in FGF7-FGFR2 or FGF10-FGFR1 signal pathway; in the two kinds of tissue, FGF7 signal was mainly sent by dFb subgroups C0, C1, and C2, and received by dFb subgroups C6 and C7. Compared with that in the normal skin tissue of healthy mouse, the expression of FGF7 protein was higher in the wound tissue of full-thickness skin defected mouse on PID 7; in the normal skin tissue, FGF7 protein was mainly expressed in the skin interstitium and also expressed in the white adipose tissue near the dermis layer; in the two kinds of tissue, FGF7 protein was co-localized with DPP4 and SCA1 proteins and expressed in the skin interstitium, co-localized with PDGFRα protein and expressed in dFbs, but was not co-localized with SMA protein, with more co-localized expression of FGF7 in the wound tissue than that in the normal skin tissue. Conclusions: In the process of wound healing of mouse full-thickness skin defect wound, dFbs are highly heterogeneous, act as potential major secretory or receiving cell populations of a variety of growth factors, and have a close and complex relationship with the growth factor signal pathways. FGF7-FGFR1 signal pathway is the main FGF signal pathway in the process of wound healing, which targets and regulates multiple dFb subgroups.

Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window / Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing / 中国实验血液学杂志

OBJECTIVE@#To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation.@*METHODS@#The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry.@*RESULTS@#A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased.@*CONCLUSION@#CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.

Application prospects of single-cell transcriptome sequencing in traditional Chinese medicine research / 中国中药杂志

Single-cell transcriptome sequencing(scRNA-seq) can be used to analyze the expression characteristics of the transcriptome at the level of individual cell, and discover the heterogeneity of gene expression in individual cell that is "diluted" or averaged in study of group organization. The scRNA-seq, with the characteristics of standardization, high-throughput, and high integration, can greatly simplify the experimental operation and significantly reduce the consumption of reagents. At the same time, a variety of cells are screened and the gene expression patterns are analyzed at the single-cell level to provide a more efficient detection technique and more rich and accurate information for drug research. In the field of traditional Chinese medicine(TCM), the scRNA-seq is still a new technology, but the individual and precision concepts embodied by scRNA-seq and the theory of TCM syndrome differentiation and treatment have reached the same effect between the micro and macro aspects. This study tried to broaden the thinking for the modernization of TCM by introducing the development of scRNA-seq technology and its application in modern drug research and discussing the application prospects of scRNA-seq in TCM research.

Single-cell analysis of angiotensin-converting enzyme II expression in human kidneys and bladders reveals a potential route of 2019 novel coronavirus infection / 中华医学杂志(英文版)

BACKGROUND@#Since 2019, a novel coronavirus named 2019 novel coronavirus (2019-nCoV) has emerged worldwide. Apart from fever and respiratory complications, acute kidney injury has been observed in a few patients with coronavirus disease 2019. Furthermore, according to recent findings, the virus has been detected in urine. Angiotensin-converting enzyme II (ACE2) has been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same as that for the severe acute respiratory syndrome. This study aimed to investigate the possible cause of kidney damage and the potential route of 2019-nCoV infection in the urinary system.@*METHODS@#We used both published kidney and bladder cell atlas data and new independent kidney single-cell RNA sequencing data generated in-house to evaluate ACE2 gene expression in all cell types in healthy kidneys and bladders. The Pearson correlation coefficients between ACE2 and all other genes were first generated. Then, genes with r values larger than 0.1 and P values smaller than 0.01 were deemed significant co-expression genes with ACE2.@*RESULTS@#Our results showed the enriched expression of ACE2 in all subtypes of proximal tubule (PT) cells of the kidney. ACE2 expression was found in 5.12%, 5.80%, and 14.38% of the proximal convoluted tubule cells, PT cells, and proximal straight tubule cells, respectively, in three published kidney cell atlas datasets. In addition, ACE2 expression was also confirmed in 12.05%, 6.80%, and 10.20% of cells of the proximal convoluted tubule, PT, and proximal straight tubule, respectively, in our own two healthy kidney samples. For the analysis of public data from three bladder samples, ACE2 expression was low but detectable in bladder epithelial cells. Only 0.25% and 1.28% of intermediate cells and umbrella cells, respectively, had ACE2 expression.@*CONCLUSION@#This study has provided bioinformatics evidence of the potential route of 2019-nCoV infection in the urinary system.

From bulk, single-cell to spatial RNA sequencing / 国际口腔科学杂志·英文版

RNA sequencing (RNAseq) can reveal gene fusions, splicing variants, mutations/indels in addition to differential gene expression, thus providing a more complete genetic picture than DNA sequencing. This most widely used technology in genomics tool box has evolved from classic bulk RNA sequencing (RNAseq), popular single cell RNA sequencing (scRNAseq) to newly emerged spatial RNA sequencing (spRNAseq). Bulk RNAseq studies average global gene expression, scRNAseq investigates single cell RNA biology up to 20,000 individual cells simultaneously, while spRNAseq has ability to dissect RNA activities spatially, representing next generation of RNA sequencing. This article highlights these technologies, characteristic features and suitable applications in precision oncology.

A review on integration methods for single-cell data / 生物医学工程学杂志

The emergence of single-cell sequencing technology enables people to observe cells with unprecedented precision. However, it is difficult to capture the information on all cells and genes in one single-cell RNA sequencing (scRNA-seq) experiment. Single-cell data of a single modality cannot explain cell state and system changes in detail. The integrative analysis of single-cell data aims to address these two types of problems. Integrating multiple scRNA-seq data can collect complete cell types and provide a powerful boost for the construction of cell atlases. Integrating single-cell multimodal data can be used to study the causal relationship and gene regulation mechanism across modalities. The development and application of data integration methods helps fully explore the richness and relevance of single-cell data and discover meaningful biological changes. Based on this, this article reviews the basic principles, methods and applications of multiple scRNA-seq data integration and single-cell multimodal data integration. Moreover, the advantages and disadvantages of existing methods are discussed. Finally, the future development is prospected.

Computational prediction and characterisation of miRNAs and their pathway genes in human schistosomiasis caused by Schistosoma haematobium

BACKGROUND Key genes control the infectivity of the Schistosoma haematobium causing schistosomiasis. A method for understanding the regulation of these genes might help in developing new disease strategies to control schistosomiasis, such as the silencing mediated by microRNAs (miRNAs). The miRNAs have been studied in schistosome species and they play important roles in the post-transcriptional regulation of genes, and in parasite-host interactions. However, genome-wide identification and characterisation of novel miRNAs and their pathway genes and their gene expression have not been explored deeply in the genome and transcriptome of S. haematobium. OBJECTIVES Identify and characterise mature and precursor miRNAs and their pathway genes in the S. haematobium genome. METHODS Computational prediction and characterisation of miRNAs and genes involved in miRNA pathway from S. haematobium genome on SchistoDB. Conserved domain analysis was performed using PFAM and CDD databases. A robust algorithm was applied to identify mature miRNAs and their precursors. The characterisation of the precursor miRNAs was performed using RNAfold, RNAalifold and Perl scripts. FINDINGS We identified and characterised 14 putative proteins involved in miRNA pathway including ARGONAUTE and DICER in S. haematobium. Besides that, 149 mature miRNAs and 131 precursor miRNAs were identified in the genome including novel miRNAs. MAIN CONCLUSIONS miRNA pathway occurs in the S. haematobium, including endogenous miRNAs and miRNA pathway components, suggesting a role of this type of non-coding RNAs in gene regulation in the parasite. The results found in this work will open up a new avenue for studying miRNAs in the S. haematobium biology in helping to understand the mechanism of gene silencing in the human parasite Schistosome.

CircPlant: An Integrated Tool for circRNA Detection and Functional Prediction in Plants / 基因组蛋白质组与生物信息学报·英文版

The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.