Severe Type 2 Inflammation Leads to High Platelet-Activating-Factor-Associated Pathology in Chronic Rhinosinusitis with Nasal Polyps-A Hierarchical Cluster Analysis Using Bulk RNA Barcoding and Sequencing.

Platelet-activating factor (PAF) is a phospholipid-derived inflammatory mediator that triggers various inflammatory conditions, including eosinophil activation and recruitment. This study aimed to evaluate the expressions of PAF-metabolism-associated genes, namely genes coding the enzymes involved in PAF synthesis (LPCAT1, LPCAT2, LPCAT3, and LPCAT4), PAF degradation (PAFAH1B2, PAFAH1B3, and PAFAH2), and the gene for the PAF receptor (PTAFR) in subtypes of CRSwNP classified by clinical- or hierarchal-analysis-based classifications. Transcriptomic analysis using bulk RNA barcoding and sequencing (BRB-seq) was performed with CRSwNP, including eosinophilic CRS (ECRS) (n = 9), nonECRS (n = 8), ECRS with aspirin-exacerbated respiratory disease (Asp) (n = 3), and controls with a normal uncinate process mucosa (n = 6). PTAFR was only upregulated in ECRS and nonECRS. In the hierarchical cluster analysis with clusters 1 and 2 reflecting patients with low-to-moderate and high levels of type 2 inflammation, respectively, cluster 1 exhibited a significant downregulation of LPCAT2 and an upregulation of PTAFR expression, while cluster 2 showed an upregulation of LPCAT1, PAFAH1B2, and PTAFR and downregulation of PAFAH2 expression. Understanding this strong PAF-associated pathophysiology in the severe type 2 inflammation group could provide valuable insights into the treatment and management of CRSwNP.

Transcriptional profiling of the developing rat ovary following intrauterine exposure to the endocrine disruptors diethylstilbestrol and ketoconazole.

Exposure to endocrine-disrupting chemicals (EDCs) during development may cause reproductive disorders in women. Although female reproductive endpoints are assessed in rodent toxicity studies, a concern is that typical endpoints are not sensitive enough to detect chemicals of concern to human health. If so, measured endpoints must be improved or new biomarkers of effects included. Herein, we have characterized the dynamic transcriptional landscape of developing rat ovaries exposed to two well-known EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), by 3' RNA sequencing. Rats were orally exposed from day 7 of gestation until birth, and from postnatal day 1 until days 6, 14 or 22. Three exposure doses for each chemical were used: 3, 6 and 12 µg/kg bw/day of DES; 3, 6, 12 mg/kg bw/day of KTZ. The transcriptome changed dynamically during perinatal development in control ovaries, with 1137 differentially expressed genes (DEGs) partitioned into 3 broad expression patterns. A cross-species deconvolution strategy based on a mouse ovary developmental cell atlas was used to map any changes to ovarian cellularity across the perinatal period to allow for characterization of actual changes to gene transcript levels. A total of 184 DEGs were observed across dose groups and developmental stages in DES-exposed ovaries, and 111 DEGs in KTZ-exposed ovaries across dose groups and developmental stages. Based on our analyses, we have identified new candidate biomarkers for female reproductive toxicity induced by EDC, including Kcne2, Calb2 and Insl3.

Profiling the Atopic Dermatitis Epidermal Transcriptome by Tape Stripping and BRB-seq.

Tape stripping is a non-invasive skin sampling technique, which has recently gained use for the study of the transcriptome of atopic dermatitis (AD), a common inflammatory skin disorder characterized by a defective epidermal barrier and perturbated immune response. Here, we performed BRB-seq-a low cost, multiplex-based, transcriptomic profiling technique-on tape-stripped skin from 30 AD patients and 30 healthy controls to evaluate the methods' ability to assess the epidermal AD transcriptome. An AD signature consisting of 91 differentially expressed genes, specific for skin barrier and inflammatory response, was identified. The gene expression in the outermost layers, stratum corneum and stratum granulosum, of the skin showed highest correlation between tape-stripped skin and matched full-thickness punch biopsies. However, we observed that low and highly variable transcript counts, probably due to low RNA yield and RNA degradation in the tape-stripped skin samples, were a limiting factor for epidermal transcriptome profiling as compared to punch biopsies. We conclude that deep BRB-seq of tape-stripped skin is needed to counteract large between-sample RNA yield variation and highly zero-inflated data in order to apply this protocol for population-wide screening of the epidermal transcriptome in inflammatory skin diseases.

Distinct Gene Set Enrichment Profiles in Eosinophilic and Non-Eosinophilic Chronic Rhinosinusitis with Nasal Polyps by Bulk RNA Barcoding and Sequencing.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease with a high symptom burden, including nasal congestion and smell disorders. This study performed a detailed transcriptomic analysis in CRSwNP classified as eosinophilic CRS (ECRS), nonECRS according to the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) criteria, and a group of ECRS with comorbid aspirin intolerant asthma (Asp). Gene expression profiles of nasal polyps and the uncinate process in CRSwNP patients and normal subjects (controls) were generated by bulk RNA barcoding and sequencing (BRB-seq). A differentially expressed genes (DEGs) analysis was performed using DESeq2 software in iDEP to clarify any relationship between gene expression and disease backgrounds. A total of 3004 genes were identified by DEGs analysis to be associated with ECRS vs control, nonECRS vs control, and Asp vs control. A pathway analysis showed distinct profiles between the groups. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) showed distinct phenotype-specific pathways of expressed genes. In the specific pathway of "cytokine-cytokine receptor interaction", the differentially expressed genes were widely distributed. This study indicates that transcriptome analysis using BRB-seq may be a valuable tool to explore the pathogenesis of type 2 inflammation in CRSwNP.

Transcriptional landscape of human keratinocyte models exposed to 60-GHz millimeter-waves.

The use of millimeter waves (MMW) will exponentially grow in the coming years due to their future utilization in 5G/6G networks. The question of possible biological effects at these frequencies has been raised. In this present study, we aimed to investigate gene expression changes under exposure to MMW using the Bulk RNA Barcoding and sequencing (BRB-seq) technology. To address this issue, three exposure scenarios were performed aiming at: i) comparing the cellular response of two primary culture of keratinocytes (HEK and NHEK) and one keratinocyte derivate cell line (HaCaT) exposed to MMW; ii) exploring the incident power density dose-effect on gene expression in HaCaT cell line; and, iii) studying the exposure duration at the new ICNIRP exposure limit for the general population. With the exception of heat effect induced by high power MMW (over 10 mW/cm2), those exposure scenarios have not enabled us to demonstrate important gene expression changes in the different cell populations studied. Very few differentially genes were observed between MMW exposed samples and heat shock control, and most of them were significantly associated with heat shock response that may reflect small differences in the heat generation. Together these results show that acute exposure to MMW has no effects on the transcriptional landscape of human keratinocyte models under athermal conditions.

The anti-androgenic fungicide triticonazole induces region-specific transcriptional changes in the developing rat perineum and phallus.

Intrauterine exposure to endocrine disrupting chemicals can interfere with male reproductive development. This can lead to male reproductive disorders such as hypospadias, cryptorchidism and reduced fertility, as well as shorter anogenital distance (AGD) - a biomarker for incomplete androgen-dependent fetal masculinization. However, it remains challenging to predict adverse in vivo outcomes based on in vitro effect patterns for many chemicals. This is a challenge for modern toxicology, which aims to reduce animal testing for chemical safety assessments. To enable the transition towards higher reliance on alternative test methods, we need to better map underlying mechanisms leading to adverse effects. Herein, we have analyzed the transcriptome of the perineum and phallus of male fetal rats and defined the impacts of exposure to an anti-androgenic fungicide, triticonazole. Previously we have shown that developmental exposure to triticonazole can induce short male AGD, but without a marked effect on the transcriptome of the fetal testes. In contrast, we report here significant changes to the transcriptional landscape of the perineum and phallus, including regional differences between these adjacent tissues. This highlights the importance of analyzing the correct tissue when characterizing mechanisms of complex in vivo effect outcomes. Our results provide a rich resource for the spatiotemporal gene networks that are involved in the development of male external genitalia, and that can be disrupted upon exposure to chemicals that prevent normal masculinization of the perineum and phallus. Such data will be critical in the development of novel alternative test methods to determine the endocrine disrupting potential of existing and emerging chemicals.