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Epithelial cytokines are involved in the orchestration of T1/T2 inflammatory patterns. We question the persistence of this trait in air–liquid interface (ALI) epithelial cultures and whether this local orientation can be related to systemic patterns (e.g., blood eosinophil counts [BECs]). We investigated alarmin release related to high versus low T2 phenotypes associated with chronic airway diseases. ALIs were reconstituted from 32 control, 40 chronic obstructive pulmonary disease, and 20 asthmatic patients. Interleukin‐8 (IL‐8; a T1‐cytokine), IL‐25, IL‐33, and thymic stromal lymphopoietin (T2‐alarmins) concentrations were assessed in subnatants at steady state and used to explain blood neutrophil and eosinophil counts. IL‐25 and IL‐8 levels were highest in asthma ALI‐subnatants, whereas IL‐33 was sparsely detected. Thymic stromal lymphopoietin levels were similar among groups. All asthma cell cultures were T1‐high/T2‐high, while chronic obstructive pulmonary disease and controls tended to be mixed. BECs were independently explained by both disease and in‐culture T2‐alarmin levels, irrespective of the T2‐alarmin considered. The epithelial ALI‐T2 signature was more frequently high in patients with a BEC > 300/mm3. Despite removal from an in vivo environment for ≥2 months, ALIs release disease‐specific cytokine "cocktails" into their subnatants, suggesting continued persistence of alarmin orientation in differentiated cell line environments. [ABSTRACT FROM AUTHOR]

This paper examines women-exclusionary discourses on the popular anime Usenet newsgroup, rec.arts.anime. By going back to pre-2000 online anime histories, this paper proposes to understand how women-exclusionary discursive practices on rec.arts.anime have contributed to shaping contemporary toxic technocultures' discursive identities, as it is admitted that forum 4chan originated from online anime fandom. By using a data set of 252 messages related to gender issues posted from 1992 to 1996, I identify 7 discursive practices that I am theorizing here under the name of negative networking: 1. Blaming female anime fans for their lack of visibility; 2. Doubting the authentic interest of women in anime; 3. Mystifying the female anime fan; 4. Harassing female anime fans; 5. Criticizing the association of feminism with anime, both as interpretive practices and as scholarship; 6. Belittling female anime fans' concerns; and 7. Denying or ignoring the challenges faced by female anime fans. I argue that the impact of these discourses must be understood as determinant in the establishment of the online anime hegemonic fan identity and its prediscourses, especially as they relate to the long-lasting marginalization of women and gender diverse anime fans. [ABSTRACT FROM AUTHOR]

Background: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air–liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery. [ABSTRACT FROM AUTHOR]

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells. • Successful SCGB1A1 sorting according to specific intracellular marker. • Preserved RNA of sufficient quantity and quality for microarray experiment. • Pathways underlying both mucus production and ciliogenesis in SCGB1A1+ cells. • Signature of basal cells for SCGB1A1- cells. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing. [ABSTRACT FROM AUTHOR]

Fruit colour is central to the sensorial and nutritional quality of strawberry fruit and is therefore a major target in breeding programmes of the octoploid cultivated strawberry (Fragaria × ananassa). The red colour of the fruit is caused by the accumulation of anthocyanins, which are water-soluble flavonoids. To facilitate molecular breeding, here we have mapped with high resolution fruit colour quantitative trait loci (QTLs) (COLOUR, scored visually as in selection programmes) and associated flavonoid metabolic QTLs (5 anthocyanins compounds together with 8 flavonols and flavan-3-ols) to specific subgenomes of cultivated strawberry. Two main colour-related QTLs were located on the LG3A linkage group (Fragaria vesca subgenome). Genetic mapping, transcriptome analysis and whole genome sequencing enabled the detection of a homoeo-allelic variant of ANTHOCYANIDIN REDUCTASE (ANR) underlying the major male M3A COLOUR and pelargonidin-3-glucoside (PgGs) QTLs (up to ∼20% of explained variance). Consistent with previously published functional studies, ANR transcript abundance was inversely related with PgGs content in contrasted progeny individuals. Genetic segregation analyses further indicated that a molecular marker designed using an 18 bp deletion found in the 5′UTR of the candidate ANR homoeo-allelic variant is effective in identifying genotypes with intense red fruit colour. Our study provides insights into the genetic and molecular control of colour-related traits in strawberry and further defines a genetic marker for marker-assisted selection of new strawberry varieties with improved colour. The QTLs detected and the underlying candidate genes are different from those described to date, emphasising the importance of screening a wide diversity of genetic resources in strawberry. [ABSTRACT FROM AUTHOR]

The effect of cultivar and environmental variations and their interaction on anthocyanin components of strawberry were assessed for six cultivars grown in five locations from North to South of Europe in two different years. To evaluate the impact of latitude- and altitude-related factors, daily mean (Tmean), maximum (Tmax) and minimum (Tmin) temperature and global radiation accumulated for 3, 5, 10 and 15 days before fruit sampling, was analyzed. In general, fruits grown in the south were more enriched in total anthocyanin and pelargonidin-3-glucoside (pel-3-glc), the most abundant anthocyanin in strawberry. Principal component analysis (PCA) provided a separation of the growing locations within a cultivar due to latitudinal climatic differences, temporary weather changes before fruit collection and cultivation technique. PCA also depicted different patterns for anthocyanin distribution indicating a cultivar specific reaction on the environmental factors. The linear regression analysis showed that pel-3-glc was relatively less affected by these factors, while the minor anthocyanins cyanidin-3-glucoside, cyanidin-3-(6-O-malonyl)-glucoside, pelargonidin-3-rutinoside and pelargonidin-3-(6-O-malonoyl)-glucoside were sensitive to Tmax. The global radiation strongly increased cya-3-mal-glc in 'Frida' and pel-3-rut in 'Frida' and 'Florence'. 'Candonga' accumulated less pel-3-glc and total anthocyanin with increased global radiation. The anthocyanin profiles of 'Gariguette' and 'Clery' were unaffected by environmental conditions. [ABSTRACT FROM AUTHOR]

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5 B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment. [ABSTRACT FROM AUTHOR]

Background: Bronchial epithelium plays a key role in orchestrating innate and adaptive immunity. The fate of ex vivo airway epithelial cultures growing at the air liquid interface (ALI) derived from human endobronchial biopsies or brushings is not easy to predict. Calibrating and differentiating these cells is a long and expensive process requiring rigorous expertise. Pinpointing factors associated with ALI culture success would help researchers gain further insight into epithelial progenitor behavior.Methods: A successful ALI culture was defined as one in which a pseudostratified epithelium has formed after 28 days in the presence of all differentiated epithelial cell types. A 4-year prospective bi-center study was conducted with adult subjects enrolled in different approved research protocols.Results: 463 consecutive endobronchial biopsies were obtained from normal healthy volunteers, healthy smokers, asthmatic patients and smokers with COPD. All demographic variables, the different fiber optic centers and culture operators, numbers of endo-bronchial biopsies and the presence of ciliated cells were carefully recorded. Univariate and multivariate models were developed. A stepwise procedure was used to select the final logistic regression model. ALI culture success was independently associated with the presence of living ciliated cells within the initial biopsy (OR = 2.18 [1.50-3.16], p < 0.001).Conclusion: This finding highlights the properties of the cells derived from the epithelium dedifferentiation process. The preferential selection of samples with ciliated beating cells would probably save time and money. It is still unknown whether successful ALI culture is related to indicators of general cell viability or a purported stem cell state specifically associated with ciliated beating cells. [ABSTRACT FROM AUTHOR]

Extending the period of fruit production is a way to substantially increase crop yield in many fruit or ornamental species. In the cultivated octoploid strawberry ( Fragaria × ananassa), the most consumed small fruit worldwide, fruit production season can be extended by selecting the perpetual flowering ( PF) cultivars. This trait is of considerable interest to growers and to the food industry. Four homoeologous loci controlling a single trait can be expected in such a complex octoploid species. However, we recently showed that the PF trait is under the control of the single dominant Fa PFRU locus ( J. Exp. Bot., 2013, 64, 1837), making it potentially amenable to marker-assisted selection ( MAS). Here, we report the successful use of a strategy, based on a selective mapping using a reduced sample of individuals, to identify nine markers in close linkage to the FaPFRU allelic variant. Thus, this strategy can be used to fine map the target homoeologous loci in other complex polyploid crop species. Recombinant analysis further enabled us to reduce the locus to a region flanked by two markers, Bx083_206 and Bx215_131, corresponding to a 1.1 Mb region in the diploid F. vesca reference genome. This region comprises 234 genes, including 15 flowering associated genes. Among these, the FLOWERING LOCUS T ( FT) is known to be a key activator of flowering. The close association between the PF trait and the Fa PFRU flanking markers was validated using an additional segregating population and genetic resources. This study lays the foundation for effective and rapid breeding of PF strawberry cultivars by MAS. [ABSTRACT FROM AUTHOR]

Impaired airway homeostasis in chronic obstructive pulmonary disease (COPD) could be partly related to club cell secretory protein (CCSP) deficiency. We hypothesize that CCSP G38A polymorphism is involved and aim to examine the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms. CCSP genotype and CCSP levels in serum and sputum were assessed in 66 subjects with stable COPD included in a 1-yr observational study. Forty-nine of them had an exacerbation. In an in vitro study, the impact on the CCSP promoter of 38G wild-type or 38A variant was assessed. BEAS-2B cells were transfected by either the 38G or 38A construct, in the presence/absence of cigarette smoke extract (CSE) or lipopolysaccharides (LPS). Cotransfections with modulating transcription factors, p53 and Nkx2.1, identified by in silico analysis by using ConSite and TFSEARCH were conducted. A allele carrier COPD patients had lower serum and sputum CCSP levels, especially among active smokers, and a decreased body mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) score. In vitro, baseline CCSP transcription levels were similar between the wild and variant constructs. CSE decreased more profoundly the CCSP transcription level of 38A transfected cells. The opposite effect was observed with p53 cotransfection. LPS stimulation induced CCSP repression in 38A promoter transfected cells. Cotransfection with Nkx2.1 significantly activated the CCSP promoters irrespective of the polymorphism. Circulating CCSP levels are associated with smoking and the CCSP G38A polymorphism. CSE, LPS, and the Nkx2.1 and p53 transcription factors modulated the CCSP promoter efficiency. The 38A polymorphism exaggerated the CCSP repression in response to p53 and CSE. [ABSTRACT FROM AUTHOR]

Obstructive lung diseases, such as chronic obstructive pulmonary disease, asthma, or non-cystic fibrosis bronchiectasis, share some major pathophysiological features: small airway involvement, dysregulation of adaptive and innate pulmonary immune homeostasis, mucus hyperproduction, and/or hyperconcentration. Mucus regulation is particularly valuable from a therapeutic perspective given it contributes to airflow obstruction, symptom intensity, disease severity, and to some extent, disease prognosis in these diseases. It is therefore crucial to understand the mucus constitution of our patients, its behavior in a stable state and during exacerbation, and its regulatory mechanisms. These are all elements representing potential therapeutic targets, especially in the era of biologics. Here, we first briefly discuss the composition and characteristics of sputum. We focus on mucus and mucins, and then elaborate on the different sample collection procedures and how their quality is ensured. We then give an overview of the different direct analytical techniques available in both clinical routine and more experimental settings, giving their advantages and limitations. We also report on indirect mucus assessment procedures (questionnaires, high-resolution computed tomography scanning of the chest, lung function tests). Finally, we consider ways of integrating these techniques with current and future therapeutic options. Cystic fibrosis will not be discussed given its monogenic nature. [ABSTRACT FROM AUTHOR]

Mucus is known to play a pathogenic role in muco-obstructive lung diseases, but little is known about the determinants of mucus rheology. The purpose of this study is to determine which sputum components influence sputum rheology in patients with muco-obstructive lung diseases. We performed a cross sectional prospective cohort study. Spontaneous sputum was collected from consecutive patients with muco-obstructive lung diseases. Sputum rheology was assessed using the Rheomuco® rheometer (Rheonova, Grenoble); the elastic modulus G′, viscous modulus G″, and the critical stress threshold σc were recorded. Key quantitative and qualitative biological sputum components were determined by cytology, nucleic acid amplification tests and mass spectrometry. 48 patients were included from January to August 2019. Among them, 10 had asthma, 14 COPD and 24 non-CF bronchiectasis (NCFB). The critical stress threshold σc predicted a sputum eosinophilia superior to 1.25% with 89.19% accuracy (AUC = 0.8762). G′ and G″ are positively correlated with MUC5AC protein concentration ((rho = 0.361; P =.013) and (rho = 0.335; P =.021), respectively). σc was positively correlated with sputum eosinophilia (rho = 0.394; P =.012), MUC5B (rho = 0.552; P <.001) and total protein (rho = 0.490; P <.001) concentrations. G′ and G″ were significantly higher in asthma patients (G' = 14.49[7.18–25.26]Pa, G'' = 3.0[2.16–5.38]Pa) compared to COPD (G' = 5.01[2.94–6.48]Pa, P =.010; G'' = 1.45[1.16–1.94]Pa, P =.006) and to NCFB (G' = 4.99[1.49–10.49]Pa, P =.003; G" = 1.46[0.71–2.47]Pa, P =.002). In muco-obstructive lung diseases, rheology predicts sputum eosinophilia and is correlated with mucin concentrations, regardless of the underlying disease. (registrar, website, and registration number), where applicable NCT04081740. • Sputum rheology is correlated with mucin content and eosinophils. • Critical stress threshold predicts sputum eosinophilia >1.25% regardless of the underlying disease. • Rheology-based diagnosis and therapeutic strategies can now be tested. [ABSTRACT FROM AUTHOR]

Abstract: Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively. [Copyright &y& Elsevier]