Interview with the ECM Award winner 2022 and introducing the new ECM members.

This article presents the interview with the ERS Early Career Member Awardee 2022 (@MathioudakisAG) and provides a brief introduction to the new ECM members https://bit.ly/3BSRgV2.

CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Physiological and biochemical responses of Limonium tetragonum to NaCl concentrations in hydroponic solution.

Introduction: Limonium (L.) tetragonum (Thunb.) A. A. Bullock, a halophyte that grows all over the southwest coast of Korea, is a medicinal plant with various pharmacological effects. The salt defense mechanism stimulates the biosynthesis of various secondary metabolites and improves functional substances. In this study, we investigated the optimal NaCl concentration for the growth and enhancement of secondary metabolites in hydroponically grown L. tetragonum. Methods: The seedlings grown for 3 weeks in a hydroponic cultivation system were treated with 0-, 25-, 50-, 75-, and 100-mM NaCl in Hoagland's nutrient solution for 8 weeks. No significant effect on the growth and chlorophyll fluorescence was observed for the NaCl concentrations below 100-mM. Results and discussions: The increase in the NaCl concentration resulted in the decrease in the water potential of the L. tetragonum leaves. The Na+ content accumulated in the aerial part increased rapidly and the content of K+, which acts as an antagonist, decreased with the increase in NaCl concentrations in hydroponics. The total amino acid content of L. tetragonum decreased compared to the 0-mM NaCl, and most of the amino acid content decreased as the NaCl concentration increased. In contrast, the content of urea, proline (Pro), ß-alanine, ornithine, and arginine was increased with an increase in NaCl concentration. The Pro content at 100-mM NaCl accounted for 60% of the total amino acids and was found to be a major osmoregulator as an important component of the salt defense mechanisms. The top five compounds identified in the L. tetragonum were classified as flavonoids while the flavanone compound was detected only in the NaCl treatments. A total of four myricetin glycosides were increased in comparison to the 0-mM NaCl. Among the differentially expressed genes, a significantly large change in Gene ontology was seen in the circadian rhythm. NaCl treatment enhanced the flavonoid-based substances of L. tetragonum. The optimum NaCl concentration for the enhancement of secondary metabolites of the L. tetragonum in the vertical farm-hydroponic cultivation system was 75-mM NaCl.

Autoantibody-mediated Macrophage Dysfunction in Patients with Severe Asthma with Airway Infections.

Rationale: Localized autoimmune responses have been reported in patients with severe eosinophilic asthma, characterized by eosinophil degranulation and airway infections. Objective: To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection. Methods: Anti-EPX (eosinophil peroxidase), anti-MARCO (macrophage receptor with collagenous structure) IgG titers, and T1 and T2 (type 1/2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and MDMs (monocyte-derived macrophages) isolated from healthy control subjects were treated with immunoprecipitated immunoglobulins from sputa with high anti-MARCO titers or nonspecific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product LPS. Measurements and Main Results: Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection), and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (odds ratio [OR], 1.79; confidence interval [CI], 1.19-2.70), IL-13 (OR, 1.06; CI, 1.02-1.12), and IL-12p70 (OR, 3.34; CI, 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective versus eosinophilic exacerbations (P = 0.01). MDMs treated with immunoprecipitated immunoglobulins (anti-MARCO+ sputa) had reduced bacterial uptake by 39% ± 15% and significantly reduced release of IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05) in response to an LPS stimulus. Conclusions: Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.

Circulating anti-nuclear autoantibodies in COVID-19 survivors predict long COVID symptoms.

BACKGROUND: Autoimmunity has been reported in patients with severe coronavirus disease 2019 (COVID-19). We investigated whether anti-nuclear/extractable-nuclear antibodies (ANAs/ENAs) were present up to a year after infection, and if they were associated with the development of clinically relevant post-acute sequalae of COVID-19 (PASC) symptoms. METHODS: A rapid-assessment line immunoassay was used to measure circulating levels of ANAs/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6 and 12 months post-recovery. Patient-reported fatigue, cough and dyspnoea were recorded at each time point. Multivariable logistic regression model and receiver operating curves were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines. RESULTS: Compared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3 months post-recovery (p<0.001). The mean number of ANA autoreactivities per individual decreased between 3 and 12 months (from 3.99 to 1.55) with persistent positive titres associated with fatigue, dyspnoea and cough severity. Antibodies to U1-snRNP and anti-SS-B/La were both positively associated with persistent symptoms of fatigue (p<0.028, area under the curve (AUC) 0.86) and dyspnoea (p<0.003, AUC=0.81). Pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and C-reactive protein predicted the elevated ANAs at 12 months. TNF-α, D-dimer and interleukin-1ß had the strongest association with symptoms at 12 months. Regression analysis showed that TNF-α predicted fatigue (ß=4.65, p=0.004) and general symptomaticity (ß=2.40, p=0.03) at 12 months. INTERPRETATION: Persistently positive ANAs at 12 months post-COVID are associated with persisting symptoms and inflammation (TNF-α) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.

Airway autoantibodies are determinants of asthma severity.

BACKGROUND: Local airway autoimmune responses may contribute to steroid dependence and persistent eosinophilia in severe asthma. Auto-IgG antibodies directed against granule proteins such as eosinophil peroxidase (EPX), macrophage scavenger receptor with collagenous structure (MARCO) and nuclear/extranuclear antigens (antinuclear antibodies (ANAs)) have been reported. Our objective was to describe the prevalence and clinical characteristics of asthmatic patients with airway autoreactivity, and to assess if this could be predicted from clinical history of autoreactivity. METHODS: We analysed anti-EPX, anti-MARCO and ANAs in 218 sputum samples collected prospectively from 148 asthmatic patients, and evaluated their association with lung function parameters, blood/airway inflammation, severity indices and exacerbations. Additionally, 107 of these patients consented to fill out an autoimmune checklist to determine personal/family history of systemic autoimmune disease and symptoms. RESULTS: Out of the 148 patients, 59 (40%) were anti-EPX IgG+, 53 (36%) were anti-MARCO IgG+ and 64 out of 129 (50%) had ≥2 nuclear/extranuclear autoreactivities. A composite airway autoreactivity score (CAAS) demonstrated that 82 patients (55%) had ≥2 airway autoreactivities (considered as CAAS+). Increased airway eosinophil degranulation (OR 15.1, 95% CI 1.1-199.4), increased blood leukocytes (OR 3.5, 95% CI 1.3-10.1) and reduced blood lymphocytes (OR 0.19, 95% CI 0.04-0.84) predicted CAAS+. A third of CAAS+ patients reported an exacerbation, associated with increased anti-EPX and/or anti-MARCO IgG (p<0.05). While no association was found between family history or personal diagnosis of autoimmune disease, 30% of CAAS+ asthmatic patients reported sicca symptoms (p=0.02). Current anti-inflammatory (inhaled/oral corticosteroids and/or adjunct anti-interleukin-5 biologics) treatment does not attenuate airway autoantibodies, irrespective of eosinophil suppression. CONCLUSION: We report 55% of moderate-severe asthmatic patients to have airway autoreactivity that persists despite anti-inflammatory treatment and is associated with exacerbations.

Comprehensive Comparison of Chemical Composition and Antioxidant Activity of Panax ginseng Sprouts by Different Cultivation Systems in a Plant Factory.

In this study, the primary (such as amino acids, fatty acids, and minerals) and secondary (including ginsenosides, phenolic acids, and flavonols) metabolites and antioxidant effects of Panax ginseng sprouts (PGSs) by different cultivation systems, such as soil-substrate cultivation (SSC) and deep-water cultivation (DWC), in a plant factory has been observed. There was no significant difference in the total fatty acid (FA) contents. Particularly, the major FAs of PGSs were palmitic acid (207.4 mg/100 g) of saturated FAs and linoleic acid (397.6 mg/100 g) and α-linolenic acid (222.6 mg/100 g) of unsaturated FAs in the SSC system. The values of total amino acids were all higher in SSC than in DWC. In the case of ginsenosides, the total protopanaxtriol product was 30.88 mg/g in SSC, while the total protopanaxdiol product was 34.83 mg/g in DWC. In particular, the values of total phenolic acids and total flavonols were 133.36 and 388.19 ug/g, respectively, and SSC had a higher content than DWC. In conclusion, the SSC system was shown to be higher in nutritional constituents and antioxidant activities in soil cultivation, suggesting that PGS with SSC has a positive effect on the quality of PGS in a plant factory.

Lasting Changes to Circulating Leukocytes in People with Mild SARS-CoV-2 Infections.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratory infections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.

Functionally Active Eosinophil Purification from Peripheral Blood.

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils.

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell-substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

The eosinophil actin cytoskeleton undergoes rapid rearrangement in response to fluid shear stress.

The regulatory processes involved in eosinophil trafficking into tissues are poorly understood; therefore, it is crucial to elucidate these mechanisms to advance the quality of clinical care for patients with eosinophil-mediated diseases. The complex interactions between eosinophil integrin receptors and their corresponding ligands on the post-capillary venules of the bronchial endothelium result in distinct modifications to the cytoskeletal architecture that occur in coordinated, temporally regulated sequences. The current study utilizes real-time confocal microscopy and time-based immunofluorescence staining to further characterize the effects of physiologically relevant fluid shear stress on this novel phenomenon of perfusion-induced calcium response. We found that the mere perfusion of fluid over adhered human eosinophils induced a release of intracellular calcium observed in conjunction with changes in cell morphology (flattening onto the coverslip surface, an increase in surface area, and a loss of circularity), suggesting a previously unknown mechanosensing aspect of eosinophil migration out of the vasculature. Although changes in morphology and degree of calcium release remained consistent across varying perfusion rates, the latency of the response was highly dependent on the degree of shear stresses. Eosinophils were fixed post-perfusion at specific timepoints for immunofluorescence staining to track proteins of interest over time. The distribution of proteins was diffuse throughout the cell prior to perfusion; however, they quickly localized to the periphery of the cell within 5 min. The actin cytoskeleton became markedly built up at the cell edges rapidly after stimulation, forming punctate dots by 4 min, suggesting a pivotal role in directed cell motility.

Short-Term Ultraviolet (UV)-A Light-Emitting Diode (LED) Radiation Improves Biomass and Bioactive Compounds of Kale.

The aim of this study was to determine the influence of two types of UV-A LEDs on the growth and accumulation of phytochemicals in kale (Brassica oleracea var. acephala). Fourteen-day-old kale seedlings were transferred to a growth chamber and cultivated for 3 weeks. The kale plants were subsequently subjected to two types of UV-A LEDs (370 and 385 nm) of 30 W/m2 for 5 days. Growth characteristics were all significantly increased in plants exposed to UV-A LEDs, especially at the 385 nm level, for which dry weight of shoots and roots were significantly increased by 2.22 and 2.5 times, respectively, at 5 days of treatment. Maximum quantum efficiency of photosystem II photochemistry (Fv/Fm ratio) began to decrease after 3 h of treatment compared to the control. The total phenolic content of plants exposed to the two types of UV-A LEDs increased by 25% at 370 nm and 42% at 385 nm at 5 days of treatment, and antioxidant capacity also increased. The two types of UV-A LEDs also induced increasing contents of caffeic acid, ferulic acid, and kaempferol. The reactive oxygen species (ROS) temporarily increased in plants exposed to the two types of UV-A LEDs after 3 h of treatment. Moreover, transcript levels of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and flavanone 3-hydroxylase (F3H) genes and PAL enzyme activity were higher in plants treated with UV-A LEDs. Our results suggested that short-term UV-A LEDs were effective in increasing growth and improving antioxidant phenolic compounds in kale, thereby representing a potentially effective strategy for enhancing the production of phytochemicals.

Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology.

Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P<0.001, n=8, Wilcoxon paired test), with high isolation purities for both eosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63+ (marker for eosinophil activation) and CD66b+ (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation assays involving degranulation markers.