Extracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages.In this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays.These particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected.Overall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors. EVs mediate the intercellular cross-talk between the producing and the target cell, through the transfer of a cargo containing different types of molecules  which display immunomodulatory properties. Being released in the extracellular environment, EVs can be recovered from any biological fluids, including milk . Milk isin vitro using IFN-γ and lipopolysaccharide (LPS). This subset presents a pro-inflammatory phenotype, and its primary role is in host defense to intracellular pathogens and in driving Th1 cellular immune responses  was chosen for milk sampling. The farm is routinely monitored and surveilled by the Department of Veterinary Medicine of Perugia University. In order to reduce problems related to interindividual variability, bulk tank goat milk was collected and immediately processed or stored at 4°C for less than 24h, avoiding any intermediate cryo-preservation in order to reduce artifacts.2.2® Centrifuge 5810R with an F34‐6‐38 rotor. This passage allows the elimination, from raw milk, of fat globules and cells/cell debris in the upper layer and in the pellet, respectively. Then ethylenediaminetetraacetic acid tetrasodium salt dihydrate  was added to the supernatant in a 1:1 ratio, left in ice for 15 minutes, and centrifuged for 1 h at 4°C at 10000 x g. To eliminate as much of the protein aggregates as possible, the recovered supernatant was further centrifuged for 1 h at 4°C at 35000 x g (Beckman Coulter Optima L‐100 XP with a 45 Ti rotor). At last, the supernatant was collected and a final ultracentrifugation for 90 minutes at 4°C at 200000 x g was used. Pellets containing mEVs were resuspended in sterile filtered (0.22 μm) phosphate-buffered saline   and stored at -80°C until use  and Western Blotting (WB). WB was employed to test the presence of EV marker proteins. Briefly, mEVs were lysed in RIPA Buffer containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS. After quantification of protein concentration by using Bradford assay, 25 μg of total proteins were resolved in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes. Blotted membranes were saturated in 0.5% of bovine serum albumin, and then incubated O.N. at 4°C with the primary antibodies against CD81 , TSG-101  and calnexin . After 2 washing steps, the appropriate secondary antibody, i.e. anti-rabbit HRP-conjugated IgG  and anti-mouse HRP-conjugated IgG , was added and incubation was carried out for 1 hour at room temperature. Immunoreactivities were highlighted by using Clarity Western ECL Substrate (Bio-Rad). The film images were acquired by using a GS-800 imaging systems scanner . A small aliquot (10 μl) of mEVs suspension was put on Parafilm for TEM analysis. mEVs were allowed to stick to formvar-coated copper grids (Electron Microscopy Sciences) for 20 minutes with the coated side facing the suspension. After being rinsed in PBS and distilled water, grids were contrasted for 5 minutes with 2% uranyl acetate. Philips EM208 transmission electron microscope with a digital camera (University Centre of Electron and Fluorescence Microscopy—CUMEF) was used for observation. The same preparations were also tested for concentration and size distribution through nanoparticle tracking analysis (NTA) and results were already reported in Mecocci et al. .2.3Five cross-bred pigs (Sus scrofa domesticus), either male or female, 6–18 months old, were used to donate blood for macrophage generation. Animals were kept at the Experimental Station of Istituto Zooprofilattico Sperimentale (IZS) of Sardinia . Animal husbandry, handling, and procedures (bleeding) were performed in accordance to the Italian Legislative Decree n.26 dated 4th of March 2014, as well as the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health (authorization n° 1232/2020-PR). Animal health status was controlled by authorized veterinarians, and samples (EDTA blood) were routinely screened for main porcine pathogens, as previously described . In deta2.46 live moMФ per well) or 4-well chamber slides  (3 × 105 live moMФ per well). After seeding, macrophages were cultured in un-supplemented fresh cRPMI at 37°C 5% CO2. 24 h later, moMФ were left untreated or differentiated into moM1, using recombinant porcine IFN-γ  and LPS , both at a concentration of 100 ng/mL , according to manufacturer’s instructions. In detail, the amounts of lactate dehydrogenase (LDH) in culture supernatants were quantified using this non-radioactive cytotoxicity assay, using a lysis solution provided by the manufacturer as a positive control. Absorbance was read at 492nm using an Epoch microplate reader  , alongside untreated control. After 24 h, cell viability was determined using Cytotox 96ky, USA) .2.6MoMФ were seeded in 4-well chamber slides and were exposed to goat milk EVs, alongside control. Before addition to the cell monolayer, EVs were labeled using the PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich), following the manufacturer’s direction. In brief, 60 μg (protein weight) EVs were resuspended in 0.5 mL dye buffer, then 2 μL of the green fluorescent dye PKH67 was diluted in 0.5 mL dye buffer and finally these were added and mixed. As a control, PBS without EVs was used: 0.2 mL of PBS was added to 0.5 mL dye buffer and 2 μL of the green fluorescent dye PKH67 and processed in parallel to EVs samples. After 5 min of incubation, 0.5 mL of PBS containing 1% bovine serum albumin  was added to stop the labeling reaction. Labeled EVs were washed by ultracentrifugation at 100,000 x g for 1 h, diluted in a complete culture medium, and added to macrophages . 24 h po2.7MoMФ or moM1 were seeded in 12 well plates and treated with EVs, alongside untreated control. 24 and 48 h post-stimulation, flow cytometry was carried on to determine the surface expression of MHC I and MHC II DR, as well as dimension , as previously described , 30. In 2.8At 24 and 48h post-treatment, culture supernatants were collected and cellular debris was removed by centrifugation (at 2500×g for 3 min). Supernatants were then stored at −80°C until analyzed. Levels of GM-CSF, TNF, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8, IL-10, IL-12, and IL-18 were determined using the Porcine Cytokine/Chemokine Magnetic Bead Panel Multiplex assay  and a Bioplex MAGPIX Multiplex Reader , following manufacturer’s instructions . The amo2.9® cDNA Syntesis Kit . RT-qPCR was assessed, on CFX96™ System  to evaluate the expression of several key immune genes, as previously described , interferon-beta (IFNB), tumor necrosis factor alfa (TNFA), Epstein-Barr virus induced 3 (EBI3), and nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65 (RELA). Primer sequences of targets and reference genes  and the corresponding reference, are reported in -ΔΔCq method (Twelve (12) well plates were used to seed MoMФ or MoM1, left untreated or stimulated with diverse EV quantities (0.6 or 60 μg) (as described in Section 2.4). After 0, 24, and 48 h, the gene expression of selected key immune genes was evaluated in harvested cells. Total RNA was isolated with an RNeasy Mini Kit  and Qiacube System , and a quali-quantitative evaluation was performed using Qubit 3.0 Fluorometer . An amount of 250 ng for each RNA sample was used as template for reverse transcription with iScriptescribed , 22. Genq method , as we pq method , 22, 27.2.10Experiments were carried out in technical duplicate  or triplicate (cytotoxic assay), using at least three different blood donor animals . Data were analyzed using GraphPad Prism 9.01 : first, data were checked for normality using the Shapiro–Wilk test, then they were graphically and statistically analyzed. Data were presented as mean and standard deviation and were analyzed using either the Student’s unpaired t-test or the non-parametric Mann-Whitney U test. The significance threshold was set at p<0.05.33.1EVs were isolated from goat milk following the procedure described in our previous studies . The preGoat milk EVs’ impact on porcine moMФ viability was assessed through a non-radiolabeled immunoassay. Cells were exposed to scalar doses of these particles , and 24h later LDH amounts in culture supernatants were measured using a cytotoxicity non-radioactive assay. Cell viability decreased only when goat mEVs were added at the highest dose: 600 μg (The impact of goat mEVs on both porcine untreated (moMФ) and classically activated macrophages (moM1) was analyzed in this study. Their immunomodulatory effects were investigated through flow cytometry, gene expression, and multiplex ELISA. Classical activation was confirmed by the upregulation of activation markers (MHC I and MHC II DR) (3.2Flow cytometry was employed to quantify the expression of MHC molecules (MHC class I and II DR) on macrophage subsets 24 and 48 h after exposure to mEVs  or extracellular  , which is a host antimicrobial peptide with antimicrobial activity against a broad range of bacteria (TLR4 (48h), TLR5 (24h), TLR9 (both at 24h and 48h) and RELA (24h). On the contrary, high doses of mEVs (60 μg) enhanced the expression of TLR3 (24h), but down-regulated the expression of DEFB1 (48h), and three TLRs: TLR4 (48h), TLR8 (24 and 48h), TLR9 (both at 24h and 48h) (TLR4 (48h) and TLR5 (48h), while higher doses (60 μg) down-regulated TLR3 expression (48h) and slightly up-regulated RELA expression (48h) . TLRs are a group of PRRs, that recognize molecules expressed by pathogens  and play a central role in initiating the host immune defense . Eight T, and 6) . Two othignaling . DEF1B ebacteria . We obseIL10 (24h), EBI3 (24h), and IFNB (48h), whereas treatment with 60 μg triggered enhanced expression of several cytokines: IL1 (24h), IL6 (24h), CXCL8 (24h), IL10 , IL12B (24h), EBI3 (24h), and IFNB (24h) (CXCL8 (0.6 μg 48h), IL10 (0.6 μg 24h), IL12A (60 μg 24h), IL12B (0.6 μg 24h), and TNF  , alongside untreated controls, and cytokines levels in culture supernatants were quantified at 24 and 48 h post-treatment. Exposure to the lowest dose (0.6 μg) did not affect cytokine contents in culture supernatants, with the exception of a small  enhancement of IL-1α, IL-1β, and IL-8 levels (4Milk-derived extracellular vesicles (mEVs) have drawn researchers’ attention during the last few years for their potential applications in the biomedical field related to their considerable theranostic properties . Diversein vitro works were carried out in mice, where goat mEVs were labeled with commercial fluorophores to create nanoprobes  and classically activated macrophages (moM1), the latter characterized by a pro-inflammatory antimicrobial phenotype . First, + T cell subsets (IL12A and IL12B) was induced in moMФ following exposure to high doses of mEVs, in association with the increased release of IL-12. The concomitant release of both IL-12 and IL-18, alongside pro-inflammatory cytokines, suggests that goat mEVs can polarize macrophages toward an M1-like phenotype in vivo, with the subsequent enhancement of IFN-γ production and the activation of both NK cells and cytotoxic T cells. Nevertheless, we observed also the EBI3 upregulation in moMФ after mEV treatment. EBI-3 is a subunit of IL-27 and IL-35 depending on the other subunit it combines with, p28 or p35 respectively (We observed that high doses of goat mEVs 60 μg) enhanced the expression of macrophage activation markers , and the induction/release of proinflammatory cytokines. We detected an increased expression/release of IL-1, IL-6, and TNF, which are indeed the most important pro-inflammatory cytokines of the innate immune response . IL-1α a subsets . Both IL subsets . IL-1β,  subsets . High do subsets  which ca subsets , 60. Eve μg enhanectively . These tectively . We mighAccordingly, we observed that porcine moMФ released two immune-suppressive cytokines (IL-1Ra and IL-10) in response to 60 μg of goat mEVs: IL-1Ra is a receptor antagonist, released to block further IL-1 activity , while Iin vitro data suggest that these nanosized structures were able to polarize porcine macrophages towards an M1-like phenotype, which can be useful to enhance defense against intracellular pathogens and malignancies , and a mild downregulation of some TLRs. The observed modest impact on moM1 represents a positive data for a clinical application of this molecules: when macrophages are already in a pro-inflammatory status (moM1), their activation seemed to be controlled, preventing potentially pathological over-response to stressors. Accordingly, we observed also the downregulation of TLR4, TLR8, and TLR9 in porcine moMФ exposed to goat mEVs. The down-regulated expression of these PRRs may indeed represent a protective mechanism, put in place to tightly regulate the macrophage activation, to prevent the development of pathological inflammatory responses or even autoimmunity . These preliminary The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Ethics Committee of the Istituto Zooprofilattico Sperimentale della Sardegna.GF, SM, KC, and ER conceived the study. GF, SM, CC, LM, FD, SZ, FF, LPao, TC, SD, AA, EC, and LPas conducted the experiments. GF, SM, AA, SD, EC, and LPas performed the formal analysis. GF, and SM wrote the first draft of the manuscript, which was reviewed and edited by KC, and ER. AO, KC, and ER provided resources and funding acquisition. All authors have read and agreed to the submitted version of the manuscript.


Boron (B) toxicity is an important stressor that negatively affects maize yield and the quality of the produce. The excessive B content in agricultural lands is a growing problem due to the increase in arid and semi-arid areas because of climate change. Recently, two Peruvian maize landraces, Sama and Pachía, were physiologically characterized based on their tolerance to B toxicity, the former being more tolerant to B excess than Pachía. However, many aspects regarding the molecular mechanisms of these two maize landraces against B toxicity are still unknown. In this study, a leaf proteomic analysis of Sama and Pachía was performed. Out of a total of 2793 proteins identified, only 303 proteins were differentially accumulated. Functional analysis indicated that many of these proteins are involved in transcription and translation processes, amino acid metabolism, photosynthesis, carbohydrate metabolism, protein degradation, and protein stabilization and folding. Compared to Sama, Pachía had a higher number of differentially expressed proteins related to protein degradation, and transcription and translation processes under B toxicity conditions, which might reflect the greater protein damage caused by B toxicity in Pachía. Our results suggest that the higher tolerance to B toxicity of Sama can be attributed to more stable photosynthesis, which can prevent damage caused by stromal over-reduction under this stress condition. Boron (B) is an essential element for plants, and is well known for its structural role in both cell walls and membranes ,2,3,4,5.Since B is a micronutrient, the range between its deficient, optimal, and toxic concentrations for plants is very narrow . Therefo2 assimilation (PN), photosynthetic electron transport rate (ETR), maximum quantum yield of chlorophyll fluorescence (Fv/Fm), and CO2 use efficiency decreased under B toxicity conditions -1-propanesulfonate), and quantified via the Bradford method using bovine serum albumin (BSA) as a standard [Maize leaves (200–250 mg fresh weight) from four separate seedlings per condition (B treatment and maize landrace) were ground to a fine powder in a mortar precooled with liquid nitrogen. Proteins were extracted with trichloroacetic acid (TCA)/acetone-phenol , solubilstandard .The cleaning of maize protein extract, protein digestion, and mass spectrometry determinations were carried out at the Proteomics Facility for Research Support Central Service (SCAI) of the University of Córdoba (Spain) as follows.w/v) SDS-PAGE gel. Protein bands were excised from the gels and, afterwards, the gel pieces were distained in 200 mM ammonium bicarbonate/50% acetonitrile for 15 min, followed by 5 min in 100% acetonitrile. Proteins were reduced by adding 20 mM dithiothreitol in 25 mM ammonium bicarbonate and incubated for 20 min at 55 °C. The mixture was cooled to room temperature, and then, free thiols were alkylated by adding 40 mM iodoacetamide in 25 mM ammonium bicarbonate for 20 min in the dark. Finally, the gel pieces were washed twice in 25 mM ammonium bicarbonate.Biological quadruplicate samples were separated and cleaned as described. Leaf protein extracts  were electrophoretically pre-concentrated in a centimeter band of 10% (v/v). Finally, the digested samples were vacuum-dried and dissolved in a mixture of 2% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid.Proteolytic digestion was performed by adding trypsin to a final concentration of 12.5 ng/µL in 25 mM ammonium bicarbonate at 37 °C overnight. Protein digestion was stopped by adding trifluoroacetic acid at a final concentration of 1% (m/z) with a 4 × 105 ion count target. Tandem MS was performed via isolation at 1.2 Da with the quadrupole. Monoisotopic precursor ions were fragmented via CID  in an ion trap, which was set up as follows: automatic gain control, 2 × 103; maximum injection time, 50 ms; and normalized collision energy, 35%. Only those precursors with charge states of 2–5 were sampled for MS2. A dynamic exclusion time of 15 s and a tolerance of 10 ppm around the selected precursor and its isotopes were used to avoid redundant fragmentations. The instrument was run in top 30 mode with 3 s cycles, meaning the instrument would continuously perform MS2 events until a maximum of 30 non-excluded precursors or 3 s, whichever was shorter.Peptide separations were performed on a nano-LC using Dionex Ultimate 3000 nano UPLC , equipped with a C18 75 μm × 50 cm Acclaim Pepmap column , at 40 °C, at a flow rate of 300 nL/min. Peptide mixtures were previously concentrated and cleaned on a 300 µm × 5 mm Acclaim Pepmap precolumn  using 2% acetonitrile/0.05% trifluoroacetic acid, at 5 µL/min, for 5 min. Peptides were eluted with a gradient of 60 min ranging from 96% solvent A (0.1% formic acid) to 90% solvent B (80% acetonitrile and 0.1% formic acid), followed by an 8 min wash at 90% solvent B and a 12 min re-equilibration at 4% solvent B. Eluted peptides were converted into gas-phase ions via nanoelectrospray ionization and analyzed on a Thermo Orbitrap Fusion mass spectrometer  operated in positive mode. Survey scans of peptide precursors were acquired over an m/z range 400–1500 at 120K resolution . Peptide quantification was carried out using MaxQuant software, via a MaxLFQ label-free quantification method [t-test and error correction . The criteria used to consider a protein as differentially expressed were as follows: (a) the protein was consistently present in at least three biological replicates per condition; (b) it had statistically significant differences  between genotypes or B treatments; and (c) it had a fold change ≥1.5 or ≤0.66667. The differentially accumulated proteins were manually categorized by function using different databases .Charge state deconvolution and deisotoping were not performed. MS2 spectra were searched using MaxQuant software v. 1.5.7.4 . MS2 spen method . In the n method . LFQ-norThe higher B content in Pachía leaves than in Sama leaves can cause greater damage to their proteins. The overexpression of several proteases, mainly cysteine protease14 and four serine proteases, can increase the degradation of damaged and misfolded proteins in Pachía. Subsequently, the over-accumulation of transcription- and translation-related proteins allows Pachía to: (1) partially replace proteins damaged by B toxicity, and (2) reduce the injury caused to ribosomes and protein synthesis by B excess by abolishing the non-viable decrease in transcription and translation processes, thus allowing Pachía to survive under this stress condition.In Sama, PAO1 over-accumulation can protect the photosynthetic apparatus. Furthermore, ZmPIFI and NDHS up-accumulation, along with the lower knockdown of several subunits of NDH and ATP synthase complexes under B excess, confers greater B toxicity tolerance to this landrace by: (1) acting as an electron safety valve that prevents stromal overreduction, and thus, decreases photosynthetic damage, and (2) providing an additional supply of ATP that contributes to repairing the photosynthetic system of Sama. On the other hand, OEE2-1 overexpression can stabilize and protect the OEC, leading to a higher photosynthesis rate in this landrace.


A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM , 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca Ciliary cells prevent the accumulation of debris and colonization by microbial pathogens via an outward-directed transportation processes2. Cilia are hair-like protrusions located on the apical side of the respiratory epithelium along the airways; they are composed of unique structural proteins, including motor proteins, like the ATP-dependent dynein3. Due to their continuous synchronized and orally directed motion, inhaled particles and complex compositions of mucus, electrolytes, and endogenous defensive substances are removed from the lower airways and are subsequently coughed up under normal physiological conditions4. While the ciliary apparatus continuously beats without external stimulation, various events can provoke an increase in ciliary beating frequency, when necessary4. Exogenous, endogenous, and paracrine effects moderate ciliary activity, and Ca2+ functions as a central second messenger. Therefore, intracellular Ca2+ concentration ([Ca2+]i) represents a keystone regulating ciliary activity because most signal transduction cascades, which contribute to the ciliary beating frequency, induce at least a temporary increase in [Ca2+]i5. Intracellular Ca2+ is foremost stored in the endoplasmic reticulum (ER) and is released following inositol trisphosphate (IP3) or ryanodine receptor (RyR) activation3. Furthermore, the ER regulates [Ca2+]i via the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), which pumps cytosolic Ca2+ ions to its internal stores, maintaining the balance of [Ca2+]i7. Mitochondria also contribute to intracellular Ca2+ homeostasis by buffering Ca2+ ions when [Ca2+]i exceeds a threshold of 500 nM9. In addition, other cell organelles contribute to intracellular Ca2+ homeostasis, such as lysosomes, which contain Ca2+ but can also regulate mitochondrial Ca2+ dynamics, and peroxisomes; however, their individual influence on [Ca2+]i in ciliary cells and mucociliary clearance remains unknown11. [Ca2+]i is not only increased by Ca2+ release from intracellular stores; extracellular Ca2+ can enter the cell via plasma membrane-bound Ca2+ channels. Although voltage-gated calcium channels and transient receptor potential channels are located at the plasma membrane together, the predominant method of extracellular Ca2+ entry into non-excitable cells occurs via store-operated Ca2+ entry (SOCE)13. SOCE is triggered by an increase in [Ca2+]i that is predominantly caused by the liberation of Ca2+ from internal stores12. Stromal interaction molecule proteins are located at the ER membrane, and they activate SOCE when Ca2+ is released13. Therefore, changes in [Ca2+]i produce fundamental metabolic and functional alterations in ciliary cells and can be influenced by different drugs administered in various settings.Mucociliary clearance of the lower airways is a pivotal physiological mechanism that protects the lung by clearing the airways of pollution and colonization by pathogens, thereby preventing infection. Together with basal, suprabasal, and goblet cells, multiciliated epithelial cells constitute the complex mucociliary clearance function of the respiratory tract16. Covalently linked theophylline and norephedrine compose cafedrine, and theodrenaline is formed from theophylline and noradrenaline in the same manner17. Both compounds are administered in a fixed 20:1 combination of cafedrine and theodrenaline as an intravenous bolus in adults, and they restore mean arterial blood pressure by increasing preload, cardiac stroke volume, and cardiac output18. The clinical effects of cafedrine/theodrenaline are mediated through β1-adrenoreceptor and α-adrenoreceptor stimulation, and nonspecific inhibition of phosphodiesterases (PDEs) is believed to enhance their response17. In contrast with synthetic vasopressors , systemic vascular resistance and heart rate remain mostly unaffected, which makes cafedrine/theodrenaline especially appealing in obstetric surgery19. Although cafedrine/theodrenaline has been widely used for decades, little is known about its pharmacodynamics or pharmacokinetics in specific end organs16. This is surprising as the unique combination of three single drugs  in a 20:1 mixture might produce different effects in vivo as recently shown in human atrial myocardium and internal mammary arteries17. However, the influence of cafedrine/theodrenaline on the [Ca2+]i of human respiratory cilia cells has not yet been investigated. The aim of this study was to evaluate the influence of cafedrine/theodrenaline on the [Ca2+]i of human tracheal epithelial cells. Cells were exposed to the clinically used 20:1 mixture of cafedrine/theodrenaline and the individual substances alone. Our experiments elucidated the effects of the individual components and identified the relevant pharmacological signaling cascades leading to alterations in [Ca2+]i. Therefore, we applied specific inhibitory substances inhibiting distinct signal transduction cascades, and we evaluated the origin of the released Ca2+. Furthermore, we used reverse transcription polymerase chain reaction (RT-PCR) to detect the messenger ribonucleic acid (mRNA) expression of key receptor subtypes influencing [Ca2+]i following the administration of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone.In Germany, intraoperative hypotension and emergency hypotensive states have been treated with a combination of cafedrine and theodrenaline  since the 1960s2, 10 mM glucose, 125 mM NaCl, 1.2 mM KH2PO4, and 1.2 mM MgSO4. NaOH was used to adjust the pH to 7.4 at 30 °C. To realize experiments in Ca2+-free solutions, CaCl2 was substituted with 1 mM ethylene glycol tetraacetic acid. The following drugs were applied during the experiments: 2-aminoethoxydiphenylborane , 2,4-dinitrophenol , cafedrine , cafedrine/theodrenaline 20:1 , caffeine , FURA-2 AM , ICI-118,551 , KCl (200 mM diluted in 66.6 µl H2O), ryanodine , and theodrenaline . The stated drug concentrations were achieved during the experiments after applying the stock solution to the buffer solution in the recording chamber. In control experiments, the buffer solution or the solvent alone was applied to rule out any contribution of the buffer, the solvent, or the mechanical application process to the Ca2+ signals.The historically established 20:1 ratio of cafedrine/theodrenaline refers to mass, not molarity. Therefore, due to the different molar masses of cafedrine (357.41 g/mol) and theodrenaline (375.38 g/mol), a 1 M solution of cafedrine/theodrenaline 20:1 with a molar mass of 7,882.98 g/mol consists of 1 M theodrenaline and approximately 21 M of cafedrine. Experiments were performed in HEPES solution consisting of 10 mM HEPES, 2.6 mM KCl, 2.5 mM CaCl2. Cells from passage one to three were seeded onto laminin-coated coverslips for [Ca2+]i measurements in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Dye loading was performed in the dark with 2.5 µM FURA-2 AM for 45 min at 37 °C. The cell-containing coverslip was then rinsed in fresh HEPES buffer and subsequently transferred to the recording chamber of an upright fluorescence microscope equipped with a 20 × immersion lens , where the coverslip was placed into a Delta T culture dish  containing 2 ml of fresh HEPES buffer. Excitation light was provided by a 50 W xenon lamp, and the microscope was equipped with a dichroic excitation longpass mirror at 400 nm. FURA-2 AM was excited at 340 nm and 380 nm while equipped with bandpass excitation filters. The emitted fluorescence was directed through a dichroic shortpass filter of 560 nm to a bandpass filter of 510 nm and was recorded with a scientific camera . Measurements of the FURA-2 AM 340/380 ratio were performed every second for 1,000 s using an automated protocol of the TiLLvisION Imaging software program . Cafedrine, theodrenaline, or 20:1 cafedrine/theodrenaline were added following a 100-s resting period to ensure adequate baseline calibration. After 800 s, 200 mM potassium chloride (KCl) was added to the buffer solution to completely invert the membrane potential, leading to massive influx of Ca2+ indicating the integrity of the analyzed cells up to the end of the respective experiment.Human tracheal epithelial cells  were cultured with the Airway Epithelial Growth Medium Kit (C-21160) containing the Airway Epithelial Growth Medium Supplement Pack  in a humidified chamber at 37 °C with air containing 5% COMale C57BL6J mice (n = 5) weighing 25–35 g (aged 12–15 weeks) were purchased from Charles River  to characterize the expression of RyR in the respiratory epithelium. All procedures involving animals were conducted in compliance with the European legislation for the protection of animals used for scientific purposes, the ARRIVE guidelines and the standards for animal experiments according to the German animal welfare law and were approved by the local committee for animal care of the regional council of Giessen, Germany . After deep narcosis using 5% isoflurane , animals were sacrificed by cervical dislocation. Tracheae, tracheal epithelium, diaphragm, cardiac muscle, and skeletal muscle were subsequently collected. To obtain isolated tracheal epithelium, the epithelial layer was gently scrubbed from the opened trachea using a hygienic swab. All tissues were stored in RNA stabilization lotion  at − 20 °C until further processing. Tissue probes were then lysed in 350 µl RNeasy Lysis Buffer  containing 3.5 µl ß-mercaptoethanol and they were subsequently homogenized in a tissue homogenizer . RNA was extracted using the RNeasy Micro Kit, and DNA removal was performed using the RNase-Free DNase set .-3 expression in whole trachea and in the respiratory epithelium alone (each n = 5) was analyzed using specific primers at temperatures shown in Table https://www.ncbi.nlm.nih.gov/tools/primer-blast, National Institutes of Health, Bethesda, MD, USA). Skeletal muscle (RyR-1), cardiac muscle (RyR-2), and diaphragm (RyR-3) served as positive controls, and H2O was used as negative control. Primer concentration was set at 0.2 µM and reactions were performed in a Mastercycler gradient . TAQ polymerase  was activated at 95° for 3 min, followed by 40 cycles consisting of a 1-min denaturation step at 95 °C, a 45-s annealing step at a primer-specific temperature  according to the manufacturer’s protocol and stored at − 20 °C until further use. RyR-1, RyR-2, and RyR50) were calculated using the Hill equation. The Mann–Whitney U test was used to compare the peak measurement points from different experiments, while the Wilcoxon rank sum test was used to compare paired variables. In general, two-tailed values of p < 0.05 were considered statistically significant, while multiple comparison problems were counteracted by adjusting the α-level according to the Bonferrroni correction. GraphPad PRISM  was used for statistical analysis and figure creation.FURA-2 340/380 ratio was recorded from at least 30 cells per experimental setup. These 30 cells were collected from at least three different coverslips each evaluating 10 cells taken from cell passages one to three. Measurements were only included in the statistical analyses when clear response of the FURA-2-fluorescence ratio (doubling of the ratio) was detected after the application of KCl at the end of the experiments. Median effective concentrations , 3.14 mM (1.12 mg/ml), and 3.45 mM (1.30 mg/ml), respectively. All three substances provoked a sharp increase in [Ca2+]i that was observed immediately, within 5 s after application . Application of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone provoked a significant increase in [Ca2+]i , was applied, baseline i peaks were significantly higher in the presence of DNP compared with the [Ca2+]i peaks without depletion of mitochondrial Ca2+ stores (both p < 0.001). [Ca2+]i did not reach baseline following the administration of cafedrine alone or theodrenaline alone. Furthermore, caffeine-sensitive Ca2+ stores, which are mainly represented by the ER, were depleted using caffeine . Subsequent application of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone did not increase [Ca2+]i  were identified by RT-PCR. RyR-1 mRNA was not detected in the whole trachea or tracheal epithelium. An overview of the RT-PCR results, including the controls, is presented in Table 2+]i, when applied to human tracheal epithelial cells. We observed sharp, transient peaks in the FURA-2 340/380 ratio, which followed a dose–response relationship and were described by the Hill equation. Herein, it should be noted that the effect curves of 20:1 cafedrine/theodrenaline and cafedrine alone showed a profile where the increase from 0 to 100% is already achieved within one log unit, which would be classically attributed to ion channel activation. In contrast, only the dose–response curve of theodrenaline clearly indicated receptor-mediated effects because the increase from 0 to 100% required two log units. However, although the formal criteria for receptor-mediated Ca2+ release were only barely met in our dose–response curves following the application of 20:1 cafedrine/theodrenaline and cafedrine alone, our following experiments clearly proved adrenergic and ryanodine receptor-mediated Ca2+ release following the application of all three substances analyzed in our experiments. Interestingly, the EC50 of cafedrine alone and theodrenaline alone were within comparable ranges; however, when applied as the clinically used 20:1 mixture, much more cafedrine was required to achieve a significant effect on [Ca2+]i, and the applied concentration of theodrenaline seemed almost negligible in the light of the calculated EC50 of theodrenaline alone. Moreover, [Ca2+]i peaks observed after the application of 20:1 cafedrine/theodrenaline were consistently lower than those observed following applications of the individual substances alone. Interestingly, clinically irrelevant high concentrations of 20:1 cafedrine/theodrenaline ultimately led to cell lysis, which was also observed following the administration of high concentrations of cafedrine or theodrenaline alone; however we never observed a higher FURA-2 340/380 ratio than that shown in our dose–response curves. Although the pharmacokinetics of cafedrine and theodrenaline remain largely unknown, the immediate response in blood pressure was attributed to theodrenaline, while the effects of cafedrine were observed after a 20-min delay20. Therefore, it has been hypothesized that cafedrine may not provoke any sympathomimetic actions alone; it may be metabolized into active metabolites that exert its clinical effects17. In our experiments, the effect observed after the application of 20:1 cafedrine/theodrenaline could only be attributed to theodrenaline alone if strong synergistic effects would apply in the presence of cafedrine. Because the concentration of theodrenaline alone in the 20:1 mixture was insufficient to provoke any significant changes in [Ca2+]i, we conclude that the foremost effect on changing [Ca2+]i in our experiments was induced by cafedrine. This assumption is supported by the observation that very high concentrations of cafedrine were applied using the 20:1 combination compared with the dose–response relationship of cafedrine alone. However, our model cannot represent the effects of potential drug metabolites, as we used isolated cells, and the drugs were directly applied to the buffer solution. Furthermore, clinical data reported a plasma concentration of 6 µg/ml after the intravenous application of one ampoule of 2 ml cafedrine/theodrenaline; therefore, all concentrations used in our experiments were significantly higher than clinically used concentrations20. However, the transient intravascular and intraepithelial concentrations immediately after injection are unknown and might be much higher than those observed after distribution to the body compartments; therefore, our data should be interpreted with caution, and further studies are necessary to evaluate the transferability of the concentrations used in our experiments.Our experiments revealed that 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone increased i rapidly returned to its baseline3. The rapid restoration of baseline [Ca2+]i is primarily explained by SERCA activity, which pumps Ca2+ into the ER after cytosolic Ca2+ release, or by mitochondrial Ca2+ buffering22. However, almost every other small cell organelle contributes to the rapid restoration of baseline [Ca2+]i, and prolonged alteration of ciliary beat frequency is induced by transient changes in [Ca2+]i. Immediately after purinergic or cholinergic stimulation, tracheal epithelial cells exhibited a transient increase in [Ca2+]i; however, ciliary beat frequency remained consistently increased24. Therefore, we conclude that analogous mechanisms are mediated after the stimulation of β-adrenergic receptors, which were the pivotal receptors involved in [Ca2+]i alterations in our experiments. The increase in [Ca2+]i completely vanished after 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone were added during non-selective adrenergic receptor inhibition. These results are in line with the clinical effect of cafedrine/theodrenaline, which increases cardiac stroke volume via β1-adrenergic receptor activation14. In-vitro studies using human atrial myocardium and coronary arteries also elucidated β1-adrenergic receptor activation as a pivotal mechanism; however, effects on α-adrenergic receptors were observed in arteries after β-adrenergic receptor inhibition25. Although these effects were attributed to theodrenaline alone, we did not observe similar effects in human tracheal epithelial cells. These observations are in line with the data reported by Weiterer et al., who reported the exclusive presence of the α1D-adrenergic receptor subtype in murine tracheal epithelium26. However, murine particle transport velocity was independent from α-adrenergic receptor activation. Therefore, we conclude that α-receptor activation might occur in human tracheal epithelial cells following the administration of cafedrine/theodrenaline, but no influence on [Ca2+]i or mucociliary clearance could be detected in light of the available in-vitro data26. The theophylline component of cafedrine and theodrenaline is believed to inhibit PDE, which should lead to the persistence of second messengers, such as cGMP and cAMP, improving cardiac inotropy14. However, only high, clinically irrelevant concentrations of cafedrine/theodrenaline were able to provoke significant inhibition of PDE in human atrial myocardium17. Although we used high concentrations of cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone, we were only able to detect β-adrenergic receptor stimulation because the increase in [Ca2+]i completely vanished in the presence of β-adrenergic receptor inhibition. When other signal transduction cascades were involved, we should have detected a persistent increase in [Ca2+]i. This finding is underlined by the knowledge that PDE inhibition might not influence ciliary beat frequency or mucociliary clearance to a clinically relevant degree, because data remain controversial regarding the alteration of mucociliary clearance following treatment with theophylline29.After the transient peak, during which Ca2+-free buffer solution, [Ca2+]i increased to a significantly lesser degree following administration of cafedrine alone and theodrenaline alone than the increase observed in Ca2+-containing buffer solution. Therefore, extracellular Ca2+ influx contributes to the rise in [Ca2+]i, which is foremost realized through SOCE in non-excitable cells following Ca2+ release from internal stores13. ORAI proteins, which are mediated by stromal interaction molecule proteins, are the most important mediators of SOCE12. Therefore, further experiments elucidating receptor expression and distinct signal transduction pathways should be performed, although their specific inhibition is complicated due to their diverse interactions and multiple targets located on the plasma membrane13. However, because the [Ca2+]i peak in the Ca2+-free buffer solution was comparable to the peak observed in Ca2+-containing buffer solution following the administration of 20:1 cafedrine/theodrenaline, the clinical relevance of SOCE following the administration of cafedrine/theodrenaline in human tracheal epithelial cells remains questionable. Internal stores were depleted to detect the intracellular stores, which released Ca2+ following the administration of cafedrine/theodrenaline. When mitochondrial Ca2+ stores were depleted, [Ca2+]i continued to increase following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone. Therefore, we conclude that cafedrine/theodrenaline does not depolarize mitochondrial membrane potential, which would lead to Ca2+ release from these stores. However, [Ca2+]i peaks following the administration of 20:1 cafedrine/theodrenaline and cafedrine alone were significantly higher than those observed without prior mitochondrial store depletion. This observation confirms the mitochondrial ability to buffer Ca2+ ions via the mitochondrial Ca2+ uniporter when [Ca2+]i exceeds 500 nM30. Interestingly, [Ca2+]i remained higher for the rest of the observation time. Because DNP administration decouples oxidative phosphorylation without altering cytosolic pH, less ATP supplying the ATP-dependent SERCA might be available in these experiments, leading to the observation of persistent [Ca2+]i baseline shift. However, SERCA was still able to handle the transient peak [Ca2+]i by altering [Ca2+]i close to its former baseline value. This finding underlines our conclusion that the elevated peak in [Ca2+]i is foremost triggered by the inhibited mitochondrial Ca2+ capacity and not by a lack of ATP, which might impair SERCA activity.When we used Ca2+]i completely vanished following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone, when RyR were inhibited, we conclude that cafedrine/theodrenaline releases Ca2+ from the ER exclusively by RyR activation. In general, Ca2+ release from the ER is achieved via IP3 receptor or RyR activation31. However, our experiments revealed equal peaks of [Ca2+]i when IP3 receptors were inhibited, and the increase in [Ca2+]i completely vanished after RyR inhibition. Therefore, we conclude that Ca2+ release following β-adrenergic receptor activation depends solely on RyR activation, and IP3 receptor-associated Ca2+ release does not occur in human tracheal epithelial cells following cafedrine/theodrenaline administration.Because the increase in i36. Local increase in [Ca2+]i can be realized through nearby RyR activation, or IP3 receptor activation; however, our data indicate that RyR activation was independent from IP3 receptor activation following cafedrine/theodrenaline administration37. Therefore, alternative RyR activation following β-adrenergic receptor stimulation must be considered in tracheal epithelial cells. β-adrenergic stimulation has been shown to increase RyR-2 activity in cardiac muscle cells via intracellular-mediated Ca2+ and Mg2+ regulation, and receptor phosphorylation38. Furthermore, protein kinase A and cAMP, which are both pivotal messengers following the β1-signal transduction cascade, have been shown to induce Ca2+ release via RyR-2 in cardiac muscles and non-excitable cells, respectively40. In addition, adrenergic receptor signaling increases nicotinic acid adenine dinucleotide phosphate and cyclic adenosine diphosphate-ribose levels, which both activate RyR-2-associated Ca2+ release41. Because RyR-3 is more readily activated by an increase in local [Ca2+]i compared with RyR-2, its activation in tracheal epithelial cells following the administration of cafedrine/theodrenaline can be achieved following RyR 2-associated Ca2+ efflux42. However, as discrepancies in receptor expression between mammals could not be excluded, further studies in human tissues should be conducted to confirm RyR expression. Local distribution, and the RyR-2-to-RyR-3 ratio in tracheal epithelial cells could be evaluated using immunohistochemistry.Consequently, RyR-2 and RyR-3 expression was revealed using RT-PCR in murine tracheal epithelium. While RyR-3 is co-expressed with RyR-1 or RyR-2 in many tissues, RyR 2 has mainly been studied in cardiac muscle cells; however, RyR-2 expression has been demonstrated in smooth muscle cells and non-excitable cells, such as pancreatic acinar cells and kidney epithelial cells2+]i and clinically administered concentrations are much lower; therefore, it is possible that we observed [Ca2+]i kinetics that do not occur when lower concentrations are used in vivo. Third, some concentrations applied to achieve the specific dose–response curves were higher than was warranted by the integrity of the observed cells; therefore, a maximum FURA-2 340/380 ratio following substance administration was set at the last valid observed value. However, concentrations applied to assess the distinct signal transduction cascades did prove the validity of the calculated dose–response curves in our experimental setting. Fourth, our measurement of [Ca2+]i using the FURA-2 340/380 ratio was not calibrated; therefore, we can only report relative alterations in [Ca2+]i, and no absolute concentrations were measured.Several limitations of our experiments must be acknowledged. First, we used isolated tracheal epithelial cells; therefore, the physiological integrity of a respiratory tract was not preserved, including a lack of basal tissues and cell–cell junctions. Therefore, physiological drug administration via capillary vessels could not be replicated, and atypical entrance  of our tested drugs was preserved. Second, we used high concentrations of cafedrine/theodrenaline, cafedrine, and theodrenaline to achieve alterations in i; therefore, further studies are needed to alter the ability of cafedrine/theodrenaline to change mucociliary clearance in clinical practice.In conclusion, we provide evidence that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone induce the release of CaSupplementary Figure 1.


Food crops are increasingly susceptible to the challenging impacts of climate change, encompassing both abiotic and biotic stresses, that cause yield losses. Root-associated microorganisms, including plant growth-promoting bacteria (PGPB), can improve plant growth as well as plant tolerance to environmental stresses. The aims of this work were to characterize bacteria isolated from soil and roots of tomato plants grown in open field.Biochemical and molecular analyses were used to evaluate the PGP potential of the considered strains on tomato plants in controlled conditions, also assessing their effects under a water deficit condition. The isolated strains were classified by 16S gene sequencing and exhibited typical features of PGPB, such as the release of siderophores, the production of proteases, and phosphorous solubilization. Inoculating tomato plants with eleven selected strains led to the identification of potentially interesting strains that increased shoot height and dry weight. Three strains were then selected for the experiment under water deficit in controlled conditions. The tomato plants were monitored from biometric and physiological point of view, and the effect of inoculation at molecular level was verified with a targeted RT-qPCR based approach on genes that play a role under water deficit condition.Results revealed the PGP potential of different bacterial isolates in tomato plants, both in well-watered and stressed conditions. The used integrated approach allowed to obtain a broader picture of the plant status, from biometric, eco-physiological and molecular point of view. Gene expression analysis showed a different regulation of genes involved in pathways related to abscisic acid, osmoprotectant compounds and heat shock proteins, depending on the treatments. Overall, results showed significant changes in tomato plants due to the bacterial inoculation, also under water deficit, that hold promise for future field applications of these bacterial strains, suggesting that a synergistic and complementary interaction between diverse PGPB is an important point to be considered for their exploitation.  Positive effects of the tomato inoculation with PGPB have been also highlighted in other works, reporting an increase in yield (Pseudomonas fluorescens (SS5) in pot significantly increased tomato root and shoot weight, length, fruit yield per plant, and total fruit yield , isolated from the rhizosphere of maize, showed the ability to colonize and to survive in the tomato roots after inoculation and to positive influence the plant growth in terms of root and shoot length, as well as fresh and dry biomass  allowed to reduce the weight fruit decrease under drought  in the frame of an in-field experiment to assess the effect of different water regimes on two tomato genotypes . To isol2PO4 1.5 g/L, NaHPO4 0.5 g/L, NH4Cl 1 g/L, NaCl 0.1 g/L). The samples were incubated at 30°C with stirring for 16 hours. Serial dilutions 10-5, 10-6 and 10-7 were then prepared in sterile water, and 100 μL of these dilutions were distributed on R2A agar (Merck®) plates incubated at 30°C for five days. The R2A medium, less rich than the classic LB, allows a broad spectrum of bacteria to grow without the fast-growing bacteria suppressing the slow-growing species.Instead, for approach (b), approximately 2 g of soil was added in 100 mL of minimum saline soil  and T , irrigated with 100% of the estimated crop evapotranspiration (water regime R1) and 10 colonies from the soil of the same tomato genotypes (L and T) irrigated with 75% of the control treatment (water regime R2). Considering the two approaches, a total of 37 single strains were obtained from soil samples.Several colonies with different phenotypes were visualized and then sticked onto fresh R2A plates. After the colonies were cleaned up to obtain 13 pure colonies from soil of tomatoes L  medium and incubated for 16 hours at 30°C. Serial dilutions  of the obtained suspension were prepared, and 100 µL of each dilution (in triplicate) were spread on R2A agar (Merck®) plates. Several colonies appeared after 4-5 days, and after repeated streaking, we obtained 25 pure colonies from R1 root samples and 22 pure colonies from R2 root samples. A total of 47 strains were obtained from root samples. All isolated strains from soils and roots (84 strains) were used for DNA extraction and taxonomic classification as already described in To isolate bacterial endophytes from plant roots, all root samples harvested in www.baua.de/abas; Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures GmbH https://www.dsmz.de/; BCCM: Belgian Coordinated Collections of Microorganisms https://bccm.belspo.be/). Thus, only the 34 bacterial isolates classified with Biohazard Level 1 were subjected to a series of in vitro assays to assess their plant growth-promoting potential. The ability to solubilize inorganic phosphate (iP) was determined by culturing the strains in NBRIP  following the Nautiyal protocol , according to the method of in vitro by inoculating them in glass tubes with 7 mL of LB medium. The tubes were incubated at 30°C for seven days without shaking. The formation of a visible layer (biofilm) at the interface between the culture medium and air indicates a potential ability to produce biofilm in vivo. The presence of the gene nifH encoding the nitrogenase reductase subunit and the widest marker gene used to identify nitrogen-fixing bacteria was looked for to determine the potential nitrogen-fixing capacity. The PCR to detect the presence of the nifH gene was performed with the two following degenerated primer pairs: 1) nifH-fwA (5’-GCIWTYTAYGGIAARGGIGG-3’); nifH-rvA (5’-GCCATCATYTCICCIGA-3’) and 2) nifH-fwA (5’-GCIWTYTAYGGIAARGGIGG-3’); nifH-rvB (5’-GCRTAIABNGCCATCATYTC-3’) using the following program: 94°C for 4’, repeated two times 94°C for 30’’, 65°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 63°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 61°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 59°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 57°C for 30’’, 72°C for 30’’, repeated 25 times 94°C for 30’’, 55°C for 30’’, 72°C for 30’’, 72°C for 2’. To detect the ability to produce the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the presence of the gene encoding acdS was also searched for, and the two following degenerated primer pairs were used: 1) acdS-fwA (5’-ATCGGCGGCATCCAGWSNAAYCANAC-3’); acdS-rvA (5’-GTGCATCGACTTGCCCTCRTANACNGGRT-3’) and 2) acdS-fwA (5’-ATCGGCGGCATCCAGWSNAAYCANAC-3’); acdS-rvB (5’-GGCACGCCGCCCARRTGNRCRTA-3’) using the following program: 94°C for 4’, repeated two times 94°C for 30’’, 66°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 64°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 62°C for 30’’, 72°C for 30’’, repeated two times 94°C for 30’’, 60°C for 30’’, 72°C for 30’’, repeated 25 times 94°C for 30’’, 58°C for 30’’, 72°C for 30’’, 72°C for 2’. For this purpose, we aligned, with the software Unipro UGENE, about 80 protein sequences (encoded by the nifH and acdS genes) from different soil microorganisms belonging above all to Actinobacteria, Firmicutes and Proteobacteria, identifying the most conserved areas and then drawing the degenerated primers on these sequences.A BLASTn analysis was performed using the 16S rRNA sequences, obtained from the 84 isolates, and revealed the presence of several strains with Biohazard level 2  were sterilized in 2.5% v/v sodium hypochlorite for 20 min, then they were rinsed five times and placed in Petri dishes on watered sterile paper. The Petri dishes were incubated at 25°C in a growth chamber for two days at dark and for other three days at light. After these five days, 62 tomato seedlings were transferred in a greenhouse to pots (0.7 L) containing quartz sand, previously sterilized at 180°C for 3h and watered with tap water. The plants were watered with tap water till a month from the transplanting, then they were watered two times a week with tap water and once a week with Hoagland solution  at half After twenty-one days from the transplanting, the plants were inoculated with plant growth promoting bacteria, previously isolated and characterized (see above). For each strain five biological replicates were considered together with seven biological replicates for the uninoculated control. All the eleven different strains reported in From 26/04/22 until 13/06/22 the plants were monitored two times a week to measure San Marzano height, stem diameter, and number of leaves. Height was measured from the shoot base to the last apical leaf, the diameter was measured with a digital caliper right under the first pair of leaves. At the same time, the portable chlorophyll meter SPAD 502  was used for the estimation of leaf chlorophyll content, one completely expanded leaf per plant was measured. On June 22, plants were removed from pots, submerged in tap water to remove the sand/coconut fiber from the root system and the shoot and root fresh weight measured. A small subsample of roots (around 700 mg) was stored at -20°C for subsequent molecular analyses. Then, roots and shoots were dried for five days at 50°C and weighed to measure the shoot and root dry mass.i.e., strains 509, 510 and 518 separately, along with all their possible combinations, i.e., 509 + 510, 509 + 518, 510 + 518, and 509 + 510 + 518. Twenty independent biological replicates (plants) were considered for each thesis, together with additional twenty uninoculated non treated control plants. The lyophilized bacteria, with the following CFU number: 1.08E+11 for 509, 1.73E+11 for 510, 9.60E+10 for 518 in 2 mL tubes, were resuspended with 1.5 mL of sterile water and vigorously vortexed, then the inoculation was performed using 750 µL of solution when the strain was used as pure. When the plants were inoculated with mixture of two strains, 375 µL of each bacterium was used, while when the mixture was of three strains, 250 µL of each bacterium was employed. After 16 days a second inoculation, following the same procedure of the first, was carried out. The plants were watered with tap water till a month and half from the transplanting, then they were watered two times a week with tap water and once a week with ½ Hoagland solution (s) measures were taken. Particularly, a LI-COR model LI-600 was used to verify the gs and the stress level at the beginning and the end of the experiment. After ten days of stress the plants reached a moderate stress level and plants were sampled following this scheme: five replicates were dried for five days at 50°C and weighed to measure the shoot and root dry mass, while the remaining were frozen in liquid nitrogen and stored at -80°C.In October 2022, San Marzano seeds (La Semiorto Sementi Srl) were sterilized as described above and, after seven days, 160 seedlings were transferred to pots filled in with sterilized quartz sand (see above). The plants were grown at 25°C temperature, having 14 hours of light and 8 hours of dark. After nineteen days from the transplanting, the plants were inoculated with three selected strains showing the best results in Experiment #1, solution . Out of SlCAC specific primers of tomato (® (Invitrogen) procedure using random primers. Reactions were carried out in the ConnectTM Real-Time PCR Detection System (Bio-Rad Laboratories), and the SYBR Green method  was used to quantify the amplification results. Thermal cycling conditions were as follows: an initial denaturation phase at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Specific annealing of primers was checked using dissociation kinetics performed at the end of each RT-qPCR run. The expression of tomato target transcripts was quantified after normalization to two established reference genes in leaves  involved in the biosynthesis of the non-volatile isoprenoid ABA (SlSnRK2) with a role in abiotic stress response (SlTAS14) (SlACO4) involved in the ethylene biosynthetic pathway (SlP5CS) (SlDREB1 and SlDREB2) were considered (SlTPS12) (SlHsp20_I and II) and a gene coding for a NAC domain protein (SlJa2)  and for each inoculum  were considered for the experiment. Total RNA was isolated by using a CTAB-based lysis buffer following the ‘pine tree method’ . The RNAnoid ABA , a gene response , a gene SlTAS14)  and a ge pathway . In addi(SlP5CS)  and two nsidered . A gene SlTPS12) , togethe (SlJa2) . Gene-sppost hoc test. Two-way ANOVA was performed to assess inoculation and water deficit effects in Experiment #2 and when ANOVA indicated that for either condition (WW and WS) or bacteria inoculum  factors or their interaction was significant, mean separation was performed according to Tukey’s HSD test at a probability level of p  ≤ 0.05; ANOVA and Tukey’s HSD test were also used to analyze variability inside conditions and inoculations. The standard deviation (SD) or error (SE) of all means was calculated. A probability level of p  ≤ 0.05 was considered for all tests. Principal component analysis (PCA), performed using R software (version 4.1.1), was used to compare both the biometric and physiological data in the different considered inocula and in each condition (WW and WS). Radar plots were obtained by R software  on biometric parameters, to allow clearer visualization and compare differences between treatments. Concerning gene expression, statistical analyses were carried out using Relative Expression Software Tool REST© 2009 v. 2.0.13 (Qiagen) . R software (version 4.1.1) was used to calculate the Pearson correlation matrix among the gene expression data.Statistical analyses were performed with R software (version 4.1.1). Data were transformed when necessary to fulfill ANOVA assumptions and one-way ANOVA was performed for the analysis of the data in Experiment #1, followed by Tukey’s HSD (Qiagen) , conside3, all can release it, except for strains 522 and 531. Strains 504, 514, 515 and 531 coded for nitrogenase iron protein 2 (nifH2), while only strain 522 coded for ACC deaminase, based on amplification of acdS.DNA amplification with primers for 16S allowed to identify the isolated bacterial strains, as reported in p 0.000475) (p 0.0137) (p > 0.05) (p 0.005) (p 4.7e-06) (p 0.000125) . The PCA, carried out on data of shoot height, stem diameter, leaf number, chlorophyll content, root/shoot dry weight and gs, recorded at the end of the stress period, showed that the control plants were separated from the inoculated ones and that there was a separation between WW and WS plants  (p 4.88e-08) and between conditions (p 0.00014). In particular, plants inoculated with strains 510 and 509 + 510 in well-watered and in stressed conditions were higher than the respective non-inoculated control plants. In addition, plants inoculated with strain 509 under stress were higher than stressed control ones (p 1.64e-08) and between conditions (p 8.86e-08). All the inoculated plants under stress were thicker than control ones (p < 2e-16) and between conditions (p 1.26e-07). In particular, all the inoculated plants under well-watered conditions had more leaves than control ones; under stress the plants inoculated with strains 510 and 509 + 510 had more leaves than stressed control ones (p 2.42 e-12), the plants inoculated with strains 509 + 510 differed on the base of the presence of the stress condition (p 9.03e-8): plants inoculated with strains 510 and 509 + 510 weighted more than control ones under well-watered conditions, while under stress plants inoculated with strains 509, 509 + 510, 509 + 518, 509 + 510, 509 + 510 + 518 had heavier shoots than stressed uninoculated controls (p 0.001318) and between conditions (p <2.2e-16). In particular, well-watered inoculated plants were not different from the control ones, while under stress plants inoculated with strains 509, 510, 509 + 510 and 509 + 518 differed from control ones, having a more weight  putatively involved in the ethylene biosynthetic pathway  and osmolytes such as proline , and genes  negatively correlated with SlDREB2 and SlACO4 , Pseudochrobactrum saccharolyticum  and Sphingobacterium canadense , respectively. All these genera have already reported to present diverse PGP traits, such as phytohormone (IAA) and siderophore production for Sphingobacterium and Pseudochrobactrum in addition to ACC deaminase activity, and phosphorous solubilization in Pseudochrobactrum  well-watered plants could instead be explained with the role of dehydrins in the plant growth under normal condition , in comparison with non-inoculated sugarcane in the same abiotic conditions  was up-regulated in presence of high temperatures. In our experiment, the gene was up-regulated in stressed plants inoculated with strain 509, while it was down-regulated in stressed plants inoculated with strains 509 + 510, suggesting a primary effect of the inoculation rather than of the stress. In rice, it has been detected the up-regulation of a HSP20 gene, together with other genes involved in ABA signaling, under drought and in presence of P. fluorescens, suggesting a role of the bacterium in inducing the systemic tolerance to drought, regulating the gene expression  and tomato roots (518) determined the regulation of genes involved in pathways related to ABA, MEP, osmoprotectant and heat shock proteins, particularly in plants subjected to water deficit. Although we cannot report data on the plant colonization microscopically showing the presence of endophytic colonization, our study confirms that a synergistic and complementary interaction between diverse PGP bacterial strains is a relevant point to be taken in account for the formulation of inocula.https://www.ncbi.nlm.nih.gov/, OP364100, OP364101, OP364102, OP364103, OP364104, OP364105, OP364106, OP364107, OP364108, OP364109, OP364110.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: EZ: Investigation, Writing – review and editing, Methodology, Writing – original draft. EF: Conceptualization, Investigation, Methodology, Writing – review and editing. LG: Data curation, Formal Analysis, Investigation, Visualization, Writing – review and editing. FB: Investigation, Writing – review and editing. FS: Data curation, Formal Analysis, Investigation, Visualization, Writing – review and editing. DF: Investigation, Methodology, Writing – review and editing. IP: Investigation, Methodology, Writing – review and editing. MC: Conceptualization, Writing – review and editing. RB: Conceptualization, Investigation, Supervision, Writing – review and editing.


Caenorhabditis elegans’ (C. elegans) transparent cuticle, along with the conservation of key genetic pathways regulating developmental and reproductive toxicology (DART)-related processes such as germ stem cell renewal and differentiation, meiosis, and embryonic tissue differentiation and growth, support this model’s potential to address the need for quicker and more dependable testing methods for DART hazard identification. Organic and inorganic forms of mercury and arsenic had different effects on reproductive-related endpoints in C. elegans, with methylmercury (meHgCl) having effects at lower concentrations than mercury chloride (HgCl2), and sodium arsenite (NaAsO2) having effects at lower concentrations than dimethylarsinic acid (DMA). Progeny to adult ratio changes and germline apoptosis were seen at concentrations that also affected gravid adult gross morphology. For both forms of arsenic tested, germline histone regulation was altered at concentrations below those that affected progeny/adult ratios, while concentrations for these two endpoints were similar for the mercury compounds. These C. elegans findings are consistent with corresponding mammalian data, where available, suggesting that small animal model test systems may help to fill critical data gaps by contributing to weight of evidence assessments.Exposures to arsenic and mercury are known to pose significant threats to human health; however, the effects specific to organic vs. inorganic forms are not fully understood.  Toxicological testing is critical for assessing the safety of chemicals we encounter daily. The U.S. Food and Drug Administration (FDA) aids in safeguarding our food supply by monitoring a broad range of chemicals ,2. UnforCaenorhabditis elegans (C. elegans), drosophila, and zebrafish ) strains were obtained from the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Purchased strains were initially grown for three generations on agar plates under continuously well-fed conditions using OP50 E. coli as a feeder organism prior to egg isolation and transfer to C. elegans Habitation Medium (CeHM) as previously described [C. elegans larvae grown in CeHM developed at the same rate as those fed OP50 E. coli [C. elegans strain aliquots were frozen at −80 °C soon after transfer to CeHM, and fresh aliquots were thawed every 6 months to avoid genetic drift in test cohorts. All cultures were fed fresh CeHM at least twice a week in biological safety cabinets using a sterile technique. Unless otherwise indicated, cultures were maintained in vented, polystyrene flasks at 20 °C on shakers in hot/cold incubators. To minimize the effects of freezing–thawing on stress resistance and gene expression, thawed C. elegans strains were allowed a minimum of three weeks growth in CeHM prior to toxicity testing. To obtain age-synchronized test cohorts, well-fed gravid C. elegans were subjected to hypochlorite treatment to isolate eggs. Eggs were allowed to hatch overnight in non-nutrient M9 buffer for 19 +/− 1 h. Only egg isolates containing <1 dauer per approximately 5000 eggs were utilized to ensure that cohorts originated from well-fed cultures and were exposed to minimal amounts of dauer pheromone. Synchronized first-larval-stage worms (L1s) were centrifuged, resuspended to approximately 1 worm per µL CeHM, and monitored for dosing at the fourth larval (L4) or young adult (yA) stage. escribed . Wildtyp E. coli ,31, and C. elegans maintenance at 19 °C allows all steps of the progeny ratio to be conducted during the daytime, and so a dedicated incubator was used  [TM, Union Biometrica, Holliston, MA, USA) with the LP SamplerTM option , extinction , and green fluorescence. Synchronized PD4251 L1 cohorts were obtained and fed as described above. OR, USA)  . Age-synchronized L4 worms were allowed to settle in 15 mL conical tubes, and were then fed with fresh CeHM at a concentration of ~1000 worms per mL. A total of 900 µL of this worm mixture was added to individual wells in 24-well plates with 100 µL of water or a 10X dosing solution of interest, and then it was incubated for 24 h at 20 °C on a shaker at 60 rpm in a hot/cold incubator. After incubation, apoptosis assay was performed via acridine orange staining on synchronized adult N2 hermaphrodites collected 24 h post-exposure (Day 1 adults), as previously described ,34. Bispn = 30–35 per condition, in 3–4 replicates). 3-Deazaneplanocin A (DZnep), a histone methyltransferase inhibitor, was used as the positive control [The exposure and GFP germline de-silencing assessments were performed as previously described . Worms wC. elegans PD4251 strain contains green fluorescent protein (GFP) transgenes encoded with nuclear and mitochondrial protein localization signals [C. elegans. First-larval-stage (L1) progeny can easily be lost during the wash steps; therefore, a method to assess progeny to adult ratios that avoids washing prior to microfluidic/laser evaluation with the COPAS was sought. The  signals . This brThe mean green fluorescence readings from nutrient media CeHM alone are ~2 COPAS specific units, while in an initial exploration of the PD4251 model for progeny ratio assessment, the mean green COPAS-specific values for PD4251 eggs, L1s, and L2s were 36, 167, and 334, respectively A–C. Thus2), the lowest observed effect levels (LOELs) for significant decreases in the progeny ratio as well as the parental ToF and EXT all occurred at 50 µg/mL (385 µM) , possibly reflecting increased levels of protein misfolding and/or degradation or changes in muscle mass and/or myo-3 gene expression with arsenic exposure. The progeny ratio LOEL for dimethylarsinic acid (DMA) of 300 µg/mL (2200 µM) was much higher than for NaAsO2, while adult ToF and EXT were not affected by DMA at the tested concentrations  A. Given 5 µM A. Gtrations B. Adult 2) decreased significantly at 4 µg/µL (15 µM), while LOELS for changes in adult ToF and green fluorescence were at 5 µg/mL (18 µM) HgCl2 (The progeny ratios for mercury chloride (HgClM) HgCl2 C. The prM) HgCl2 D. The LOC. elegans germline via fluorescence microscopy. C. elegans were exposed to water  elicited a significant increase in germline apoptosis when compared to the control  meHgCl, elicited a significant increase in apoptotic germline nuclei when compared to the water control  in the germline [C. elegans were exposed from the mid-L4 stage (50–52 h post-L1 feeding) for 24 h, encompassing the window from the L4 stage to Day 1 of adulthood (when gonadogenesis has been completed) [2 at 20–40 µg/mL (155–310 µM) induced a significant de-silencing effect in the germline with a 22–24% increase from the control  DMA. In mercury-exposed worms, HgCl2 induced a significant de-silencing effect at 2.5 µg/mL (9 µM) (19.72 ± 4.46 SEM) but failed to do so at double the concentration  . Arsenicmpleted) . NaAsO2 ntration C, likelyC. elegans study assessed progeny to adult ratios, germline apoptosis, and germline epigenetic regulation for DMA and NaAsO2, along with inorganic HgCl2 and organic meHgCl  was the same as the LOEL for reduced gravid adult body size and optical density . Therefore, increased green fluorescence could reflect changes in muscle cells, greater protein stability with DMA exposure relative to NaAsO2, or altered behavior or muscle tone during laser assessment.The progeny ratio LOEL for DMA was 300 µg/mL (2200 µM), a 6-fold concentration increase relative to the NaAsO2 LOEL B, but thal delay . These s2 significantly decreased progeny ratios at 4 µg/µL (15 µM), while the LOEL for meHgCl was much lower at 0.25 µg/mL (1 µM). There was a good deal of variability from one experiment to the next with both forms of mercury, resulting in large standard errors  [Regarding mercury, HgCld errors C,D. As w similar ,59,60. Iy HgCl2) .C. elegans completed gonadogenesis and reached reproductive age [C. elegans as developing oocytes exit the pachytene stage of meiotic prophase [2 had a dose-dependent increase in germline apoptosis, with a LOEL of 50 µg/mL (385 µM); higher NaAsO2 concentrations altered gonadal morphology, making assessment difficult. At eight times the LOEL concentration of NaAsO2, DMA had a germline apoptosis LOEL of 400 µg/mL (2900 µM). For NaAsO2, germline apoptosis, progeny ratio, and parental morphology were statistically altered at the same concentration of 50 µg/mL, five times the concentration required for developmental delay and developmental hypoactivity in our previous study [2 on mice, where the concentration that induced oocyte apoptosis was close to the maternally toxic concentration [To assess the reproductive-related effects of arsenic and mercury on the germline, exposures to inorganic and organic arsenic and mercury were conducted during the window in which tive age . The gerprophase . NaAsO2 us study . This isntration .2) before germline and overall morphology was affected. There are few rodent studies that directly assess the effects of oral mercury exposures on female germ cells, as most studies focus on male endpoints [C. elegans germline at a time. As this is not a higher-throughput assessment, worms were only included if the germline remained visible, although morphology changes were evident. A more thorough investigation could compare germline size and apoptotic nuclei ratios, while here we propose that counts on healthier germline nuclei could indicate a specific effect as opposed to general toxicity.In contrast to arsenic, both assessed forms of mercury had LOELs at the highest concentrations that could be tested (2 µg/mL (11 µM) meHgCl, and 3 µg/mL (8 µM) HgClndpoints . One stundpoints . This coC. elegans gonad, at a reproductive relevant stage. Histone modifications fluctuate during germ cell development, and thus play a key role in the establishment of chromatin environment and gametogenesis [C. elegans [C. elegans assays have demonstrated epigenetic alterations, including to histone modifications, directly induced by NaAsO2 and meHgCl exposures, as well as in their offspring [C. elegans can be potentially employed as a model for the more rapid assessment of effects on the germline epigenome. We also evaluated the potential effects on the germline epigenome. In a previous gene expression assessment, our lab demonstrated that the direction of expression of genes involved in transcriptional regulation was toward the condensation of chromatin and the repression of transcription, potentially affecting biological processes including reproduction and transgenerational epigenetic regulation . The preogenesis . Studiesogenesis ,66. The  elegans ,68,69 an elegans ,70. Rodeffspring ,74,75,76ffspring ,78,79. H2 disrupted germline histone regulation at a LOEL of 20 µg/mL (155 µM), while inorganic DMA had a LOEL of 100 µg/mL (750 µM) for this endpoint. Compared to the progeny ratio and germline apoptosis, the germline epigenome assay is more sensitive for arsenic, suggesting potential arsenic specific effects on the epigenome. In mercury-exposed worms, inorganic HgCl2 disrupted germline histone regulation significantly at a LOEL concentration of 2.5 µg/mL (9 µM), while the organic meHgCl LOEL was at a 5-times-lower concentration than the inorganic form (0.5 ug/mL (2 µM)). For developmental delay and germline histone regulation, the LOELs were similar for both forms of mercury tested. We evaluated a disruption in histone regulation with the use of an epigenetic reporter strain . The hisFor the histone regulation assay, the worms were individually evaluated, and although this is a quicker method than in vivo mammalian studies, it is also a laborious experiment that shows high sensitivity. The transgenic strain that was utilized has been available for over two decades, and the field could be much improved with upgraded transgenics, allowing for a truly higher-throughput assay making use of a biosorter, as was the case for the progeny output assays. C. elegans toxicity screening; however, much larger panels of chemicals with known effects on mammals need to be tested in order to move toward using this model for regulatory purposes.This work contributes to the understanding of accuracy and fit-for-purpose categories for C. elegans results for exposures to mercury and inorganic arsenic reflect what is known from mammalian oral exposures, while less-studied DMA was far less toxic for the reproductive-related endpoints assessed here. Germline histone regulation was altered at arsenic and mercury concentrations that also induced developmental delay in juvenile C. elegans. For arsenic, but not mercury, germline histone regulation was altered at concentrations below those that affected the progeny/adult ratios. For these endpoints, organic and inorganic forms of arsenic and mercury were ranked meHgCl > HgCl2 > NaAsO2 > DMA. Where comparable mammalian oral toxicity data were available, concordant effects suggest that C. elegans data have the potential to complement existing in vivo studies. C. elegans data can be a useful component in integrated assessments that include other NAMs and small model organisms, potentially contributing to weight of evidence assessments, hazard ID, and regulatory decision making.This study demonstrates the importance of assessing multiple endpoints to identify specific effects at lower concentrations that do not induce general toxicity. Overall,