Agricultural systems are facing the negative impacts of erosion and water scarcity, directly impacting the hydro-mechanical behavior of soil aggregation. Several technologies have been proposed to reduce hydro-mechanical soil-related problems in agriculture. Biopolymer-based hydrogels have been reported to be a great tool to tackle these problems in soils. In this study, we investigated the hydro-mechanical behavior of different soils media treated with Ca-bacterial alginate hydrogel. We used an unconfined uniaxial compression test, aggregate stability test and hydraulic conductivity measurements to investigate the mechanical and hydraulic behavior of treated soils media. Our results from unconfined uniaxial compression test showed that yield stress  increased in treated soils with higher kaolinite and water content , compared with untreated coarse quartz sand . Furthermore, we found that temperature is an important factor in the gelation capacity of our hydrogel. At room temperature, HCM3 displayed the higher aggregate stability, almost 5.5-fold compared with treated coarse quartz sand (HCM1), while this differential response was not sustained at warm temperature. In general, the addition of different quantities of kaolinite decreased the saturated hydraulic conductivity for all treatments. Finally, bright field microscopy imaging represents the soil media matrix between sand and clay particles with Ca-bacterial alginate hydrogel that modify the hydro-mechanical behavior of different soils media. The results of this study could be helpful for the soil-related problems in agriculture facing the negative effects of climate change. Poor aggregation of soil particles due to climate change  increase soil degradation, creating a negative feedback loop that threatens food production ,2,3. SoiAzotobacter vinelandii [Hydrogels in agriculture have been proved to be potential tools to modify the hydro-mechanical behavior of soils by (1) changing and improving the fluid mechanics of the fluid phase, (2) increasing the water-holding capacity and (3) improving the mechanical behavior of soils under different environmental stresses ,13,14. Hnelandii . It belonelandii ,12,21. Tnelandii ,19,20,22nelandii . HoweverHere, we aimed to study the effect of Ca-bacterial alginate hydrogel over the hydro-mechanical behavior of three types of soils composed by coarse quartz sand and kaolinite and subjected to different content of water and temperatures. We used unconfined uniaxial compression tests, falling head permeability tests, aggregate stability tests and bright field microscopy to analyze the mechanics and hydraulics of the different treatments. With this information, we try to provide information that could be used in agricultural systems that are facing the side effects of climate change .+, K+, Mg+2, Ca+2 and Al+3 are the main cations adsorbed on the clay surfaces. These phenomena generate the cation exchange, a very important process in the soil solution that affects the nutrient balance and the flocculation-dispersion processes of soil colloids and soil aggregates [Three types of soil-porous materials with different particle size fractions were used in this study. The percent of fractions was estimated according to the dry weight of samples. Soils were composed by: (1) quartz sand (100% fraction); (2) quartz sand and kaolinite ; and (3) quartz sand and kaolinite . To assemble the different soils, quartz sand  was used, as it is highly susceptible to mechanical compressive stresses and highly permeable to water, mainly due to its lack of aggregate stability ,23. Secogregates . A standgregates .Azotobacter vinelandii ATCC 9046  was cultivated in a medium under atmospheric N fixation conditions, utilizing sucrose as carbon source. The fermentation stage was carried out in a 30 L bioreactor  at 300 rpm, 1 vvm, 30 °C and constant pH 7.0 controlled with 2 N NaOH [4EDTA at 0.1 M. and 3 mL of 1 M NaCl solutions. The final mix was centrifuged for 10 min at 7650 g. The alginate was recovered from the supernatant using cold propan-2-ol with a 1:3 volume, and then filtered with a 0.45 µm Millipore filter paper (MF-Millipore®) t. Later, the filtered precipitate was dried at 60 °C until reaching a constant weight. Then, the dried precipitate was ground and weighed again. The final solid product had a molecular weight (MW) of 453 ± 42 kDa [To recover bacterial alginate several stages were used. Bacterial strain of 2 N NaOH . 30 mL o± 42 kDa . Finally−1, until achieving sixty-four scans with a 4 cm−1 resolution [−1 [−1, respectively [−1, guluronic/mannuronic ratio was calculated by division.A mix of techniques were implemented to quantify the mannuronic and guluronic acid contents of bacterial alginate obtained from cultivation and bioprocessing. In this case, we used Fourier transform infrared (FTIR) spectroscopy, accompanied with the total attenuated reflectance technique. To achieve the correct quantification of the mannuronic and guluronic acid, bacterial alginate samples were freeze-dried before FTIR measurements. Using a Jasco FT/IR-4600 spectrometer , several FTIR spectroscopy analyses were carried out from 500 to 4000 cmsolution . Once thtion [−1 . Bonds oectively ,28. UltiAll the experiments were carried out in the Plant & Soil Biophysics and Biomechanics lab at the Pontificia Universidad Católica de Valparaíso. Six soil porous materials with triplicates were used in this study: CM1 = control consisting of quartz sand (100% fraction); CM2 = control consisting of quartz sand with kaolinite ; CM3 = control consisting of quartz sand with kaolinite ; HCM1 = consisting of quartz sand (100% fraction) mixed with Ca-bacterial alginate; HCM2 = consisting of quartz sand with kaolinite  mixed with Ca-bacterial alginate; and HCM3 = consisting of quartz sand with kaolinite  mixed with Ca-bacterial alginate.2 as Ca+2 source. One of the best characteristics of Ca(OH)2 is its higher solubility in water compared with other Ca+2 sources  [2 at 0.5 M, which is equivalent to 37.04 g L−1, was hydrated with filtered (FI) water using a magnetic stirrer , and the resultant solution with free Ca+2 molecules, released from the reaction, created the eggbox complex with the linear chain polysaccharide of the bacterial alginate , we used Ca(OH)and CaO) . Ca(OH)2alginate . Thus, Cw/w) ratio of the soil. At 800 g of growing media, 0.8 g of bacterial alginate was applied in a water-soluble solution that is equal to 0.1% (w/w). For unconfined uniaxial compression test, samples of 90 g of the different soils were weighted in custom-made plastic devices that served as cylindrical molds  at 2 mm deformation to record with accuracy the elastic behavior of the treated soils. The data were then transformed into Mega Pascals (MPa) and axial strain.A texture analyzer  equipped with a compression plate of 100 mm of diameter  was used over cylindrical specimens of 2.8 cm in diameter × 5.5 cm height, with a cylindrical area of 6.1544 cm gravity . Three lrT) were obtained by the average of 3 repetitions per treatment (n = 3) (rT) [The yield strength, yield strain, elastic modulus (reatment . The You (n = 3) . To calc 3) (Tr)  was usedTo evaluate the distortion  of the different elastic zones of soils, we can estimate the shear modulus (G) linking aterial) ,31,32.V is the volume of the material (m3). Besides, U can be estimated only for linear elastic materials. On the other hand, for non-elastic materials and plastic materials, U must be determined from the area under the stress-strain diagram .Finally, to estimate the amount of energy that must be introduced into the system  to create deformation, the strain energy (U) was calculated , as follw/w) ratio of the soil porous material. For incubation, each experimental unit was exposed to 20 cycles  of wetting and drying (dry wight). Wetting until saturation and drying parameters were defined gravimetrically. After incubation, mechanical breakdown by the hand shaking method were applied for each experimental unit. The samples were dried in the oven  at 60 °C for 24 h, and then the soil media were screened by seven sieves . After drying, the mass was recorded. Finally, the mean weight diameter (MWD) was calculated as an index of soil media aggregation using the following [i is the weight of dry growing media in the sieve i (g) and Wi is the average diameter of the pores of the adjacent sieve meshes (mm).The pre-wetting method was used after the mechanical breakdown by shaking. Using the previous methodology, two experiments were carried out: the samples were subjected to (1) room temperature ; and (2) a dry environment . This was done to evaluate the role of the temperature over the intra and inter-interactions in the different soil media. The experimental unit consisted of plastic pots of 250 g of growing media with four replications. In addition, the Ca-bacterial alginate hydrogel treatment was applied at a concentration of 0.1% . To verify the flow regime, we estimated the Reynolds number for each treatment as [−3), v is the flow speed (m s−1), Dp is the diameter of particles of the growing media (mm) and μ is the dynamic viscosity of the fluid (Pa·s). Finally, to evaluate the relevance of the viscous forces versus surface tension of the Ca-bacterial alginate hydrogel filaments and its effects on the different composite materials, the Ohnesorge number was used [−3), −1) and L belongs to the length scale of filaments of Ca-bacterial alginate hydrogel between soil particle surfaces (m).The falling head permeability method was implemented following the ASTM D5084-16a protocol . After stment as (6)Re=ρvwas used ,36(7)OhTo observe the internal matrix of the different soil-hydrogel composite materials, bright field light microscopy imaging was used. The eggbox effect of the Ca-bacterial alginate hydrogel matrix absorbs and retains the fluids that are in the solution. Thus, to identify the internal structures of the different composite materials, we used a blue ink-water solution to create contrast at the light source. Small samples of the different composite materials were fixed on glass slides and put under the microscope. Using a 200 µm micropipette, the blue ink-water solution with a gravimetric ratio of 1:2 was mixed with the soil samples ,37. FinaAnalysis of variance (ANOVA) was performed using R version 4.0.0 statistical computing environment  with the aid of the CAR software package . The Shap = 6.98 × 10−6 and p = 2.20 × 10−16, respectively). HCM3 75 and 100 WC displayed a 3-fold higher yield stress compared with HCM1 50 WC and CM1 50 WC (p = 0.0002462). Young´s modulus (E) of HCM3 75 WC and HCM2 75 WC treatments showed higher values (3-fold higher) than the HCM1 100 WC and CM2 50 WC  treatments (p = 0.0002462). HCM3 75 WC and HCM2 75 WC treatments displayed the higher Young´s modulus values compared to HCM1 100 WC and CM2 50 WC treatments (p = 5.25 × 10−13). HCM3 100 WC showed the most elastic resilience, reaching 1.6-fold (4.76 × 10−11 ± 2.89 × 10−12 J SE) compared with CM3 100 WC (2.93 × 10−11 ± 4.49 × 10−12 J SE)  was analyzed, and we found significant differences among treatments , compared with lower-value treatments CM2 50 WC, HCM2 50 WC, CM1 100 WC and HCM1 50 WC . These treatments consisted of soil media with 5% or without kaolinite, and low water content .We used unconfined uniaxial compression test to quantify the mechanical behavior of the different soil porous materials on the stress-strain relationship, elastic properties, rigidity properties, strain energy and relaxation time. Significant differences were found in the stress and strain behavior among treatments (M1 50 WC . The resM1 50 WC . Strain M1 50 WC . Signifieatments . Regardieatments . Neverth−1 SE) almost 5.5-fold and 3.6-fold, compared with HCM2 (0.26 ± 0.08 g mm−1 SE) and HCM1 (0.31 ± 0.1 g mm−1 SE), respectively. Significant differences of aggregate stability were found among room temperature treatments (p = 0.009512) . No significant differences in aggregate stability were found among warm temperature treatments (p = 0.5012).Mechanical breakdown/disruption of soil aggregates were used to test the aggregate stability of the different composite materials with Ca-bacterial alginate hydrogel. In previous studies, we evaluated the aggregate stability of coarse quartz sand with and without hydrogel . In the .009512) A. Nevert.009512) B. Higheractivity . Moreoveactivity . HCM1, HK). Soil composite materials with Ca-bacterial alginate hydrogel were subjected to the same environmental conditions of the aggregate stability test. As previously explained, the temperature plays a key role in the crosslinking of hydrogels. We measured how these two environmental conditions  affected the saturated hydraulic conductivity of the different soils’ media. It is important to highlight that all treatments laid into Darcy´s flow regime . Furthermore, estimated Oh numbers of CM1, CM2, and CM3 were of ~0.16, representing the liquid bridges between the soil particles. HCM1, HCM2 and HCM3 displayed Oh numbers of ~83.4, caused by the incorporation of Ca-bacterial alginate hydrogel to the soil matrix.To quantify the effects of the Ca-alginate hydrogel over the different soil media, we used the falling head permeability test to measure saturated hydraulic conductivity (−6 ± 1.28 × 10−7 SE m s−1), almost 2.3-fold and 4.4-fold compared with HCM2 (2.39 × 10−6 ± 2.64 × 10−7 SE m s−1) and HCM3 (1.25 × 10−6 ± 3.01 × 10−7 SE m s−1), respectively. HCM3 achieved to reduce K substantially, compared with HCM1 (77.1%) and HCM2 (56.3%). Significant differences of saturated hydraulic conductivity were found among room temperature treatments (p = 1.769 × 10−6) (K compared with HCM1 at room temperature (4.64 × 10−6 ± 9.97 × 10−7 SE m s−1 and 5.47 × 10−6 ± 1.28 × 10−7 SE m s−1). Additionally, HCM1 reached almost 1.8-fold and 4.5-fold compared with HCM2 (2.57 × 10−6 ± 4.24 × 10−7 SE m s−1) and HCM3 (1.05 × 10−6 ± 1.92 × 10−7 SE m s−1), respectively. However, HCM2 showed a reduction of K of 55.4% compared with HCM1. HCM3 showed the more prominent reduction of K (77.3%) compared with HCM1, and 40.5% compared with HCM2. Significant differences of saturated hydraulic conductivity were found among warm temperature treatments (p = 0.009856)  C,D. The .009856) A,B.We used bright field light microscopy imaging to observe, identify and capture all the details of the different composite materials with Ca-bacterial alginate hydrogel . Composite materials without Ca-bacterial alginate hydrogel  showed direct contact between the sand particles A, and thNevertheless, the addition of Ca-bacterial alginate hydrogel changes the structure of the composite materials at the particle level, showing a three-dimensional network indicated by black arrows D–F. FurtAvailability of important belowground resources for plants such as water and nutrients deeply depend on soil physical properties that are currently threatened by the side effects of climate change phenomena  . In the rT). The analysis of stress-strain relationship revealed that HCM3 75 and 100 WC had a strength value increased by 68%, compared with HCM1 50 WC and CM1 50 WC, the lower-yield stress treatments (rT) measurement to have a better depiction of soil mechanics [rT can exhibit different behavior; for example, if we are working with and ideal elastic material rT rT rT analysis is relevant to understand the shrinkage and swelling phenomena in soil media, since the change in volume of soil media is in function to its water content and the swelling properties of clay minerals [soil plasma , which alter the soil porosity and structural behavior under mechanical stress. Shrinkage and swelling phenomena cause crack forming in agricultural soils, generating problems with the soil structural resilience and the snapping of roots [rT behavior curves. The results showed that CM3 100 WC displayed the higher value (15.34 ± 0.88 s SE), almost 4-fold compared with HCM1 50 WC (4.94 ± 0.42 s SE). For the same soil media but with Ca-bacterial alginate hydrogel , rT had a value of 12.59 ± 0.54 s SE, showing a reduction of 18% in rT. However, when the clay content in the soil media decreased from 10% to 5%, or the water content decreased from 100 to 75 or 50, the magnitude of rT decreases. rT behavior of different soil media with 10% clay content could be due to the small particle size (<2 µm), high surface area, which alters the internal structure of the different soil matrix, and greater water adsorption [To understand the mechanical behavior of the different soils treated with Ca-bacterial alginate hydrogel and different water contents, two types of mechanical measurements were carried out: (1) Unconfined uniaxial compression test (UCS); and (2) Aggregate stability test ,12. Betweatments . This daechanics . Tr can or Tr ≈0 . Tr analminerals ,42. Agrihenomena . The claof roots . It is iof roots . In our sorption ,46.+2 reactivity [Temperature, as well as water content, changes the hydro-mechanical behavior of hydrogels within different porous media ,16. Highactivity . Moreoveactivity . In our activity . Here, wactivity ,48. Furtactivity . The filactivity ,49. Howeactivity ,51. Climactivity ,53. The activity ,55. In tAggregate stability and hydraulic conductivity data shown here might be explained by the reinforcement mechanism of Ca-bacterial alginate hydrogel exerted among the soil particles. The mechanism is based on the strong adhesion of the biopolymer with the soil particles ,56,57. TIn this study, we investigated the hydro-mechanical behavior of different soils media treated with Ca-bacterial alginate hydrogel. Our results highlight that yield stress  increases in treated soils with higher kaolinite and water content , compared with untreated coarse quartz sand . We also found that temperature is an important factor in the gelation capacity of several hydrogels. At room temperature, HCM3 displayed the higher aggregate stability, at almost 5.5-fold compared with treated coarse quartz sand (HCM1). In general, the addition of different quantities of kaolinite, a typical clay particle found in different agricultural regions around the world, decreased the saturated hydraulic conductivity for all treatments. Further studies are still needed to come up with the best bacterial alginate-based hydrogel formulation, as well as experiments that test lower temperatures and pH during the gelation process. Furthermore, we think future studies should explore the interaction of these polymers with plants and microorganisms, and under stresses such as drought. Water scarcity and soil erosion are serious threats to humanity, and we believe the results from this study could be useful to growers and decision makers.


The simple hypoxic cell culture method presented here is technically reliable and experimentally safe. As it can be performed in any standard incubator, it is suitable for use at both low and higher biosafety levels.We present a new and straightforward method by which standard cell culture plates can be sealed off from ambient air and be placed under controlled hypoxic cell culture conditions without costly or highly specialized materials. The method was established on a murine cell culture system using the dendritic cell line JAWS II but can be readily adapted to other cell cultures. The procedure was designed to be easy to implement in cell culture laboratories with standard incubators and requires only readily available materials, resources, and consumables, such as six-well plates, degassed culture medium, CoCl However, it is well known that the partial pressure of oxygen in the blood decreases significantly from the arteries to the cells in the tissues [2 of 80–100 mmHg is found in the pulmonary vessels, it decreases to 40–60 mmHg in the arterioles and is only between 1 and 20 mmHg (0.1–3%) in the cells. [2 serves the mitochondria as a terminal electron acceptor for energy production, and one important question in biological research is how cells adapt to low O2 concentrations (hypoxia), which can develop in cells/organs during inflammation [The oxygen concentration of normal ambient air is 21% (paOmmHg 0.1–% in the ammation ), cancerammation . As a spammation . Hypoxiaammation . Howeverw/w)), sodium salts (≤20% (w/w)), and activated carbon (≤20% (w/w)) [2 in the entire volume of the 6-well plate (100 cm3). Another well of the plate contains a reversible resazurin/resorufin-based oxygen indicator [2 is already reduced. Sterile “plastic spacers” (placed between the lid and the 6-well plate) guarantee that the entire cell culture vessel with all six round chambers has a uniform hypoxic atmosphere. To maintain the hypoxic cellular state when opening the 6-well plate after culturing, 100 µM CoCl2 is added preventively to the medium and all other solutions and buffers. This stabilizes the cellular hypoxia regulator HIF-1α and the cellular downstream processes controlled by it [This article describes a methodical procedure  in which% (w/w)) , which indicator  ;15 mL conical tubes ;2-Mercaptoethanol ;50 mL conical tubes ;6-well plate ;Acrylamide/bis—Rotiphorese Gel 30 ;AgelessEye (oxygen indicator) ;Amphotericin B ;Anti-GAPDH antibody ;Anti-HIF-1α antibody ;Anti-mouse IgG-HRP ;Anti-rabbit IgG-HRP ;Anti-PDK1 antibody ;APS (Ammonium peroxodisulfate) ;Blotting filter paper MN 218 B ;Bromophenol blue ;2  Merk, Darmstadt, Germany; Cat. No.: 232696-5G);CoClFCS , 0.2 µm sterile filtered ;Formaldehyde, 37% ;Glycerin ;Glycine ;HCl (hydrogen chloride), 37% ;HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) ;Image-iT Green Hypoxia Reagent ;IMDM (Iscove’s Modified Dulbecco’s) Medium ;JAWS II (American Type Culture Collection (ATCC), Manassas, U.S.A., Cat. No.: CRL-11904);KCl (potassium chloride) ;2PO4 (potassium hydrogen phosphate) ;KHL-lactate-assay-kit ;MINI-Vertical Spacer, 0.75 mm ;Mouse GM-CSF (granulocyte-macrophage colony-stimulating factor), recombinant protein ;2HPO4 (sodium hydrogen phosphate) ;NaNaCl (sodium chloride) ;Nitrocellulose membrane, 0.45µm ;Oxygen absorber ;Penicillin–streptomycin (5000 U/mL) ;Pierce ECL Western Blotting Substrate ;Pipette Tips, 1–200 μL ;Pipette Tips, 200–1000 μL ;Polyoxyethylene (20)-sorbitan monolaurate (Tween 20) ;Ponceau S ;Prestained protein ladder ;™ Gold antifade reagent with DAPI (mounting medium) ;ProLongProtease inhibitor  ;Scalpel ;SDS (sodium dodecyl sulfate) ;Skim milk powder ;TEMED  ;Transparent foil ;Trichloroacetic acid (TCA)-Deproteinization Kit—Deproteinizing Sample Preparation Kit II ;Tris ;Trypan blue ;Trypsin-EDTA (Ethylenediaminetetraacetic acid) solution ;Urea ;Vacuum bags ;X-ray film, UV/blue sensitive Super RX-N .Bio-Rad Trans-Blot Cell ;2-130);Biological safety cabinet—Berner Flow Safe ;Centrifuge—Hettich Rotina 380R ;Digital camera—Panasonic DMC-TZ18 Lumix ;ELISA Reader—Sunrise Remote ;Film developer—Compact 2 ;Fluorescence microscope—Zeiss Axiovert 200 with ApoTome unit ;Heating block—Thermomixer comfort 5355 ;Hypoxia Cabin—Whitley H35 Hypoxystation ;2 ;Incubator—Sanyo MCO-19AIC COTS100-F);Inverted microscope—ECLIPSE TS100-F ;Microscope LED illumination source—CoolLED pe-200 ;Neubauer counting chamber ;pH meter—Hanna Checker ;Pipette 100–1000 µL ;Pipette 20–200 µL ;Pipette 2–20 µL ;Power supply—Bio-Rad PowerPac Universal Power Supply ;Rocking platform—Heidolph Duomax 1030 ;Ultrasound cleaning unit—Elmasonic S40 ;Vacuum device—Univapo 150 H & Unijet II Refrigerated Aspirator ;Vacuum sealer- Kitchenboss ;Vertical electrophoresis chamber-Mini-PROTEAN Tetra Cell .5 cells per well in degassed HEPES-buffered complete IMDM (with or without 100 µM CoCl2) in four wells of a 6-well plate. This first step ensures efficient cell adherence and adaptation. CoCl2 blocks any degradation of the hypoxia factor HIF-1α under normoxic conditions [Seed 1 × 10nditions  when the2, add 0.3 g of oxygen absorber to the fifth well and a single AgelessEye indicator to the sixth well of the 6-well plate . This fluorogenic compound is live cell-permeable and emits a green fluorescence in hypoxic environments.Put the assembled cell culture plate into a vacuum bag. Insert the bag’s open end into the vacuum sealing machine and evacuate the air within the experimental unit to a low vacuum .2, 7.5% CO2, 90.5% N2) in control experiments.Place the assembled and evacuated hypoxic cell culture plate into a standard incubator and cultivate the cells for 5 h  at 37 °C. As a control, a 6-well plate with corresponding cells (in the presence and absence of Image-iT Green Hypoxia Reagent) can be cultivated with complete IMDM in parallel for the same time under normoxic conditions. Further, for initial validation of the new hypoxic cell culture method, we additionally used a Whitley H35 hypoxystation .After 5 h of hypoxic cultivation, remove the hypoxic cell culture device from the incubator, cut the vacuum bag at the sealed end, and remove the 6-well plate. When exposed to oxygen, the AgelessEye will turn from pink to purple and then blue (≥0.5%).To distinguish living from dead cells, dispense 500 µL trypsin-EDTA solution into one of the culture vessels to completely cover the cells and place in the incubator at 37 °C for up to 5 min. This allows the cells to detach from the culture plate surface. This can be checked with an inverted microscope. When this is complete, all cells will be in suspension. Add complete IMDM (Iscove’s Modified Dulbecco’s Medium) containing FCS  to the cell suspension to inhibit further tryptic activity. Mix one part of 0.4% trypan blue solution with one part cell suspension. Allow mixture to incubate for 3 min at room temperature and analyze by light microscopy. Do this for later analysis time points  as well.Image the cells that were pretreated with Image-iT Green Hypoxia Reagent in the vessel using the fluorescence microscope with excitation/emission 488/520 nm (a standard FITC/GFP (Fluorescein Isothiocyanate/Green Fluorescent Protein) excitation/emission filter set is recommended). If necessary, the fluorescent cells can also be fixed with 2% formaldehyde. Fluorescence of the fixed cells lasts max. 24 h . If cells are also grown on coverslips, they can be viewed at higher magnification with an appropriate fluorescence microscope  after respective fixation and embedding in mounting medium.After 48 h of cultivation under norm- and hypoxia, monitor the cells for increased glycolysis via an L-lactate assay.Remove the medium from the culture vessel by aspiration and wash cells with cold PBS.Dispense 500 µL trypsin-EDTA (Ethylenediamine Tetraacetic Acid) solution into the culture vessel and place it in the incubator at 37 °C for up to 5 min to detach the cells from the culture plate surface. When this is complete, add complete IMDM containing FCS to the cell suspension to inhibit further tryptic activity.Subsequently, determine the cell number by a Neubauer counting chamber.g for 5 min and resuspend them in PBS at a concentration of 1 × 105 cells/100 µL.Wash the detached/harvested cells by centrifugation at 200× g for 5 min, resuspend the cell pellet in 200 µL of lactate assay buffer provided with the kit.After further centrifugation of 300 µL cell suspension at 200× Homogenize the cells quickly by pipetting up and down ten times.g in a cold centrifuge to remove insoluble material.Centrifuge for 5 min at 4 °C and 1000× Deproteinize the resulting supernatants using a protein precipitating TCA (Tricarboxylic acid) kit according to the manufacturer’s protocol.For the further steps of the L-lactate assay, follow the detailed protocol of the manufacturer. The colorimetric response of the assay was measured with an ELISA (Enzyme-Linked Immunosorbent Assay) reader at a wavelength of 450 nm.For the preparation of cell lysates used for SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)/immunoblot analysis, detach the cells with a trypsin-EDTA solution as described above.g for 5 min at 4 °C.Collect the detached cells in a centrifuge tube and centrifuge at 400× g for 5 min at 4 °C. Subsequently, determine the cell number by a Neubauer counting chamber and resuspend the cell pellet in HIF-lysis buffer at a concentration of 1 × 105 cells/100 µL.Resuspend the resulting cell pellet in 1 mL PBS centrifuge again at 400× g and 4 °C for 30 min. Nuclei and cell debris will form a pellet so that the lysate supernatant can be transferred to a new reaction tube.Perform the cell lysis on ice for 30 min. Centrifuge the lysed cells at 1000× For denaturation and complexation with SDS, add one volume of 2× SDS sample buffer to the cell lysates and boil for 10 min in a heating block at 95 °C.Load the polyacrylamide gel with the respective samples ) and a molecular weight marker (prestained protein ladder).Run the gel at 100 V until the dye front migrates from the stacking into the running gel (15 min) and increase to 200 V until the dye front reaches the bottom of the gel (45 min).Remove the gel from the apparatus, spacers, and glass plates and equilibrate it by soaking in transfer buffer for 2 min.Prepare the nitrocellulose membrane by wetting it in transfer buffer for 30 s. Handle the membrane carefully, ideally with rounded tweezers, to avoid scratching or puncturing the surface.Soak blotting filter papers and sponges in the transfer buffer for 5 min.Starting on the side facing the cathode, sequentially assemble the following components: sponge, filter paper, gel, nitrocellulose membrane, filter paper, sponge. Gently remove any air bubbles with a roller or serological pipette. Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.Place the completed transfer stack into a transfer cassette and perform wet transfer according to the manufacturer’s instructions for the blotting apparatus.After transfer, rinse the membrane briefly in distilled water. Gently mark the position of the molecular weight ladder bands with a pencil for size detection. Using a scalpel, cut the membrane horizontally at the level of the 40 and 70 KDa markers of the prestained protein ladder.v/v) Tween 20/5% (w/v) milk powder solution for 1 h at room temperature with constant rocking.Stain the membrane with Ponceau S for 30 s and then rinse briefly with distilled water to visualize protein bands and confirm that the protein transfer was successful. Wash away Ponceau S with several washes in PBS until the membrane is clear. Incubate membrane in PBS/0.1% (v/v) FCS.Dilute the primary antibodies to the working concentration  in PBS/0.1% Tween 20/10% .v/v) Tween 20 solution three times for 10 min each with gentle rocking.Wash the membrane with PBS/0.1% (v/v) Tween 20 for 1 h at room temperature with gentle rocking .Incubate the membrane with secondary antibody  in PBS/0.1% (v/v) Tween 20 three times for 10 min each with gentle rocking.Wash the membrane in PBS/0.1% (Prepare the enhanced ECL (chemiluminescence) substrate just before use according to the manufacturer’s instructions.According to the manufacturer’s suggestions, incubate the membrane in the substrate .Carefully remove the membrane from the detection reagent and sandwich it between layers of plastic .Expose the membrane to autoradiography film in a dark room.After the exposure is complete, place the film into the developer and wait until it is processed.Once the film has been developed, overlay it back on your blot to mark the position of the protein ladder with a marker.2) at all times, indicating a constant low oxygen concentration in the sealed culture plate. Moreover, trypan blue staining showed 91, 89, and 85% intact live cells after 5, 48, and 72 h of hypoxic incubation, respectively. This corresponds well to the survival rates obtained for cells cultured in the commercial hypoxystation. Microscopic analysis of the hypoxic cultivation conditions in the sealed hypoxic cell culture plates was performed for live JAWS II cells using the Image-iT Green Hypoxia Reagent, which sustains its fluorescence when cells/tissue return to normal oxygen levels. During incubation of the sealed hypoxic cell culture plate in a standard incubator at 37 °C, the AgelessEye hypoxia sensor was used to visually monitor oxygen levels. Inspection at the beginning, middle, and end of the cell cultivation revealed that the sensor’s color remained light pink  after opening the hypoxic cell culture plate for further downstream work/analyses under normal ambient air conditions. Immunoblot analyses showed a readily detectable HIF-1α-stabilization for the cells from the hypoxic culture plate, which was almost the same intensity as that found for hypoxic cells cultured in the hypoxystation  and cannot fully replace all the capabilities of a commercial hypoxystation, it opens new possibilities for the simple and inexpensive experimental application of hypoxic cell culture conditions in pilot experiments. A further application of this approach is cell culture work performed under higher biological safety levels (such as BSL3 and 4) where sophisticated hypoxia chambers are less likely to be available, and where their use might not be ideal for a variety of practical and safety reasons.Although the presented method has experimental limitations , 190 mM glycine. Store at room temperature.Complete IMDM with 25 mM HEPES and 20% FCS: Add 2.95 g of HEPES and 100 mL of FCS to 400 mL of IMDM (supplemented with penicillin–streptomycin (100 U/mL)), amphotericin B (2.5 µg/mL), and GM-CSF (5 ng/mL mouse GM-CSF). Adjust to pH 7.4 with HCl.v/v) SDS. Store at room temperature.Electrophoresis buffer: 25 mM Tris–HCl (pH 7.5), 190 mM glycine, 10% , 1% SDS, 10% glycerin, and one tablet of cOmplete protease inhibitor per 50 mL of lysis buffer.v/v) Tween 20, 5% (w/v) milk powder. Prepare freshly (short-term storage on ice).Immunoblot blocking buffer: 1× PBS, 0.1% (v/v) Tween 20. Store at room temperature.Immunoblot washing buffer: 1× PBS, 0.3% (v/v) SDS; 10% (v/v) glycerin; 0.25% (w/v) bromophenol blue; 7% (v/v) 2-mercaptoethanol, and one tablet of cOmplete protease inhibitor per 50 mL of loading buffer.Loading buffer: 10 mM Tris–HCl (pH 6.8); 6.6 M urea; 1% . Sterilize the solution by autoclaving for 30 min and degas the buffer by sonication : For 1000 mL of 1× PBS, start with 800 mL of distilled water. Then, add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Naw/v) Ponceau S dissolved in 1% (v/v) acetic acid.Ponceau S solution: 0.5% (v/v) Tween 20, 10% (v/v) fetal calf serum (FCS). Prepare fresh (short-term storage on ice until use).Primary and secondary antibody incubation buffer: 1× PBS, 0.1% : 3.3 mL; Tris–HCl : 2.5 mL; 10% (v/v) SDS: 100 µL; TEMED: 10 µL; 10% (v/v) ammonium persulfate (APS): 32 µL.Separating gel (10%): Mix in the following order: H2O: 6.1 mL; acrylamide/bis : 1.3 l; Tris–HCl : 2.5 mL; 10% (v/v) SDS: 100 µL; TEMED: 10 µL; 10% (v/v) APS: 100 µL.Stacking gel (4%): Mix in the following order: HTransfer buffer: 25 mM Tris–HCl (pH 7.5), 190 mM glycine. Store at room temperature.Trypan Blue Solution: Dissolve 0.4 g of Trypan Blue in 80 mL of 1× PBS and bring to a slow boil. Cool to room temperature and add PBS to a final volume of 100 mL. Store at room temperature.


Results showed significant differences in some agronomic traits between the two wheat genotypes, i.e., greater leaf size and area, and a higher ratio of productive tillers to total tillers in RC875 when compared with Wyalkatchem. The CO2 response curve showed Wyalkatchem was more severely affected by low P than RAC875 at the booting stage. The relative ratio of the photosynthetic rate at low P to adequate P was also higher in RAC875 at the booting stage. Photochemical quenching (qP) in RAC875 was significantly higher when compared with Wyalkatchem at the booting stage. Maintaining CO2 fixation capacity under low P and higher qP would be associated with P efficiency in RAC875 and measuring qP could be a potential method to screen for P efficient wheat.Maintaining carbohydrate biosynthesis and C assimilation is critical under phosphorus (P) deficiency as inorganic P (Pi) is essential for ATP synthesis. Low available P in agricultural soils occurs worldwide and fertilizer P sources are being depleted. Thus, identifying biosynthetic traits that are favorable for P use efficiency (PUE) in crops is crucial. This study characterized agronomic traits, gas exchange, and chlorophyll traits of two wheat genotypes that differ in PUE. RAC875 was a P efficient genotype and Wyalkatchem was a P inefficient genotype. The plants were grown in pots under growth room conditions at two P levels; 10 mg P kg Phosphorus (P) is an essential macronutrient for higher plants and an important fertilizer for crops, but P fertilizer sources are being depleted . Upon apPhosphorus deficiency significantly reduces crop grain yield and biomass . This nuvia carbon dioxide (CO2) fixation. The absorbed light has three potential fates: (1) it is used to drive photosynthesis, (2) released as heat, and (3) reemitted as fluorescence (PSII), photochemical quenching (qP), and electron transport rate (ETR) are indicators for the efficiency of photosynthesis, while non-photochemical quenching (NPQ) is an indicator of dissipated heat  Tzvel] showed that low P supply reduced photosynthetic rate (Pn), maximum quantum efficiency of PSII (Fv/Fm), ΦPSII, and ETR, and in parallel, low P activates the NPQ mechanism to release excess light energy and results in increased NPQ  (A number of studies have investigated how plants adapt to low P soils and improving our understanding of the mechanisms involved in phosphorus use efficiency (PUE) . These stration) . HoweverAs reported in previous studies, RAC875 is a P efficient wheat genotype, while Wyalkatchem is a P inefficient wheat genotype . Also, i–1 soil (in the form of KH2PO4) along with added basal nutrients with the final amount in the soil as described in –1 soil, respectively] were prepared. A quantity of 420 mL of the nutrient solution was added to preweighed, bagged, dry soil (4.2 kg) and mixed thoroughly by hand.Two wheat genotypes RAC875 (P efficient) and Wyalkatchem (P inefficient) were used for this experiment. Plants were grown in round pots (18.5 cm deep × 17.5 cm top diameter × 16.0 cm base diameter) filled with 4.2 kg of a washed sand . The plants were grown at two P rates, 10 and 30 mg P kg® (>18 MΩ resistivity) water, placed in Petri dishes lined with moistened filter paper, sealed with parafilm, and kept in a dark place at room temperature for 3 days. Two germinated seeds were sown into each pot, with four replicates of each treatment. The pots were kept in a controlled environment growth room with the following conditions: 20°C/10°C, 13-h/11-h day/night cycle, and light intensity of 700 μmol m–2 s–1 at the leaf level. Light source was a combination of fluorescent and incandescent lights. The pots were arranged in a randomized design and were rotated every 3–7 days to minimize the effect of light gradients within the growth room. One plant was thinned from each pot at 10 days after sowing (DAS). The plants were watered to 10% of the dry soil weight every 2–3 days.Wheat seeds were washed thoroughly with Milli-Q–2s–1 (90% red and 10% blue) with an aperture of 6 cm2. All photosynthetic measurements were taken at a constant airflow rate of 500 μmol s–1. The CO2 concentration supplied was 400 μmol mol–1 and the temperature (Tleaf) was 22°C. The chamber humidity was controlled by setting the leaf vapor pressure deficit (VDPleaf) at 1. The measured leaf area was adjusted by the method developed by Gas exchange was measured using a portable photosynthesis system  at the vegetative and booting stage . The meaDark respiration was measured on dark-adapted leaves after 60 min wrapped with aluminum (Al) foil. The same setting parameters were used for the chamber as detailed above with no light illumination.2 response curves were generated at the booting stage  on two replicates of each treatment for both genotypes. The light intensity was set at 1,500 μmol m–2s–1 (90% red and 10% blue) and the airflow rate was set at 500 μmol s–1. The temperature at the leaf surface (Tleaf) was set at 22°C. The CO2 concentrations were set at 400, 300, 200, 100, 50, 0, 400, 400, 600, 800, 1,000, and 1,200 μmol mol–1 using the default method of the instrument. The measurements were taken using the Auto Program and the logging time was set from 1 to 2 min.The CO–2s–1, duration 1,000 ms, output rate of 100 Hz, and margin of 5 points. In this process, minimal fluorescence (Fo) was measured before flash and maximal fluorescence (Fm) was measured when flashed (–2s–1 (90% red and 10% blue) and dark mode rate, light mode rate, and flash mode rate were set to 50 Hz, 50 kHz, and 250 kHz, respectively. The flash was rectangular with the red target 8,000 μmol m–2s–1, duration 1,000 ms, output rate 100 Hz, and margin 5 points. The dark pulse parameters were set at 25 μmol m–2s–1 for far red target, 5 s for time that actinic light is off, 1 s for time that far red is off after actinic is off, 1 s for time that far red off after actinic is off, and 5 for number of data points before and after. The maximal fluorescence (Fm’) was taken at the saturated flash period, then the steady state fluorescence (Fs) was measured immediately before the flash, and minimal fluorescence (F’o) was measured after the flash and removal of actinic light  and were taken on both dark-adapted and light-adapted leaves. For the dark-adapted measurement, the leaves were adapted to dark by wrapping with Al foils for 1 h before the measurement. The measurement was taken with the actinic light off and dark mode rate set to 50 Hz. The flash was rectangular with the red target 8,000 μmol m flashed . The varic light . Photoch–1, CO2 concentration was set at 400 μmol mol–1, and the leaf temperature at 22°C. The humidity of chamber was controlled by setting VDPleaf = 1. Measurements were made at the vegetative stage (42 DAS for both genotypes) and booting stage .The airflow rate was set at 500 μmol sLeaf area (LA) was measured using the method described by P < 0.05). Simple effect and interaction analysis were then performed to compare differences between genotypes at each P supply for these traits using the Phia package developed by P < 0.05). Simple interactions and simple effects were performed to analyze the interactions and effects for two variables at a specific level of the third variable . Two-way ANOVA analysis was used to analyze the effect of genotype (G) and P supply (P) on agronomic traits , gas exchange parameters at the booting stage, and chlorophyll fluorescence parameters at the vegetative and booting stage were performed by a two-way ANOVA. Main effect differences were analyzed using a list significant different (LSD) test with Bonferroni correction (variable . Simple  methods .P < 0.001) affected by P supply and tiller number also significantly (P < 0.001) varied according to genotype and growth stage (Tiller number was significantly (th stage . Tiller P < 0.01), G × P (P < 0.05), G × GS (P < 0.001), and P × GS (P < 0.001) interactions for the tiller number were observed (P < 0.05 with the family error rate and Dunn’s method corrections) G × P interaction occurring at 69 DAS, but not at all the other growth stages. Tiller production responding to P was observed from 23 DAS where both wheat genotypes had more tiller numbers at adequate P when compared to low P (P < 0.05). The response was slightly less at 25 and 27 DAS, and then more obvious at older stages. Indeed, at 51 DAS, tiller numbers produced by plants grown under adequate P was 37 ± 14 on average, about 1.50-fold higher than under low P, while at 23 DAS, the number of tillers under adequate P was 10 ± 2, just about 1.14-fold higher than under low P (Significant G × P × GS (observed . Separater low P .P < 0.05 with the family error rate and Dunn’s correction) greater tiller number than RAC875 at all later growth stages (P < 0.001) and 13 ± 1 (P < 0.001) productive tillers greater than RAC875 that had 7 ± 0 and 9 ± 1 under low P and adequate P, respectively  greater ratio of productive tillers to total tillers  reduction in leaf width and leaf area but not in leaf length for the youngest leaf, low P did not affect leaf width, leaf length, and leaf area for the second youngest leaf (P < 0.001) greater leaf width, leaf length, and leaf area for the youngest and the second youngest leaf, when compared to Wyalkatchem  reduced grain yield and the reduction occurred more so in Wyalkatchem (P < 0.05) grain yield than Wyalkatchem under low P, while no significant difference in grain yield between the two genotypes was observed under adequate P (Low P significantly (lkatchem . RAC875 equate P .P < 0.05) impacted transpiration rate, stomatal conductance, and intercellular CO2 concentration. In contrast, P supply significantly (P < 0.05) affected photosynthetic rate, while it had no significant effect on transpiration rate, stomatal conductance, and intercellular CO2 concentration (P < 0.05) effect on photosynthetic rate, but not on the other three gas exchange parameters (P < 0.05) G × GS interaction for intercellular CO2 concentration was observed  higher transpiration rate and stomatal conductance than RAC875. Transpiration rate and stomatal conductance in Wyalkatchem were 0.0075 ± 0.0005 and 0.83 ± 0.07 mol H2O m–2s–1, respectively, while these values in RAC875 were 0.0064 ± 0.0010 and 0.71 ± 0.16 mol H2O m–2s–1  reduced the photosynthetic rate to 22.4 ± 2.8 μmol CO2 m–2s–1 in comparison with 26.1 ± 3.1 μmol CO2 m–2s–1 at adequate P. At this stage, on an average, photosynthetic rate at 34 DAS (25.8 ± 3.0 μmol CO2 m–2s–1) was significantly (P < 0.05) higher than that at 41 DAS (22.7 ± 3.3 μmol CO2 m–2s–1)  .P < 0.05) P × GS interaction occurred for intercellular CO2 concentration, therefore further separate two-way ANOVAs at each growth stage were used and the result showed genotype significantly affected intercellular CO2 concentration at 41 DAS (P < 0.05 with both family error rate and Dunn’s corrections), while genotype did not impact this parameter at 34 DAS. Wyalkatchem had greater intercellular CO2 concentration at 41 DAS than RAC875 (A significant (n RAC875 .P ≤ 0.001) affected transpiration rate, stomatal conductance, and intercellular CO2 concentration (P < 0.01) effect on the four gas exchange parameters. No significant G × P interactions occurred for transpiration rate, stomatal conductance, and intercellular CO2 concentration, while a slight (P = 0.062) G × P interaction for photosynthetic rate was observed. This explained the greater reduction in photosynthetic rate under low P in Wyalkatchem compared with RAC875 at the booting stage  higher transpiration rate, stomatal conductance, and intercellular CO2 concentration than RAC875  variation in the dark respiration between genotypes was observed (P < 0.05) greater dark respiration (−1.30 ± 0.21 μmol CO2 m–2s–1) than Wyalkatchem (−0.95 ± 0.15 μmol CO2 m–2s–1).Dark respiration was measured at the booting state and the results showed that P supply did not significantly affect dark respiration, while significant  affected Fo, Fm, Fv, and Fv/Fm, while it had a slight (P = 0.066) impact on ΦPSII and ETR at the vegetative stage and had no significant effect on these two parameters at the booting stage (P < 0.05) effect on qP.Six chlorophyll fluorescence parameters including Fng stage . At the o, Fm, and Fv, while it had no impact on Fv/Fm, ΦPSII, and ETR. At this stage, no significant P × G interactions were found for these six chlorophyll fluorescence parameters  greater Fo, Fm, and Fv than Wyalkatchem at both vegetative and booting stages (P < 0.05) greater Fv/Fm than RAC875  higher than that in Wyalkatchem at booting stage  reduced Fo, Fm, and Fv  reduced Fo, Fm, and Fv for RAC875 but not for Wyalkatchem  P × G interaction occurred for this parameter. Further interaction analysis showed low increased (P = 0.063) NPQ 1.5-fold in Wyalkatchem, while low P did not increase NPQ in RAC875  had agronomic traits that may explain its ability to succeed on P deficient soils such as a larger leaf area, lower relative leaf number per tiller, and a higher ratio of productive tillers to total tillers. RAC875 also had some differences in gas exchange and chlorophyll fluorescence traits that might help to contribute to its PUE. This discussion will explore how wheat genotypes respond to P levels and how contrasting agronomic, gas exchange and chlorophyll traits between the two wheat genotypes may help to explain PUE in RAC875.Wyalkatchem produced a large number of tillers during the vegetative stage and many tillers were not productive. In contrast, RAC875 produced less tillers during the vegetative stage and it had a lower tiller mortality. It seems that RAC875 and Wyalkatchem have different developmental approaches. RAC875 produced a lower leaf number but with greater leaf area, while Wyalkatchem had higher leaf number with smaller leaf area . Johnson2 fixation . Indeed, low P reduces the efficiency of photosynthetic electron transport and results in a decreased ATP production  may reflect the maximum potential of plant photosynthesis  and adequate P (30 mg P kg–1 soil), while the experiment conducted by 2PO4) and treatment (0.3 mM KH2PO4). No P present in the control could result in severe effects on the photosynthetic apparatus and thus lead to a significant reduction in ΦPS2, qP, and ETR. A study in peanut also showed that with more extreme P stress, the greater reduction in these parameters  qP at the booting stage and slightly higher (P = 0.066) ΦPS2, ETR at the vegetative stage than Wyalkatchem  NPQ in Wyalkatchem but not in RAC875  increased NPQ in the inefficient wheat. Thus, a study on a large wheat population would confirm if low P significantly increases NPQ under low P in inefficient genotypes. Also, elevated CO2 through climate change and P deficiency are current issues  for PUE. Further research should be conducted on measuring qP of a large wheat population to classify P efficient wheat and P inefficient wheat based on this parameter. The COt issues  and the P = 0.063) under low P in Wyalkatchem but not in RAC875. Low P was less affected on photosynthetic rate on RAC875 at the booting stage as well as the greater in qP might contribute to higher PUE in RAC875.This work has revealed that the P efficient wheat, RAC875, had favorable agronomic, gas exchange, and chlorophyll fluorescence traits in comparison with the P inefficient wheat, Wyalkatchem. RAC875 had greater leaf area per leaf and RAC875 was also more efficient in producing productive tillers than Wyalkatchem. Regarding gas exchange, low P showed more impact on Wyalkatchem than RAC875 at the booting stage. Regarding chlorophyll fluorescence, photochemical quenching (qP) was higher in RAC875 than in Wyalkatchem, while NPQ increased (The original contributions presented in the study are included in the article/VN and JS designed the research. VN implemented the experiments, performed the data analysis, and wrote the manuscript. LP was involved in editing the manuscript and analyzing the data. JS made the revision of the manuscript. All the authors approved the final revision to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Candida albicans and Candida glabrata infection, two of the most important agents contributing to human fungal infections. Phagocytic cells, such as neutrophils, constitute the first line of host defense mechanisms, and the release of neutrophil extracellular traps (NETs) represent an important strategy to immobilize and to kill invading microorganisms, arresting the establishment of infection. The purinergic system operates an important role in the homeostasis of immunity and inflammation, and ectophosphatase and ectonucleotidase activities are recognized as essential for survival strategies and infectious potential of several pathogens. The expression and unique activity of a 3′-nucleotidase/nuclease (3′NT/NU), able to hydrolyze not only AMP but also nucleic acids, has been considered as part of a possible mechanism of microbes to escape from NETs. The aim of the present study was to evaluate if yeasts escape from the NET-mediated killing through their 3′NT/NU enzymatic activity contributing to NET-hydrolysis. After demonstrating the presence of 3′NT/NU activity in C. albicans, C. glabrata, and Saccharomyces cerevisiae, we show that, during neutrophils-Candida interaction, when NETs formation and release are triggered, NETs digestion occurs and this process of NETs disruption promoted by yeast cells was prevented by ammonium tetrathiomolybdate (TTM), a 3′NT/NU inhibitor. In conclusion, although the exact nature and specificity of yeasts ectonucleotidases are not completely unraveled, we highlight the importance of these enzymes in the context of infection, helping yeasts to overcome host defenses, whereby C. albicans and C. glabrata can escape NET-mediate killing through their 3′NT/NU activity.Host innate immunity is fundamental to the resistance against  Candida, Candida albicans and Candida glabrata, are considered important opportunistic agents of fungal infections accounting for approximately 90% of invasive infections in North America  agar plates, harvested by centrifugation, and resuspended in phosphate-buffered saline (PBS) (pH 7.4). Whenever necessary, yeasts were pre-labeled with 1 μM Oregon Green 488 (Invitrogen), for 30 min at 30°C with continuous gentle shaking in the dark, and then washed twice with PBS containing 100 mM glycine, or heat-killed (HK) at 95°C, for 30 min. Yeast cells were counted in a Neubauer chamber and adjusted to the desired cell concentration.9 cells) were incubated for 1 h at room temperature (RT) in 0.5 ml of reaction mixture containing 116 mM NaCl, 5.4 mM glucose, 50 mM HEPES-MES-Tris buffer (pH 4.0), and 5 mM of 5′AMP (Sigma-Aldrich) or 5 mM of 3′AMP (Sigma-Aldrich) as substrates. Additional tubes with substrates incubated with the ectonucleotidase inhibitor ammonium tetrathiomolybdate  were also prepared. The reaction was stopped by the addition of 1 ml of 25% charcoal in 0.1 M HCl. Then, the mixture was centrifuged and 0.1 ml of the supernatant was added to 0.1 ml of Fiske Subbarow reactive mixture. The absorbance of the released Pi was measured spectrophotometrically at 650 nm. Ecto-3′ and ecto-5′-nucleotidase activities were calculated by subtracting the nonspecific 3′AMP and 5′AMP hydrolysis in blanks; the concentration of Pi released in the reaction was determined using a standard curve of Pi.Ectonucleotidase activity was determined by the rate of inorganic phosphate (Pi) released, as previously described . Briefly2 atmosphere. The PMN-rich supernatant was carefully aspirated into a new tube, diluted in PBS solution and centrifuged 5 min at 878g, 4°C. The supernatant was discarded and the remaining pelleted RBC were lysed with RBC lysis buffer . The tube content was mixed by inversion, and lysis was then stopped with addition of PBS solution at 1:1 ratio. After centrifugation for 10 min, 319g, 4°C, the supernatant was carefully discarded and the white pellet with PMN cells was resuspended in RPMI 1640, 1% PenStrep antibiotic mix (Sigma-Aldrich), and counted in a Neubauer chamber.Human polymorphonuclear cells (PMNs) were obtained from whole-blood samples of healthy young volunteer donors and collected in a lithium heparin vacutainer. Using a well-established methodology, blood samples were carefully transferred to falcon tubes and diluted in PBS solution at 1:1 ratio, followed by dextran sedimentation (3% dextran solution in PBS) of red blood cells (RBC), during 90 min at 37°C in a 5% CO6 cells/well) were seeded in a 12-well plate treated with poly-l-lysine (with or without 16 mm glass coverslips) and allowed to sediment for 1 h at 37°C in a 5% CO2 atmosphere. Sedimented neutrophils were then washed with PBS and incubated with C. albicans, C. glabrata, or S. cerevisiae , and with the NETs inducer phorbol 12‐myristate 13‐acetate , 100 nM, at 37°C, in a 5% CO2 atmosphere.Fresh human neutrophils (1 × 10At the end of each infection period (1 or 3 h), coverslips were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at RT and washed. Cells were stained with Wheat Germ Agglutinin (WGA), tetramethylrhodamine conjugate , washed, and neutrophils nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at RT. Coverslips were mounted on glass slides with DAKO mounting medium (DakoCytomation Fluorescent Mounting Medium). For each experiment, duplicates for each condition were performed, and five digital images were obtained per coverslip with a Zeiss Axio Observer Z1 fluorescence microscope, with Plan-ApoChromat 20× and 63×/1.40 immersion objectives. Zeiss Zen lite and Image J software were used to analyze the images.To check the neutrophils viability/membrane integrity, a Trypan Blue exclusion test was performed. Briefly, the infection assays were performed as mentioned above and, after incubation, the cells were washed and scraped, and the obtained cell suspension mixed with a 4% Trypan Blue solution. Viable and non-viable cells were counted in a Neubauer chamber.C. albicans, C. glabrata, and S. cerevisiae viability was evaluated using a colony forming unit (CFU) assay. Neutrophils (2.5 × 105 cells/well) were seeded in 96-well plates coated with poly-l-lysine and incubated with yeasts for 1 h at 37°C in a 5% CO2 atmosphere. The supernatants were collected, corresponding to the non-internalized yeasts (“out” condition). The adhered neutrophils were then lysed with 0.5% Triton X-100 in sterile distilled water and scrapped. The yeasts recovered in this fraction corresponded to the adhered and internalized yeasts (“in” condition). Serial dilutions were performed and each condition was spread in YPD agar plates, with colonies counting after 3 days at 30°C.2 atmosphere with the NETs inducer PMA (100 nM). NET-enriched supernatants (pH 7-8) were collected and incubated with C. albicans, C. glabrata, or S. cerevisiae to evaluate the ability of yeasts to degrade NETs through their 3′NT/NU enzymatic activity. Yeast cells (1 × 106 cells/well), treated or not with the 3′NT/NU inhibitor TTM (100 μM), were incubated with the NET-enriched supernatants for 3 h at 37°C. After this period, some samples were also treated with EcoRI and HindIII restriction enzymes (20 U/ml) for 30 min. The supernatants of each condition were collected and centrifuged at 1,520g for 10 min at 4°C, concentrated in a freeze-drying apparatus and run in a GreenSafe pre-stained 1% agarose gel.Human neutrophils prepared as described above were incubated for 3 h at 37°C in a 5% COData analysis was performed using GraphPad Prism software (Version 7.04). Data are presented as means ± SEM. Statistical differences were determined, depending on the analysis, using one- or two-way ANOVA with Bonferroni post-hoc test or unpaired t test for analysis of two groups. Results with P < 0.05 were considered statistically significant.C. albicans, C. glabrata, and S. cerevisiae  and ecto-5′-nucleotidase (5′NT) activities in revisiae 2. YeastC. albicans, C. glabrata, and S. cerevisiae cells has been associated to the escape of other microorganisms from NETs control , C. glabrata (Nɸ+CG), and S. cerevisiae (Nɸ+SC) were able to activate neutrophils and trigger NETs release, with this process being more evident at 3 h p.i. Surprisingly, during Nɸ+CA interaction, the activation of neutrophils and NETs release could be observed, but the NETs (in blue) poorly colocalize with C. albicans cells (in green); free/not trapped by NETs yeast were found more frequently, contrary to what was observed with C. glabrata and S. cerevisiae  presented the highest viability values for yeasts not adhered or not phagocytized by neutrophils and/or retained in NETs (“out” columns), when compared with the other yeast species, and a statistical inferior number in its own adhered/phagocytized/retained viable cells , thus corresponding to a higher ability to escape neutrophils’ immune responses. C. glabrata (CG) and S. cerevisiae (SC) presented lower CFU counts, being more effectively controlled by neutrophils, with no statistical differences between adhered versus non-adhered viable cells. In more prolonged interactions, at 3 h p.i.  and C. glabrata (CG) presented higher viability counts of adhered/phagocytized/retained cells when compared with non-adhered ones , and those values were again more evident in C. albicans (CA). Being the less virulent species used, S. cerevisiae (SC) presented lower viability counts in all conditions tested, being more prone to neutrophil elimination.Once yeasts have stimulated neutrophils to release NETs, the viability of both cell types is expected to be affected, and this was quantified next. A Trypan blue exclusion test revealed that the number of viable neutrophils at 1 and 3 h p.i. was always statistically higher than the number of non-viable neutrophils for all the conditions tested Figure 4C. albicans (CA), C. glabrata (CG), and S. cerevisiae (SC) to digest nucleic acids and, consequently NETs , C. glabrata (CG), and S. cerevisiae (SC) displayed more pronounced DNA smears and correspondent integrated densities when compared with neutrophils not exposed to yeasts (p<0.01), thus proving the ability of yeasts to degrade NETs. However, when yeast cells were incubated with the 3′NT/NU inhibitor, TTM, which has no impact in neutrophils viability  and C. glabrata , as demonstrated by smaller gel smears and lower integrated density quantification. This profile was less evident in S. cerevisiae . Moreover, C. albicans, the most virulent yeast species, showed a decreased yeast viability when the cells were incubated with TTM  and used to assess the ability of tly NETs Figure 6C. albicans and C. glabrata both induce proinflammatory immune responses, which involve the recruitment of phagocytic cells, especially neutrophils. These cells are valuable for their clearance, early in the course of infection , turning C. albicans more prone to neutrophils control and elimination. Under these physiologic conditions, a pH of 7 to 8 was measured in NET-enriched supernatants, resembling pH values found in human blood. Although alkaline pH was associated to lower 3′NT/NU enzymatic activities in all yeasts tested, the complexity of these interactions seems to overcome this feature. However, we might speculate that, under acidic conditions, where the 3′NT/NU activity profile could be at its maximum, the yeasts’ enzymatic activity could be more pronounced. Moreover, whenever heat-killed yeasts were used, no degradation of NETs samples were obtained, and upon interaction with neutrophils, instead of NET formation, a more pronounced phagocytic profile was found. This indicates that neutrophils’ NETs extrusion upon contact with yeasts is dependent on Candida metabolism and not only on its recognition , through the Centro 2020 Regional Operational Programme under project CENTRO-01-0246-FEDER-000010, CENTRO-01-0145-FEDER-000012-HealthyAging2020, and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalization and Portuguese national funds The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FUT2, responsible for secretor status, MCPyV with variants in STING1, involved in interferon induction, and WUPyV with a functional variant in MUC1, previously associated with risk for gastric cancer. These results provide insights into the genetic control of a family of very prevalent human viruses, highlighting genes and pathways that play a modulating role in human humoral immunity.Human polyomaviruses are widespread in humans and can cause severe disease in immunocompromised individuals. To identify human genetic determinants of the humoral immune response against polyomaviruses, we performed genome-wide association studies and meta-analyses of qualitative and quantitative immunoglobulin G responses against BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel cellpolyomavirus (MCPyV), WU polyomavirus (WUPyV), and human polyomavirus 6 (HPyV6) in 15,660 individuals of European ancestry from three independent studies. We observed significant associations for all tested viruses: JCPyV, HPyV6, and MCPyV associated with human leukocyte antigen class II variation, BKPyV and JCPyV with variants in  First dse pairs . Serologse pairs . In mostpatients .A better understanding of the genetic control of the human immune response against polyomaviruses may provide new insights into pathologic mechanisms. We therefore searched for associations between human genetic variation and both differences in IgG levels and serostatus against five human polyomaviruses: BKPyV, JCPyV, human polyomavirus 6 (HPyV6), Merkel cell polyomavirus (MCPyV), and WU polyomavirus (WUPyV), using genome-wide association study (GWAS) and meta-analysis in a total of 15,660 healthy individuals of European ancestry from three studies from Switzerland, the United Kingdom, and Germany.2.2.1P < 2 × 10−16), JCPyV (P = 4.56 × 10−03), MCPyV (P = 5.77 × 10−03), and WUPyV (P < 2 × 10−16) and lower IgG levels against HPyV6 (P = 3.51 × 10−15), JCPyV (P < 2 × 10−16), and MCPyV (P = 1.07 × 10−05) in female participants.We included 3,689 individuals from CoLaus study , 2,363 from GRAS Data Collection , and 9,351 from the UKB , resulting in a total of 15,660 individuals with available genotyping data, serological data, and covariates. All tested polyomaviruses had high seroprevalence in the three samples, with highest observed seroprevalence for WUPyV (96 per cent), followed by BKPyV (91 per cent), HPyV6 (84 per cent), MCPyV (73 per cent), and JCPyV (55 per cent) . In all 2.2λ ranged from 0.99 to 1.01 across the tested cohorts, indicating proper control of population stratification , FUT2 (for BKPyV and JCPyV), STING1 (for MCPyV), and MUC1 (for WUPyV) regions  or a continuous, quantitative approach  to search for human genetic determinants of the antibody response to BKPyV and JCPyV in the three cohorts, to HPyV6 and WUPyV in CoLaus and GRAS, and to MCPyV in GRAS and UKB . We did fication . In tota regions .2.3P = 9.62 × 10−14), rs112463084 (P = 2.18 × 10−20), and rs147658078 (P = 8.09 × 10−20), respectively. To further dissect the association signals, we imputed four-digit HLA alleles and tested them for association with anti-HPyV6, anti-JCPyV, and anti-MCPyV VP1 IgG levels. The regional association plots are shown in P-values are listed in Significant associations were observed in the HLA class II region on chromosome 6 for HPyV6, JCPyV, and MCPyV , with th2.4FUT2 gene on chromosome 19 for BKPyV and JCPyV  antigens that can be found in secretions and fluids (P = 6.40 × 10−16) and JCPyV  are in high linkage disequilibrium , with the common FUT2 nonsense variant rs601338 . The homozygous genotype for the rs601338 minor allele (AA) is classically referred to as the ‘non-secretor’ variant, as individuals homozygous for this null allele do not secrete blood group antigens at epithelial surfaces  production by infected cells to protect them and neighboring cells from local infection (P = 5.95 × 10−15), a strong eQTL for STING1 in the GTEx data set, whose G allele was associated with a stronger IgG response against MCPyV VP1 , with the splicing variant rs4072037 (P = 4.08 × 10−30) located in the second exon of the MUC1 gene. The rs4072037 T allele was associated with a stronger IgG response against WUPyV VP1. The same allele was previously associated with an increased risk of gastric cancer  and JCPyV (λ = 0.98), while MCPyV (λ = 0.90) and BKPyV (λ = 0.72) showed varying degrees of deflation (P = 4.31 × 10−46) and MCPyV   and STING1 for MCPyV . Associations with FUT2 and HLA variants were not replicated for BKPyV and HPyV6, respectively. In addition, the case–control analyses did not reveal any additional associated region, suggesting that there are no SNPs purely associated with susceptibility.We then investigated whether the genetic architecture of antibody levels against human polyomaviruses is similar to that of serostatus of the same polyomaviruses. Serological status was defined based on the manufacturers’ defined cutoff of 250 MFI . Individuals with levels of IgG antibodies above the threshold were considered seropositive (cases), while those below the threshold were seronegative (controls) . Case–coeflation . Chicago× 10−29) . Stronge3.Human polyomaviruses are highly prevalent and can cause severe disease in immunocompromised individuals. They present a wide range of tissue tropisms and appear to cause neurologic, renal, and skin diseases . While tGenes within the HLA region are prime candidates for susceptibility to viral infection and regulation of the immune response. HLA has been associated with multiple infectious diseases such as HIV, hepatitis B, hepatitis C, and tuberculosis . In thisFUT2 variation (rs601338 G>A) plays a role in the determination of antibody levels against BKPyV and JCPyV VP1. The FUT2 enzyme catalyzes the addition of a fucose residue to type 1 glycoprotein chains to form ABH blood group antigens that are released in secretions and at mucosal surfaces  had significantly higher anti-MCPyV IgG levels. A recent study by Kennedy et al. reported an association between the same variant and the intensity of the immune response following primary smallpox vaccination (STING1 gene leads to a decrease in IFN-α expression levels. This result substantiates a previous finding showing reduced IFN-β transcription for carriers of the R232H variant (G = 0.28) relative to African populations , suggesting a possible role of infection-driven selection in this genomic region.Our analyses confirm the implication of the cination . The sam variant . EvidencMUC1, the gene encoding the Mucin1 protein. There is no established role of WUPyV in disease thus far. However, it was detected in the trachea of immunocompromised children, in close proximity to MUC5AC-expressing cells  and binary (case–control) phenotypes, with comparable statistical strength. The same variable genetic loci in HLA class II, Here, we report the first meta-analyses aiming to identify human genetic determinants of the humoral immune response against multiple polyomaviruses. Statistically combining data from CoLaus, GRAS, and a subset of the UKB allowed us to run analyses on a total of 15,660 individuals of European ancestry. This provided greater statistical power and a more accurate estimate of the underlying effects, also reducing the risk of false-negative results. Some limitations need, however, to be considered. First, we did not have serological data for all five polyomaviruses in each cohort. Second, the meta-analyses relied on summary statistics derived from independently computed GWAS, which might result in biases due to differences in the design, analysis, and conduct of the individual studies. Third, our analyses were restricted to individuals of European ancestry, which limits the generalizability of our results. Finally, as mentioned above, individuals with very low IgG levels were excluded from the quantitative association analyses, due to the impossibility of distinguishing them from truly seronegative  study participants; this resulted in reduced statistical power.HLA class II, FUT2, STING1, and MUC1 genetic variants with the intensity of the humoral IgG response to multiple human polyomaviruses. Together, these results demonstrate the modulating contribution of host genetic variation to the individual response against some of the most prevalent human viruses.In summary, we here report strong associations of 4.4.14.1.1Cohorte Lausannoise (CoLaus) is a population-based prospective study that started in 2003 in Lausanne, Switzerland (www.cer-vd.ch), approved the baseline CoLaus study , and written consent was obtained from all participants.The tzerland . It incl4.1.2The Göttingen Research Association for Schizophrenia (GRAS) data collection consists of 2,363 immunocompetent adults of European ancestry, comprising 1,147 anonymized blood donors  and 1,216 individuals with psychiatric diagnoses  . All stu4.1.3The UK Biobank (UKB) is a population-based prospective study whose recruitment process has been described previously . Briefly4.2H. pylori antigens  and four EBV antigens  was measured in CoLaus.To assess humoral responses to polyomaviruses, serum samples were independently shipped by the three studies to the Infections and Cancer Epidemiology Division at the German Cancer Research Center  in Heidelberg. Seroreactivity against BKPyV, HPyV6, JCPyV, MCPyV, and WUPyV viral capsid protein 1 (VP1) was measured at serum dilution 1:1,000 using multiplex serology based on glutathione-S-transferase–VP1 fusion capture immunosorbent assays combined with fluorescent bead technology . Antigen4.34.3.1et al. were applied (P < 10 × 10−7), resulting in 697,923 SNPs kept for phasing and imputation.DNA samples from 5,399 CoLaus participants were genotyped for 799,653 SNPs using the BB2 GSK-customized Affymetrix Axiom Biobank array. Quality control procedures described by Anderson  applied . A totalhttps://imputation.sanger.ac.uk). The imputed data set (N = 89 million SNPs) consisted of (1) SNPs imputed with HRC reference panel only, (2) SNPs imputed with the merged reference panel only, and (3) SNPs with the highest imputation information (INFO) score if imputed with both reference panels. To filter out poorly imputed or rare genotypes, SNPs with INFO score <0.8, minor allele frequency (MAF) <1 per cent, or significant deviation from HWE (P < 10 × 10−7) were excluded, resulting in a final number of 9,031,264 SNPs available for association analysis. We also imputed 111 four-digit classical HLA alleles using SNP2HLA and the T1DGC Immunochip/HLA reference panel  r1.1 reference panel . Genotypce panel .Genome-wide association analyses were performed using mixed models in GCTA . HLA ass4.3.2P < 5 × 10−07). We then used linear regression models in PLINK v1.9 to test for association between ∼6 million SNPs and IgG responses using a continuous, quantitative approach  . The quality control steps have been described previously . Imputatsamples) . The firsamples) .4.3.3Genotyping and imputation of UKB individuals have been fully described by 4.4P-value, sample size, and direction of effect. The Bonferroni-corrected significance level threshold of P = 5.6 × 10−09 (P = 5 × 10−08 divided by the eight GWAS performed) was applied. Similarly, for HLA alleles, we determined significance thresholds correcting for the number of alleles, the viruses tested, and the two study designs (P = 4.3 × 10−05).Meta-analyses of the GWAS results obtained in the different cohorts were performed using a fixed-effect model in METAL . For BKP4.5FUT2 secretor status was determined from the rs601338 genotype, where the wild-type rs601338 (G) encodes the secretor allele, whereas A is the nonsense ‘non-secretor’ allele. Individuals with homozygous major (GG) or heterozygous (GA) genotypes were considered as secretors and compared with homozygous minor (AA) non-secretors .4.6A total of twelve RNA samples from gastrointestinal tissue (200–225 ng each) were obtained from the Genotype-Tissue Expression (GTEx) project . Samplesveab058_SuppClick here for additional data file.