Cell monolayers underpin the discovery and screeningof new drugsand allow for fundamental studies of cell biology and disease. However,current cryopreservation technologies do not allow cells to be storedfrozen while attached to tissue culture plastic. Hence, cells mustbe thawed from suspension, cultured for several days or weeks, andfinally transferred into multiwell plates for the desired application.This inefficient process consumes significant time handling cells,rather than conducting biomedical research or other value-adding activities.Here, we demonstrate that a synthetic macromolecular cryoprotectantenables the routine, reproducible, and robust cryopreservation ofbiomedically important cell monolayers, within industry-standard tissueculture multiwell plates. The cells are simply thawed with media andplaced in an incubator ready to use within 24 h. Post-thaw cell recoveryvalues were >80% across three cell lines with low well-to-wellvariance.The cryopreserved cells retained healthy morphology, membrane integrity,proliferative capacity, and metabolic activity; showed marginal increasesin apoptotic cells; and responded well to a toxicological challengeusing doxorubicin. These discoveries confirm that the cells are “assay-ready”24 h after thaw. Overall, we show that macromolecular cryoprotectantscan address a long-standing cryobiological challenge and offers thepotential to transform routine cell culture for biomedical discovery. To mitigate the damagecaused during cryopreservation, a cryoprotective agent (CPA) is essential,with DMSO (dimethyl sulfoxide) being the most widely used cryoprotectantfor nucleated mammalian cells.2 Duringtypical slow-freezing cryopreservation (1 °C min–1), as cellular dehydration occurs, DMSO replaces lost water to reducecell death from excessive dehydration and IIF.4 DMSOalso minimizes osmotic shock by reducing electrolyte concentrationin residual unfrozen solution during freezing.5Convenient, ready-to-use from the freezer,cell-based assays couldaccelerate and simplify the discovery of pharmaceutically active compounds,biocompatibility testing, assay development, and discovery of cellsignaling and disease pathways. Cryopreservation enables the long-termstorage of cells to prevent phenotypic drift associated with continuousculture and allows the distribution of standardized cells around theglobe. However, the cold stress of cryopreservation can lead to celldeath as described by Mazur’s two-factor hypothesis.8 however for most laboratory-basedapplications (such as toxicity or uptake screening) cells are usedas adherent monolayers, thus, creating a mismatch between the formatused for storage and that used to generate data. From suspension,cryopreserved cells must be thawed, seeded, and propagated throughseveral flasks until enough cells can be plated into well plates forthe desired application. Hence, a cell biologist is required to devotea large portion of their time to cell preparation, approximately 1–3weeks depending on the cell proliferation rates. In essence, moretime is spent “handling” the cells rather than conductingresearch or “value-adding” activities with the cells.The routine handling of cells is also plagued with significant single-useplastic waste,9 which presents environmentalconcerns. Furthermore, exploring different cell lines  can require time, financialresources, and extensive cell culture knowledge to establish, suchas optimum confluency for activity and substrate specificity.13Mammalian cells are typically cryopreserved in suspension,whichoften gives high yields,15 However, DMSO is unable to cryopreserve cells in the monolayer format,with typically ≤30% of cells recovered after freeze/thaw andthe presence of cell debris, which could compromise assay results.16 IIF is a particular problem for the cryopreservationof cells as monolayers (and spheroids), compared to suspension cells,as cell–cell contacts promote the propagation of intracellularice,19 which is usually fatal and contributes to the lowcell recovery rates .20 To increase post-thaw cell recovery, IIF canbe reduced by directional freezing21 ormanually inducing nucleation at warmer temperatures, for example usingN2 ice mist,22 or pollen extracts23 however reproducibly inducing nucleation isdifficult using manual methods and chemically defined nucleating agentsare not available.A (deceptively) simple solution to the abovementioned challengeswould be to cryopreserve cells directly in microwell plate substrates.In an ideal world, cells could be removed from cold storage in an“assay-ready” format, thawed, and used 24 h post-thaw,removing weeks of laboratory researcher time, facilitating high-throughputscreening, and ensuring phenotypically identical cells. Furthermore,highly desirable characteristics of adherent cells could be preserved,including neuron networks and tight cellular junctions.27 and synthetic31 small molecules and macromoleculeshave been explored, which areinspired by extremophiles32 and capableof inhibiting ice recrystallization (IRI) and modulating ice growthand formation.33 IRI has been shown tobenefit red blood cell cryopreservation;35 however, themagnitude of benefit during monolayer or suspension cryopreservationof nucleated cells is limited.38 Protective osmolytes such astrehalose and l-proline can mitigate some damage during monolayerfreezing,36 increasing the recovery ofNeuro-2a cell monolayers from 13 to 53%39 and HepG2 cells from 13 to 42%.40 Matsumuraand Hyon reported the cryoprotective properties of polyampholytes,polymers bearing mixed anionic/cationic side chains, using carboxylatedpoly(ε-lysine) (PLL) and 10% (v/v) DMSO, which performed twiceas well as DMSO alone for freezing of L929 and rat bone marrow mesenchymalstem cells as monolayers.41 The mechanismof protection of polyampholytes is not yet clear, but there is evidencethat salts are trapped by a matrix surrounding the cells to minimizeosmotic damage, while ensuring sufficient dehydration to prevent spontaneousIIF.43 Polyampholytes may also interact/protectthe cell membrane,44 similar to how antifreezeproteins can protect liposome models.45 COOH-PLL has also been used to preserve oocytes,46 chondrocyte sheets,47 and humanstem mesenchymal stem cells.49 However, in these systems,vitrification is often required to achieve high cell recovery, necessitatingsignificant volumes of organic solvent cryoprotectants, such as 6.5M ethylene glycol.48 The broad applicabilityof the ampholyte scaffold has been shown by deploying backbones withmixed charged side-chains, with reports suggesting that either excessanionic50 or 1:1 anionic/cationic groups yield thestrongest cryoprotective activity.53 Bailey et al. introduceda synthetically scalable polyampholyte based on the ring open polymerizationpoly.54 The use of a maleic anhydride-containing polymerensures a 1:1 ratio of cationic to anionic groups, unlike heterogeneousmaterials based on random copolymerization52 or carboxylation of PLL.41 This polyampholyteenhanced the post-thaw recovery of a panel of cell lines  compared to DMSO alone and the suspension cryopreservationof stem cells.55 Despite these advances, thereis still no demonstration that cells frozen as monolayers are “assayready”, whereby they can be removed from the freezer and, withminimal user intervention, be ready to use. To achieve this, an in-depthanalysis of cellular health and function is required and the celllines selected should be of crucial importance to toxicological orscreening assays (such as liver cells) to have a significant impact.57To address the challenges of cryopreservation,naturalalt-maleic anhydride) and dimethylaminoethanol enables near-quantitative recovery of intact and viable cellmonolayers 24 h following removal from a −80 °C freezer,with minimal processing and handling. High cell recovery was demonstratedfor adherent cell lines that are widely used in the drug discoveryfield including A549, HepG2, and Caco-2 cells. Banked “assay-ready”cells were frozen at multiple cell densities, designed for adaptabilitytoward desired applications, and could be stored for over 1 monthin a conventional −80 °C freezer. A large panel of post-thawproliferation, metabolic, membrane integrity, apoptosis, cell cycle,and morphological analyses confirmed that cell monolayers are healthy,function as normal, and are “assay ready”. Finally,a model drug screening test was undertaken using doxorubicin, a chemotherapeuticdrug. Our work demonstrates that adherent cells can be cryopreservedso that they are “assay ready” direct from the freezerwith minimal handling, facilitating the drug discovery process andreducing the significant time burden associated with routine cellculture.Herein, we demonstrate that the polyampholyte derived frompoly (Mn ≈ 80 kDa),tetrahydrofuran (THF), dimethylamino ethanol, deuterium oxide (D2O), type I solution from rat tail, doxorubicin hydrochloride(98%), Eagle’s minimum essential media (MEM), fetal bovineserum (FBS) (non-USA origin), MEM nonessential amino acid (NEAA) solution(100×), 0.4% Trypan blue, dimethyl sulfoxide Hybri-Max, and RNaseA from bovine pancreas were purchased from Merck .Dulbecco phosphate-buffered saline (DPBS), Ham’s F-12K (Kaighn’s)medium (F-12K), Antibiotic–Antimycotic (100×) (PSA), Trypsin–EDTA(0.25%), CellEvent Caspase-3/7 Detection Reagent, Live/Dead Viability/CytotoxicityKit for mammalian cells, phenol-free DMEM/F-12, and propidium iodidewere purchased from Fisher Scientific . Resazurintablets were purchased from Scientific Laboratory Supplies . P450-Glo CYP3A4 Assay and P450-Glo CYP2C9 Assay were purchasedfrom Promega .Poly relative to residual nondeuterated solvent resonances(D2O 1H: δ = 4.71 ppm). Bruker Topspin3.5 Software was used to process and export spectra.–1 with a spectral resolution<2 cm–1, wavenumber accuracy of 0.05 cm–1, and wavenumber reproducibility of 0.005 cm–1.Gain, aperture, scan speed, and filter were all set to auto. AgilentMicroLab Software, version B.05.4, was used to process and exportspectra.FTIR spectra were acquired usingan Agilent Cary 630 FTIR spectrometer equipped with a single reflection diamond ATR accessorywith a 45° angle of incidence, a 1 mm diameter sampling surface(200 μm active area), and a rotating pressure clamp (applyingmaximum pressure). Scans (128) were obtained of dried, crushed samplesbetween 4000 and 650 cmAn Olympus CX41 microscope equipped witha UIS-2 20×/0.45/∞/0–2/FN22 lens and blue and greenexcitation lasers were used for imaging. Image processing was completedusing ImageJ (v1.52).–1 (10 μmcore) and thresholding maintained at 10,000. BD CSampler Plus software(v 1.0.34.1) was used for data collection and processing. FlowJo X10.0.7r2  was used for all statistical analysisand plotting of flow cytometry data.A BD Accuri C6, equipped with 488 (solid)and 640 (diode) nm lasers and 533/30, 585/40, and 670 LP filters,was used for flow cytometry experiments. Instrument calibration wascompleted using BD Biosciences CS&T beads. Maintenance was completedwith BD Biosciences cleaning solutions. All samples were analyzedwith a flow rate of 14 μL minData were plotted and analyzedusing Graphpad Prism 9 with a one-way analysis of variance (ANOVA)followed by a comparison of experimental groups with the appropriatecontrol group (Tukey’s post hoc test).58 Cells were seededon a 14 mm round glass coverslip at a density of 1 × 106 cells mL–1. The coverslip of adhered cells wasplaced on a quartz crucible containing cryoprotectant solution (5μL), consisting of polyampholyte (40 mg mL–1) and 10% DMSO, which was subsequently placed on a Linkam BCS 196cryostage . Cells wereincubated at 20 °C for 10 min, to achieve equilibrium, frozenat −40 °C at 1–5 °C min–1, and warmed to RT at 20 °C/min. Lynksis 32 software was usedto control the cryostage parameters. All images were captured usingan Olympus CX41 microscope equipped with a UIS-2 20×/0.45/∞/0–2/FN22lens (Olympus Ltd.) and a Canon EOS 500D SLR digital camera. Imageprocessing was completed using ImageJ (v1.52).Intracellular ice growth was assessedas described previouslys–1, and sodium chloride solution, with an osmolality of 2000 mOsmolkg–1. Solutions containing cell culture base media,10% FBS, 10% DMSO, and/or 40 mg mL–1 of polyampholytewere subsequently measured.An Osmomat 3000  freezing point depression osmometer was used to test osmolality.The two-point instrument calibration was completed with distilledwater, possessing an osmolality of 300 mOsmol kg54 Poly with an average Mn ≈ 80 kDa (10 g) was stirred in THF (100 mL) and heatedto 50 °C until dissolved. After dissolution, dimethylamino ethanol(∼10 g) was added in excess, with the mixture turning fromclear colorless to a pink waxy solid. Following 30 min, the waxy solidwas dissolved in water (100 mL) and left to stir overnight. The remainingTHF was removed under vacuum, and the resulting solution was purifiedin dialysis tubing  for 72 h with6 water changes. The resulting solution was freeze-dried to form awhite solid, which was analyzed using 1H and 13C NMR (in D2O) and IR spectroscopy. Polyampholyte solutionsin D2O were analyzed after 3 months to determine potentialdegradation routes.The polyampholytewas synthesized as described by Bailey et al., at a larger scale.1H NMR  δppm: 4.71 ppm , 4.38 ppm , 3.81 ppm , 3.67 ppm , 3.39 ppm , 3.18 ppm , 2.86 , 2.82 ppm (N–(CH3)2, s), 1.77 ppm , 0.78 ppm . 13C NMR  δppm: 66.6 ppm , 58.8 ppm (HCOO–CH2), 55.3 ppm(CH2–N(CH3)2),43.1 ppm (CH–COOH), 42.8 ppm (CH3)2–N. IR ν cm–1:3650–2940 cm–1 ,1724 cm–1 , 1560 cm–1 , 1342 cm–1 , 1225 cm–1 .–1 penicillin, 100 μg mL–1 streptomycin, and250 ng mL–1 amphotericin B (1% PSA). Human liverhepatocellular carcinoma cells  were culturedin Eagle’s MEM supplemented with 10% FBS, 1% NEAA, and 1% PSA.Human Caucasian colon adenocarcinoma cells were cultured in MEM media supplemented with 20% FBS and 1% PSA. Cellswere incubated at 37 °C and 5% CO2 and passaged every3–4 days, before reaching 70–80% confluency. Cells weredissociated using a balanced salt solution containing trypsin (0.25%)and EDTA (1 mM).Human Caucasian lung carcinoma cells  were cultured in Ham’s F-12K (Kaighn’s) medium(F-12K) supplemented with 10% FBS and 100 units mL–1 of polyampholytedissolved in cell media (either F12-K or MEM), 10% DMSO, and 10% FBSfor 10 min. Polyampholyte solutions were subsequently removed, andthe 24 well plates were positioned on a Corning XT CoolSink 96F andplaced in a −80 °C freezer overnight. The thermoconductiveblock ensures well-to-well temperature consistency (no “edgeeffect”), while eliminating the air gap between the bottomof the well plate and the temperature source. As a control, cellswere also cryopreserved as monolayers, in the same manner, with 10%DMSO alone. Cells were removed from the −80 °C, immediatelythawed with warm complete cell media (37 °C), and placed in theincubator for 24 h. For variable density freezing, the freezing processwas described above, however, cells seeded were serially diluted 2-foldacross each column . Phase contrastimages of nonfrozen and frozen cells were taken on an Olympus CX41microscope equipped with a UIS-2 20×/0.45/∞/0–2/FN22lens  and a Canon EOS 500D SLRdigital camera. Images were processed using ImageJ (v1.52). Followingimaging, cells were dissociated with trypsin (0.25%) and EDTA (1 mM),and the suspended cells were stained with trypan blue to count membraneintact cells. Percentage cell recovery was calculated by comparingcell counts immediately before and 24 h after thawing.A549 (300k/well), HepG2(400k/well), and Caco-2 (150k/well) cells were seeded on 24 well plates. HepG2 and Caco-2 cells were seeded ontocollagen  coated plates.Cells were incubated to allow attachment to the substrate . Cell media was removed, and cellswere incubated at RT with 40 mg mLVarious assays were completed asa demonstration of perfect cell heath following freeze-thaw, alongwith immediate assay-readiness status 24 h post-thaw. The previouslydescribed cryopreservation method was used for the banking of cellsused throughout.A549 (0–35k cells/well)and HepG2 (0–15k cells/well) cells frozen at multiple densitieswere thawed with warm complete cell media (37 °C). After 24 h,the media was replaced with resazurin solution (500 μL), whichwas prepared by dissolving 1 tablet  in 50 mL of phenol-free DMEM/F-12. Absorbance measurementswere obtained at 570 and 600 nm every 30 min/1 h for 4 h to monitorthe reduction of resazurin to resorufin by viable cells. Fluorescencemeasurements were also recorded using a BioTek Synergy HT microplatereader with a 530/25 nm excitation and 590/35 nm emission filter.Nonfrozen cells were also treated with resazurin solution at similardensities to compare against frozen cells. Cells were counted andthe exact cell density was plotted against either resazurin reduction(absorbance measurements) or normalized fluorescence intensity, wherebyfluorescence measurements were normalized to readings obtained withthe highest cell density. Resazurin solutions without any cells wereused to provide background measurements.A549 and HepG2 cells frozen as confluentmonolayers were thawed with warm complete cell media (37 °C)and, 24 h post-thaw, were stained with ethidium iodide (2 μM)and calcein (2 μM) in phenol-free DMEM/F-12 (100 μL) for40 min at RT. Cells were imaged with an Olympus CX41 microscope usingphase contrast and blue  and green (ethidium) excitationlasers. Nonfrozen cells were also stained to provide control images.ImageJ was utilized to count live  and dead (red) cells todetermine the percentage of live, membrane intact, cells.A549 and HepG2cells frozen as confluent monolayers were thawed with warm completecell media (37 °C). Following 10 min of incubation, the mediawas replaced with complete media supplemented with CellEvent Caspase-3/7Detection Reagent (5 μM). Images were taken at 1, 2, 4, and24 h time intervals on an Olympus CX41 microscope with a phase contrastchannel and a blue excitation laser. Cells were counted using ImageJ,and values were reported as percentage caspase positive cells relativeto the total number of cells. Nonfrozen cells were also stained withCellEvent Caspase-3/7 Detection Reagent to provide a baseline reading.HepG2 cells were frozen at a density of37.5–150k cells per well, as described above, for 24 h. Cellswere thawed with warm complete cell media (37 °C) and incubatedfor 48 h. To determine innate CYP450 activity, CYP3A4 and CYP2C9 weresubsequently measured using the corresponding Promega CYP450 kits.Briefly, media were replaced with a culture medium containing a luminogenicCYP substrate, either CYP3A4/Luciferin-IPA  or CYP2C9/Luciferin-H . TheCYP substrate was also added to empty wells as a background measurement.The culture medium containing the CYP substrate (25 μL) wastransferred to an opaque white 96 well plate, and luciferin detectionreagent (25 μL) was added for 20 min at RT. Luminescence wasmeasured on a BioTek Synergy HT microplate reader. The CYP activityof nonfrozen cells was also measured for comparison. Nonfrozen andFrozen HepG2 monolayers were also treated with rifampicin , 24 h post-thaw, to induce further CYP activity andmeasured as described previously.A549 and HepG2 cells were cryopreservedat a density of 20k and 50k cells/well, respectively, in 24 well plates.Cells were thawed with warm complete cell media (37 °C) and allowedto incubate for 24 h. Cells were counted daily until ∼80–90%confluency was reached, with trypan blue used to stain for membraneintact cells, and proliferation rates were determined.–1 of polyampholyte, as described above, andthawed with warm complete cell media (37 °C) after 24 h of storagein a −80 °C freezer. Cells were washed with DPBS, 24 hpost-thaw, and incubated with doxorubicin (200–0 μg mL–1) for a further 24 h. Cells were washed with DPBS,and resazurin solutions (500 μL) were added to the cells. Absorbancemeasurements were obtained at 570 and 600 nm every 30 min/1 h for4 h to monitor the reduction of resazurin to resorufin by viable cells.Control cells untreated with doxorubicin were also treated with resazurinsolutions to provide a maximum resazurin reduction value of viablecells, and resazurin solutions without any cells were used to providebackground measurements.A549 and HepG2 cellswere seeded at the optimum density determined for resazurin assayspreviously explored , on24 well plates, taking into consideration proliferation rates andduration of drug screening experiments. Cells were frozen with 40mg mL6 cells mL–1. Cells were centrifuged and resuspendedin cold 70% ethanol (250 μL), added dropwise while vortexing,and fixed for 30 min at 4 °C. Cells were centrifuged at 300g for 5 min, and the pellet was washed with DPBS. Centrifugationwas performed again, and the resulting pellet was resuspended in asolution of DPBS containing 20 μg/mL PI and 100 μg/mLRNase A for 30 min.59 Nonfrozen cells werealso stained for comparative cell cycle measurements. Flow cytometrywas performed on a BD Accuri C6 equipped with a 488 nm laser and 585/40filter, and 50,000 events were recorded. BD CSampler Plus software(v 1.0.34.1) was used for data collection and processing. Cell cycleanalysis was completed using ModFit LT v4.0.A549, HepG2, and Caco-2 cells frozenas confluent monolayers were thawed with warm complete cell media(37 °C) and incubated for 24 h. Cell dissociation was completedusing trypsin (0.25%) and EDTA (1 mM) to harvest 1 × 10A549 andHepG2 cells were frozen at a density of 200k cells per well, as describedabove, and stored for 1 month in a −80 °C freezer or for5 days in dry ice, to determine the potential for long-term storageand transportation, respectively. Cells were thawed with warm completecell media (37 °C). After 24 h, cells were dissociated with trypsin(0.25%) and EDTA (1 mM), and the suspended cells were stained withtrypan blue to count membrane intact cells. Percentage cell recoverywas calculated by comparing cell counts immediately before and 24h after thawing.alt-maleic anhydride) with dimethylamino ethanol, to producea polymer with 1:1 cationic/anionic groups, which has been previouslydemonstrated to allow the cryopreservation of A549 monolayers.54 The proposed workflow for the cryopreservationand recovery of cell monolayers is displayed in –1 of polyampholyteand 10% (v/v) DMSO) at RT for 10 min. The CPAs were subsequently removedso that only a thin coating remains, and the well plate was placedonto a thermoconductive block in a −80 °C freezer. Aspart of the process, the excess cryoprotectant is removed before freezingto reduce any potential cytotoxicity from the cryoprotectants, increasefreezing rates and facilitate the thawing process. Acker and Mcgannpreviously confirmed that rapid freezing by removing CPA is beneficialto monolayers, and could promote innocuous  IIF.60 Once required for use, the plates were simplyremoved from the freezer, and cells were rapidly thawed with warmcomplete cell culture media and placed in an incubator ready to usethe following day (24 h). Rapid thawing was completed to minimizethe growth of ice crystals in the solid state and cell denaturationdue to high electrolyte concentrations.61 A post-thaw recovery time of 24 h was selected, as shorter times(4 h) can exaggerate both cell recovery and viability, and programedcell death pathways can take 12–24 h to complete,63 leading to significant false positive results when discovering innovativecryoprotectants.6 For example, a previouspolyampholyte was shown to have high immediate post-thaw viabilitybut no viable cells after 24 h.52Our primary aim is to cryopreservecells in a format that is readyto use directly from a −80 °C freezer, with no additionalprocessing steps, in particular, to remove the need for routine cellhandling, Figure S5. The abnormally high cell recovery values for DMSO alone, comparedto previously reported literature values (≤30%),16 reflect the optimization of the freezing protocols,which, together with the selection of cryoprotectant, is crucial forthe successful cryopreservation of nucleated cells. The minimal handlingrequired to obtain high post-thaw cell recovery values shows the remarkablepotency of polyampholyte as a cryoprotectant, transforming sub-usefulcell recovery levels to near-quantitative. For comparison, prolinepreconditioning of A549 cells only achieves a post-thaw cell recoveryof ∼50% for the cryopreservation of monolayers36 and the post-thaw recovery of HepG2 monolayers cryopreservedin trehalose supplemented solutions is 42%.40 Thus, this polyampholyte can protect cell lines that are routinelydifficult to cryopreserve for the future banking of cell libraries.As a note, HepG2 and Caco-2 cells required collagen-coated platesto ensure high cell recovery values  by promoting adhesion to the substrate during freezing. To demonstratereproducibility, two independent researchers were able to freeze six24 well plates containing cell monolayers with no impact to cell recoveryvalues, Figure S7.To evaluate the cryoprotective effect of this polymer,A549 , HepG2 (human hepatocellularcarcinoma), and Caco-2  cells wereemployed, all of which are widely used as model cell lines. The seededcells were counted immediately before freezing and 24 h post-thawto determine the percentage of cells recovered, as shown in Rather than aggregatedcell data across the entire plate, the cellrecovery values for a selection of individual wells have also beenreported  to exaggerate any variancebetween wells, 60 However, for cell viabilityassays, a linear response between cell seeding density and signaloutput  is required to ensurethat cell viability can be accurately determined. Thus, the abilityto cryopreserve cells at multiple cell seeding densities is crucialfor assay development and toxicological screening assays. A resazurinreduction metabolic assay was completed on nonfrozen and frozen A549and HepG2 cells, plated at varying seeding densities, to determinethe quantity of cells required to freeze to ensure a linear outputwith resazurin reduction to resorufin and, thus, allow its use fordrug screening, S10, and S11. Both cell lines wereable to metabolize resazurin to resorufin 24 h post-thaw and offereda linear response between cell density and absorbance/fluorescence.Although the metabolic activity of A549 cells was undisturbed by thefreezing process, HepG2 metabolic activity was halved 24 h post-thaw.The reduction in metabolic activity is expected, halving (or more)of metabolic activity has also been observed for osteoblasts (MG-63),64 ovarian tissue,65 and mesenchymal stem cells post-thaw.66 Despite this, both A549 and HepG2 cells remained functional foruse in cell viability assays required for drug screening, see below.Varying cell seeding densities (12.5k–100kcells per well)also had no influence on the cryopreservation outcome, with over 75%of cells recovered 24 h post-thaw, Figure S8).Again, cells appeared healthy regardless of cell density and wereready for use within the 24 h recovery time provided. Cryopreservationwith DMSO alone resulted in spherical cell morphologies associatedwith unhealthy cells. The injury phenotype has previously been characterizedas intracellular freezing damage, indicated by the dark cytosol anddisruption of intercellular networks.21 Standard rapid freezing methods increase the likelihood of IIF byreducing cell dehydration.1 Cryomicroscopyimages of A549, HepG2, and Caco-2 cells, Figure S12, confirmed the presence of IIF during freezing (darkeningof the cytosol); however, cells preincubated with both polyampholyteand DMSO have a lower surface area compared to DMSO alone , suggesting that polyampholytecan aid in cellular dehydration as part of their mechanisms of action.Thus, the polyampholyte is not acting to completely remove IIF butto minimize the damage caused by it, so that the intracellular iceformed can be considered innocuous.60Optical microscopy images taken of nonfrozen cellsand cells frozenwith both polyampholyte and DMSO, shown in 67 Confluent monolayers wereobserved in all other positions for both A549 and HepG2 cells.Optical microscopy images were also taken of post-thawconfluentA549 and HepG2 cells from different positions within a single well,to illustrate the low intra-well variance obtained using polyampholyteand DMSO, 69 A549 and HepG2 cells, both nonfrozen and post-thaw, were stainedwith calcein  and ethidium iodide to visualize the presence of membranedamage, To evaluate the quality of the cells post-thaw,a panel of furtherassays was undertaken. Membrane integrity is a general measure ofpost-thaw recovery and is a vital assessment for the health of livercell lines, as enzyme leakage would be detrimental to enzyme activityassays used in hepatotoxicity screening for the identification ofbioactive drugs and their potential mechanisms.72 Hypothermic solutions and general caspase and ROCKinhibitors (apoptotic inhibitors) have been used to reduce post-thawapoptotic cells.74 Due to the importance of apoptosis in cryopreservation outcomes,the induction of executioner caspases 3 and 7 (cysteinyl aspartate-specificproteases) in post-thaw confluent A549 and HepG2 cell monolayers wasmonitored by fluorescence microscopy to visualize the appearance ofapoptotic cells over time, S14. Minimal caspase-3/7positive cells (green) were observed in microscopy images at all timepointswithin the 24 h imaging period. The percentage of caspase-3/7 positivecells relative to the total number of cells present was quantified, 70 Our system offerscryopreserved monolayer cells with substantially lower apoptosis ratesfor the cell lines selected, thus the introduction of apoptosis inhibitorswould provide minimal benefits. The post-thaw recovery time selected(24 h) is underpinned by these results, to enable apoptotic eventsto occur and minimize the potential for false-positive results thatoccur when immediate post-thaw recovery/viability is reported.6Programed cell death  pathwayswere also investigatedas cryopreservation can activate intrinsic, extrinsic, and calpainapoptosis pathways.75Membrane permeability and apoptosis measurementsoffer an assessmentof immediate cell health post-thaw; however, growth curve measurementsare required to determine if cells are healthy long-term. Nonfrozenand post-thaw cells were counted daily, until 80–100% confluencywas reached, to determine if the proliferation times of monolayerfrozen cells were affected by the freeze/thaw process and have beenreported in 76 Previous cell recovery and viability measurementsconfirm that, after freezing, cells maintain a “healthy”state, but gaining information into cell cycle population distributionsoffers additional insight into proliferation capacity,77 apoptotic cells,78 and cells undergoing repair processes.79 Cell cycle population distributions were measured by univariateanalysis of cellular DNA following propidium iodide staining, flowcytometry, and deconvolution of the cellular DNA content frequencyhistograms.77 Histograms can be found inthe Supporting Information, Figure S15,and the cell cycle populations have been reported in 80 and retain proliferative capacity.77 A549and Caco-2 cells retained similar cell cycle population distributionsbefore and after freezing (24 h post-thaw), whereas clear differenceswere observed with HepG2 cells which could reflect the changes inproliferation rates between nonfrozen and frozen HepG2 cells previouslyobserved. Regardless, proliferative capacity was retained for allcell types, with minimal indicators of apoptosis found, confirmingthat cells are functional for use following freeze/thaw.Cryopreservation may significantly alter cell cyclepopulationsup to 48 h post-thaw; however, there are limited reports on this.–1) for 24 h. Resazurin solutions were subsequently added, and thereduction of resazurin to resorufin was measured at 570 and 600 nmto calculate percentage cell viability and generate dose–responsecurves, 10 of doxorubicinconcentrations and % cell viability, with plateaus at the bottom andtop of the response, allowing the accurate determination of doxorubicin’sIC50 value (the concentration of doxorubicin required toreduce cell viability to 50%). HepG2 cells are the most widely usedline for the toxicological screening of new drugs and are currentlydeployed in automated/high throughput systems.57 The ability to generate dose–response curves and calculateIC50 values using monolayer cryopreserved cells confirmstheir suitability for drug screening applications, finding biologicallyactive compounds, and providing a way to increase automation throughminimal cell-handling procedures.A primary aim of this research program was to providea scalablemethod of freezing cells in the monolayer format for use in screeningapplications, eliminating the need for extensive cell culturing andhanding. Obtaining dose–response information, which involvesthe core principles of pharmacokinetics and pharmacodynamics, is essentialfor drug screening, to determine the required drug dosage and frequency,as well as its therapeutic index. Thus, a proof-of-concept drug screeningexperiment was carried out with doxorubicin (a chemotherapeutic) usingthe previously optimized resazurin reduction cell viability assay.The drug screening workflow is illustrated in Figure S16. CYP enzymes are vital in drug metabolism,with CYP3A4 being responsible for 50% of all drug metabolism.81 Thus, many drug screening applications relyon monitoring the inhibition of CYP enzymes, which could slow drugmetabolism time resulting in undesired drug–drug interactions.83 Although insignificant, CYP3A4 activity of cells post-thaw was slightlysuppressed post-thaw compared to nonfrozen cells, whereas CYP2C9 levelsremained either unaffected or marginally higher. The innate expressionof CYP enzymes was low for HepG2 cells and remained low even withthe use of an inducer (rifampicin), Figure S17, which is consistent with previous studies.84 The selection of future cells with greater response to CYP inducers,with improved predictability for human hepatotoxin detection, willbe explored to expand our library of cryopreserved monolayer cells.However, the data presented here clearly demonstrates that HepG2 monolayerscan be cryopreserved in an assay-ready format, potentially enablingthe first direct from storage to automated screening platform.HepG2 cytochrome p450 (CYP) activity was also assessedbetweennonfrozen and monolayer preserved cells 24 h post-thaw, 85 no reports were foundon the medium-to-long term storage of cells cryopreserved as monolayersin a −80 °C freezer. Confluent A549 and HepG2 monolayerswere cryopreserved as previously described and stored for 1 monthin a −80 °C freezer. Optical microscopy images were takenof the confluent monolayers directly before freezing and 24 h post-thaw, 2 methods. Cells were also stored in dry ice forover 5 days, with no significant decrease in cell recovery , which provides a route for the transportationof cryopreserved plated cell monolayers.To ensure that a sustainable supply of monolayer cryopreservedcell lines is possible, the long-term storage of cells is required.Although cells cryopreserved in suspension can be stored long-termat temperatures less than or equal to −80 °C, retaining>90% viability post-thaw,Here we introduce truly “assay-ready”cryopreservedadherent cells, ready to be used directly from a freezer within industrystandard microplates, removing the need for standard handling, manipulations,management, and other time- and resource-expensive activities associatedwith cell culture and biomedical testing. This overcomes a historicalchallenge and perception that cells cannot be stored and distributedin monolayer format. The key component to enable such reproduciblemonolayer freezing was the use of a synthetic polyampholyte which,when used alongside the standard CPA DMSO, leads to dramatic increasesin post-thaw cell yield and viability of up to 93%, compared to ∼50%using DMSO alone. Initial data suggests that the polyampholyte supportsthe cellular dehydration process during freezing, reducing the damagingeffects of IIF. In addition to the macromolecular cryoprotectant,the freezing and thawing conditions were optimized to minimize well-to-wellvariance, ensuring homogenous cell recovery within and between microplates.Successful monolayer cryopreservation was demonstrated for A549, HepG2,and Caco-2 cells, representing a diverse range of commonly used celllines within biomedical research and toxicology testing. To confirmthat the cells were functional and healthy, a large panel of assayswas undertaken. Post-thaw membrane damage and necrosis indicatorswere kept minimal and only a small increase in cells displaying apoptosismarkers was observed. Cell cycle analysis and post-thaw growth curvesconfirmed that cells retained proliferative capacity. CryopreservedA549 and HepG2 cell monolayers were subjected to a model assay developmentand toxicological challenge, to demonstrate that the cells are truly“assay ready” 24 h post-thaw and can be used for thetesting of pharmaceutically active compounds. Overall, we have demonstratedthat cells can be cryopreserved as “assay-ready” monolayers,in a scalable and versatile manner, which will enable the developmentof new screening technologies, enhance automation, and reduce theexperimental burden on researchers.Supporting Information and any additional datacan be found at http://wrap.warwick.ac.uk.The research data supportingthis publication is found in the


Lonicera japonica Thunb. under cadmium (Cd) treatments were investigated by assessing the plant growth and photosynthetic characteristics. Under Cd treatments without electric fields, the parameters of plant growth and photosynthetic characteristics increased significantly when exposed to 5 mg L−1 Cd, and decreased slightly when exposed to 25 mg L−1 Cd, showing an inverted U-shaped trend, which confirmed that low concentration Cd has a hormesis effect on L. japonica. Under electric fields, different voltages significantly promoted the inverted U-shaped trend of the hormesis effect on the plant, especially by 2 V cm−1 voltage. Under 2 V cm−1 voltage, the dry weight of the root and leaf biomass exposed to 5 mg L−1 Cd increased significantly by 38.38% and 42.14%, and the photosynthetic pigment contents and photosynthetic parameters were also increased significantly relative to the control, indicating that a suitable electric field provides better improvements for the hormesis responses of the plant under Cd treatments. The synergistic benefits of the 5 mg L−1 Cd and 2 V cm−1 electric field in terms of the enhanced hormesis responses of growth and photosynthetic characteristics could contribute to the promoted application of electro-phytotechnology.“Hormesis” is considered a dose–response phenomenon mainly observed at hyperaccumulator plants under heavy metals stress. In this study, the effects of electric fields on hormesis responses in  With rapid population growth and intensive human activities, large quantities of chemical contaminants, especially heavy metals released into the environment, have recently attracted global attention ,2. HeavyIt is known that numerous studies focus on the toxicity of high-dose environmental contaminants ,32,33,34Lonicera japonica Thunb. (Japanese honeysuckle) is a popular ornamental plant and has become established in temperate and tropical regions worldwide in the past 150 years [L. japonica has a strong tolerance and good accumulation capability for Cd in plant tissues , and it is recognized as a new woody ornamental Cd-hyperaccumulator [L. japonica were stimulated by hormesis under low concentrations of Cd stress [L. japonica as a model plant to investigate the effect of different electric fields on the hormesis responses of the growth, photosynthetic pigment composition and photosynthesis of the plant. The specific objectives are to explore whether an electric field can improve the hormesis responses of the plant under different concentrations of Cd stress. It will contribute to a better understanding of the hormesis response strategy of hyperaccumulators and promote the application of electro-phytotechnology.50 years . It is c50 years . Our preumulator ,55. Mored stress ,55,56,57L. japonica were collected from a non-contaminated experimental field and cultivated in sterilized sand by culture medium. The culture medium was Hoagland solution containing the following composition (mmol L−1): Ca(NO3)2·4H2O 5.00, MgSO4·7H2O 2.00, KNO3 5.00, KH2PO4 1.00, H3BO3 0.05, ZnSO4·7H2O 0.80 × 10−3, MnCl2·4H2O 9.00 × 10−3, CuSO4·5H2O 0.30 × 10−3, (NH4)6Mo7O24·4H2O 0.02 × 10−3, Fe-EDTA 0.10 [The experiment was carried out in a greenhouse of Shenyang Agricultural University . Seedlings of DTA 0.10 ,58. The 2·2.5H2O  was added into the culture medium and the Cd treatments were 0, 5 and 25 mg L−1. Subsequently, an electric field (EF), which contains a pair of graphite electrodes (10.0 cm in length) associated with a DC power supply , was daily 6 h and referred to 0, 1, 2 and 3 V cm−1 according to Liu et al. (2022) [After the plants were cultivated for 8 weeks, CdCl. (2022) . The EF-2-EDTA for 15 min and then washed with deionized water to remove Cd adhering to the root surface [The harvested plants were washed with tap water, and the roots of the plants were immersed in 20 mM Na surface . The pla3/HClO4 . The plant Cd concentrations in L. japonica were determined with a flame atomic absorption spectrophotometer  after microwave digestion.Dried plant materials were ground to fine powder by a grinder. The powders were digested with a concentrated acid mixture of HNOv/v) ethanol at 4 °C in darkness until the tissues became white. The extracting solution absorbance at 649, 665 and 470 nm was measured. The contents of chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll  and carotenoid (Car) were calculated by a modified method according to Lichtenthaler and Wellburn (1983) and Lichtenthaler (1987) [The photosynthetic pigments were measured by the uniform and similar leaf samples. The leaf samples (0.2 g) were cut into small pieces, and then soaked in 25 mL 95% (r (1987) ,61.2 concentration (Ci). During different treatments, the parameters inside the leaf chamber  were maintained constant at 1000 μmol m−2 s−1 PPFD, 25 ± 0.3 °C and 380 ± 5 μmol CO2 mol−1. The upper second fully expanded leaves were used for the determination according to the method of Pandey et al. (2003) [The photosynthetic parameters were determined by a portable photosynthesis system  under different treatments. The photosynthetic parameters consisted of net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO. (2003) .p < 0.05 level. The least significant difference (LSD) test was used to determine the multiple comparison between treatments.All measurements in the study were replicated three times. The data analyses were performed as the means ± SD. The statistical analysis of variance was carried out with SPSS 22.0. The significant difference was presented at the L. japonica under different treatments were shown in 1–T3, L. japonica had an increased trend, which ranged from 11.11, 137.43 to 419.05 mg kg−1. Under the T4–T6 treatments , a slight increase of plant Cd concentrations in L. japonica under different concentrations by Cd stress was observed by 1 V cm−1 voltages, which ranged from 12.31, 198.92 to 654.65 mg kg−1. Under the T7–T9 treatments , the plant Cd concentrations in L. japonica under different concentrations by Cd stress had a significant increased trend compared with the T1–T3 treatments (under Cd treatments without electric field), which ranged from 13.23, 358.30 to 1440.00 mg kg−1. Under the T10–T13 treatments , the plant Cd concentrations in L. japonica under different concentrations by Cd stress had a more significant increase compared with the T1–T3 treatments (under Cd treatments without electric field), which ranged from 13.99, 414.58 to 1630.84 mg kg−1. It was demonstrated that the concentrations of several heavy metals  in plants were promoted because of the application of electric fields [L. japonica exposed to different concentrations Cd compared with T1–T3 treatments (under Cd treatments without electric fields). The significant increase of plant Cd concentrations under the electric field were observed when the plants were exposed to different concentrations of Cd, especially exposed to high concentrations (25 mg L−1) Cd. Under different concentrations of Cd treatments, the plant Cd concentrations in L. japonica were increased significantly by 2 V cm−1 voltage and 3 V cm−1 voltage, which reached 1440.00  and 1630.84 mg kg−1 , which were 3.44 and 3.89 times of the T3 treatment (V0-Cd25), respectively. The positive effect of the electric field may be correlated with the variety of metal ions polarity and cell membrane properties in plants [The Cd accumulations in c fields ,63. In tn plants ,65. The n plants .L. japonica under different treatments is displayed in 1–T3 treatments (under Cd treatments without electric fields), the dry weight of the root biomass exposed to 5 mg L−1 Cd increased significantly by 10.12% relative to the T1 treatment (V0-Cd0), and had a slight decrease when exposed to 25 mg L−1 Cd, indicating an inverted U-shaped curve, which confirmed that low concentration Cd has the hormesis effect on the root growth of L. japonica. The results correspond to our previous studies, which showed that the growth characteristics, photosynthetic pigments contents, relative water contents and other physiological parameters of Cd treatments all significantly indicated an inverted U-shaped dose–response curve, confirming that the hormesis effect of low concentration Cd occurred in L. japonica [4–T12 treatments (under electric field), the dry weight of root biomass had an increased trend compared with the T1–T3 treatments (under Cd treatments without electric fields), which showed that different voltages significantly promoted the inverted U-shaped trend of dry weight of root biomass in L. japonica, especially by 1 V cm−1 voltage and 2 V cm−1 voltage. Under 1 V cm−1 voltage and 2 V cm−1 voltage, the dry weight of root biomass exposed to 5 mg L−1 Cd increased significantly by 20.54% and 38.38% relative to the T2 treatment (V0-Cd5), which investigated that the medium voltage (2 V cm−1) has more improvements to the hormesis effect of low concentration Cd on the plant growth of L. japonica. He et al. (2017) have also reported that the dry weight of root biomass in maize under a drought environment was enhanced by a pulsed electric field, which could be derived from the improvement of the respiration metabolism and substance transformation through the pulsed electric field [−1 Cd increased by 15.12%, 34.30% and 10.47% relative to the T3 treatment (V0-Cd25), which confirmed our previous study, indicating the electric field-enhanced tolerance of L. japonica responded to high concentrations of Cd.It is well known that the biomass of plants is an important highly sensitive indicator responding to heavy metal or other abiotic stresses ,33. The japonica ,55,56,57ic field . Under d1–T3 treatments (under Cd treatments without electric fields), the dry weight of leaf biomass exposed to 5 mg L−1 Cd increased significantly by 17.78% relative to the T1 treatment (V0-Cd0), and decreased slightly when exposed to 25 mg L−1 Cd, showing a similar inverted U-shaped trend of the hormesis effect with the dry weight of root biomass. Wiewiórka (2013) observed that a high-intensity electric field had limited impacts on the growth of tomatoes in a hydroponic culture [4–T12 treatments (under electric field), the dry weight of leaf biomass had an increased trend compared with the T1–T3 treatments (under Cd treatments without electric field), which indicated that different voltages significantly promoted the inverted U-shaped trend of the dry weight of leaf biomass in L. japonica, especially by 1 V cm−1 voltage and 2 V cm−1 voltage. Under 1 V cm−1 voltage and 2 V cm−1 voltage, the dry weight of leaf biomass exposed to 5 mg L−1 Cd increased significantly by 15.09% and 42.14% relative to the T2 treatment (V0-Cd5). Compared with the results of the dry weight of root biomass above, it indicated that the medium voltage (2 V cm−1) more significantly enhanced the hormesis effect of low concentration Cd on the leaf biomass than the root biomass in L. japonica, which could be the reason that plant organs in L. japonica have different sensitivity and tolerance mechanisms when responding to environmental stress. In summary, a medium strength electric field (2 V cm−1) could improve the hormesis responses of plant growth in L. japonica under different treatments. This is in accordance with those earlier studies that reported that the electric field stimulated the plant growth and productivity though regulating the different levels of plant growth hormones [In contrast, under the T culture . Howeverhormones ,64,66.L. japonica are presented in 1–T3 treatments (under Cd treatments without electric fields), the contents of Chla, Chlb, Total Chl and Car exposed to 5 mg L−1 Cd increased significantly by 5.99%, 7.56%, 6.55% and 7.41% relative to T1 treatment (V0-Cd0), and had a decrease exposed to 25 mg L−1 Cd, which showed an inverted U-shaped curve, indicating low concentration Cd has the hormesis effect on the photosynthetic pigment composition of L. japonica. The results confirmed that low concentration Cd could have a stimulatory effect on plant growth, the reasons of which may be the promoted dry matter accumulation and seedling biomass through the increased photosynthetic pigment contents [L. japonica was exposed to 25 mg L−1 Cd without electric fields (T1–T3 treatments), the contents of Chla, Chlb, Total Chl and Car in the plant showed the decreased trend, which could have resulted from the substitution of chlorophyll Mg2+ in photosynthetic pigment composition by Cd2+ [2+ in the photosynthetic pigment composition are replaced spontaneously by Cd2+ and cause the degradation of photosynthetic pigments and even the inhibition of photosynthesis. Under the T4–T12 treatments (under electric fields), the contents of Chla, Chlb, Total Chl and Car had an increased trend compared with the T1–T3 treatments (under Cd treatments without electric fields), which showed the electric field could improve the Cd-induced degradation of photosynthetic pigments and stimulate the protective mechanism in L. japonica. It was observed that the different voltages significantly promoted the inverted U-shaped trend of the contents of Chla, Chlb, Total Chl and Cars, especially by 1 V cm−1 voltage and 2 V cm−1 voltage. Moreover, different photosynthetic pigments have different sensibilities to environmental stress [−1 voltage, the contents of Chla, Chlb, Total Chl and Cars exposed to 5 mg L−1 Cd increased significantly by 13.48%, 6.25%, 10.89% and 13.79% relative to the T2 treatment (V0-Cd5); by comparison, under 2 V cm−1 voltage, the contents of Chla, Chlb, Total Chl and Cars exposed to 5 mg L−1 Cd increased significantly by 24.78%, 23.44%, 24.30% and 22.41% relative to the T2 treatment (V0-Cd5), which indicated that a medium voltage (2 V cm−1) better promotes the hormesis effect of low concentration Cd on the photosynthetic pigment composition of L. japonica. The phenomenon is in agreement with the hormesis responses of plant growth in L. japonica under different treatments, which mainly result from electric field-induced uptake increase of Fe, Mg or other trace elements [−1 under the electric field, the contents of Chla, Chlb, Total Chl and Cars exposed to 5 mg L−1 Cd had no significant increases relative to the T2 treatment (V0-Cd5), the contents of which were 2.45 mg g −1FW, 1.29 mg g −1FW, 3.74 mg g −1FW and 1.86 mg g −1FW, respectively. The results indicated that a suitable electric field could have better improvement for the hormesis responses of photosynthetic pigment composition in L. japonica to Cd stress.The measured results of photosynthetic pigment composition including chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll  and carotenoid (Car) in leaves of contents ,69,70. W by Cd2+ . With thl stress . Under 1elements ,73. When2 concentration (Ci) in L. japonica under different treatments are evaluated in 1–T3 treatments (under Cd treatments without electric fields), when the plants were exposed to low concentration (5 mg L−1) Cd, the contents of Pn, Gs, Tr and Ci in L. japonica had a significant increase compared with the T1 treatment (V0-Cd0), which were 15.85 ± 0.91 μmol m−2 s−1, 0.38 ± 0.01 mol m−2 s−1, 2.52 ± 0.05 mmol m−2 s−1 and 383.25 ± 10.78 μL L−1, respectively. It is shown that the significant hormesis effect on Pn promoted the gas exchange and transpiration in L. japonica in the form of the increased Gs, Tr and Ci, the reasons of which may result from the stimulating impact of low concentration Cd on the Rubisco contents [4–T12 treatments (under electric fields), when the plants were exposed to low concentration (5 mg L−1) Cd, the contents of Pn, Gs, Tr and Ci in L. japonica were all increased significantly by 1 V cm−1 voltage , 2 V cm−1 voltage  and 3 V cm−1 voltage , respectively. Under the T4–T12 treatments (under electric fields), different voltages significantly promoted the inverted U-shaped trend of the contents of Pn, Gs, Tr and Ci, especially by 2 V cm−1 voltage. Under different EF-Cd treatments, the maximum value of Pn, Gs and Tr reached 22.95 ± 0.98 μmol m−2 s−1, 1.19 ± 0.05 mol m−2 s−1 and 3.33 ± 0.08 mmol m−2 s−1, and under low concentration (5 mg L−1) Cd treatment, the contents of Pn, Gs, Tr and Ci were all increased significantly by 2 V cm−1 voltage . This is in agreement with the dry weight of root and leaf biomass, which showed that the combination of low concentration (5 mg L−1) Cd and medium voltage (2 V cm−1) was useful to improve the photosynthesis capacity and plant growth. The photosynthesis responses, in terms of Pn, Gs, Tr and Ci in L. japonica under different treatments, also have a good correlation with the change trend of the photosynthetic pigment composition. Several researchers observed that Cd stress had a negative impact on plant photosynthesis, which is probably traceable in the decreased chlorophyll biosynthesis and thylakoids or the inhibited plant growth [−1) Cd treatment, the contents of Pn, Gs, Tr and Ci in L. japonica were promoted significantly by electric fields relative to the T3 treatment (V0-Cd25), which is probably associated with the adaptive mechanisms of hyperaccumulators responding to external stress [Photosynthesis, as the basis of all plant growth and crop yield, is undoubtedly the most important biological process and is very susceptible to environments contaminated by Cd . The phocontents . Under tt growth ,75,76,77l stress ,80.L. japonica is a good model plant to investigate the effect of different electric fields on the hormesis responses of the growth, photosynthetic pigment composition and photosynthesis of plants. In the study, under the T1–T3 treatments (under Cd treatments without electric fields), the parameters of plant growth (dry weight of root and leaf biomass), photosynthetic pigment composition  and photosynthesis  increased significantly when exposed to 5 mg L−1 Cd (p < 0.05), and had a slight decrease when exposed to 25 mg L−1 Cd, showing an inverted U-shaped trend, which confirmed that low concentration Cd has a hormesis effect on L. japonica. Under the T4–T12 treatments (under electric field), different voltages significantly promoted the inverted U-shaped trend of the hormesis effect, especially by 2 V cm−1 voltage, which indicated that a suitable electric field better improves the hormesis responses of growth photosynthetic pigment composition and photosynthesis in L. japonica to Cd stress. The present results will be useful to explore the underlying mechanisms of the hormesis effect of Cd stress on hyperaccumulators for electric field-assisted phytoremediation.Based on the previous study, it is shown that


Background and Objectives: The erector spinae plane block (ESPB) is a multimodal opioid-sparing component, providing chest-wall analgesia of variable extent, duration, and intensity. The objective was to examine the ESPB effect on perioperative opioid usage and postoperative rehabilitation when used within a Nociception Level (NOL) index-directed anesthetic protocol. Materials and Methods: This prospective, randomized, controlled, open-label study was performed in adult patients undergoing on-pump cardiac surgery in a single tertiary hospital. Eighty-three adult patients who met eligibility criteria were randomly allocated to group 1  and group 2  and received general anesthesia with NOL index-directed fentanyl dosing. Preoperatively, group 2 also received bilateral single-shot ultrasound-guided ESPB (1.5 mg/kg/side 0.5% ropivacaine mixed with dexamethasone 8 mg/20 mL). Postoperatively, both groups received intravenous paracetamol (1 g every 6 h). Morphine (0.03 mg/kg) was administered for numeric rating scale (NRS) scores ≥4. Results: The median  intraoperative fentanyl and 48 h morphine dose in group 2-to-group 1 were 1.2 (1.1–1.5) vs. 4.5 (3.8–5.5) µg·kg−1·h−1 (p < 0.001) and 22.1 (0–40.4) vs. 60.6 (40–95.7) µg/kg (p < 0.001). The median (IQR) time to extubation in group 2-to-group 1 was 90 (60–105) vs. 360 (285–510) min (p < 0.001). Two hours after ICU admission, 87.5% of ESPB patients were extubated compared to 0% of controls (p < 0.001), and 87.5% were weaned off norepinephrine compared to 46.5% of controls (p < 0.001). The median NRS scores at 0, 6, 12, 24, and 48 h after extubation were significantly decreased in group 2. There was no difference in opioid-related adverse events and length of stay. Conclusions: NOL index-directed ESPB reduced intraoperative fentanyl by 73.3% and 48 h morphine by 63.5%. It also hastened the extubation and liberation from vasopressor support and improved postoperative analgesia. Cardiac surgery with sternotomy is associated with significant perioperative nociception of musculoskeletal, visceral, and neurogenic origins . OpioidsNeuraxial techniques and paravertebral blocks are pivotal ERAS instruments in thoracic surgery, but concerns about bleeding complications have traditionally hindered their adoption in cardiac surgery. Recently, ultrasound-guided chest wall fascial plane blocks have emerged as an efficient analgesic alternative . Technic5 transverse process and the erector spinae muscle, resulting in multimetameric somatic and visceral analgesia [Fascial blocks must match surgical procedures to maximize clinical gains and avert complications . A bilatnalgesia . Severalnalgesia ,8,9,10. nalgesia . TM  monitor can track several nociception-related parameters, including heart rate, heart rate variability, pulse wave amplitude, level and fluctuations of galvanic skin response, skin temperature, and movement, to derive the Nociception Level (NOL) index, a real-time measure of the individual’s response to nociception [Objective real-time intraoperative nociception monitoring may help prevent overdosing or underdosing opioids and personalize nociception control. The PMD-200iception . The NOLiception , and heliception . In addiiception . By exteThe authors of this randomized controlled trial compared NOL index-directed general anesthesia (NDGA) to NDGA and ESPB combined to see if adult patients undergoing cardiac surgery with sternotomy could benefit from ESPB in terms of reducing perioperative opioid consumption. In addition, metrics of analgesic quality, fast-tracking, vasopressor usage, and adverse events were studied.ClinicalTrials.gov (accessed on 26 July 2019) (Identifier: NCT04338984).This prospective, single-center, open-label, randomized controlled trial was performed from December 2019 to May 2021 in adult patients undergoing on-pump cardiac surgery. It was approved by the Institutional Review Board for Biomedical Research of Prof. Dr. C.C. Iliescu Emergency Institute for Cardiovascular Diseases, Bucharest, Romania (2019.07.26/18750) and registered with After obtaining written consent, patients that met the following criteria were included in the study: an age range of 18–75 years, elective surgery, and sinus rhythm. The exclusion criteria were as follows: allergy to medications used in the study; body mass index larger than 35; abnormal coagulation profile; emergency or redo surgery; American Society of Anesthesiologists class 4 or higher; pharmacological, or mechanical preoperative cardiocirculatory support; and severe left ventricular dysfunction .A physician unassociated with the study enrolled the patients. Following enrolment, patients were allocated one-to-one using a random permuted block generator  into two groups: group 1 (Control) received NDGA, and group 2 (ESPB) received bilateral single-shot US-guided ESPB followed by NDGA .TM monitor.Patients were explained the 11-point pain intensity numeric rating scale (NRS), where zero indicates no pain and 10 indicates the worst possible pain. Upon arrival to the operating room, oxygen was administered by face mask and standard monitoring was applied, including electrocardiography with automated ST segment analysis, pulse oximetry, and non-invasive blood pressure cuff. After skin asepsis with chlorhexidine 2%, a 16-gauge peripheral intravenous cannula was inserted in the dorsum of the right hand. Skin asepsis was repeated and, under local anesthesia with lidocaine 1%, a 20-gauge left radial arterial cannula was inserted for invasive arterial pressure monitoring. A bispectral index (BIS) sensor was applied to the patients’ foreheads to monitor the depth of anesthesia. The NOL index probe was also attached to all patients on the middle right finger. A period of two minutes devoid of noxious stimuli was ensured to allow calibration of the PMD-2005–T6 transverse process, identified as a flat, squared-off acoustic shadow flanked by an ill-defined pleural line. A 22-gauge 80 mm needle  was inserted in-plane, caudocranially, at a 30° angle, across a three-layered muscle bundle  until it reached the T5–T6 transverse process. Correct positioning was certified by hydro-location with saline 2–3 mL, and, after negative aspiration of blood, 1.5 mg/kg 0.5% ropivacaine mixed with dexamethasone 8 mg/20 mL was administered on each side . The probe was positioned parasagittal, 2–3 cm lateral from midline, over the Tach side . ClassicAnesthesia induction was performed with propofol 1.5 mg/kg, fentanyl 5 µg/kg, and rocuronium 0.6 mg/kg, followed by protective volume-controlled mechanical ventilation. Afterward, a triple-lumen central venous line, a nasal temperature probe, a urinary catheter, and end-tidal carbon dioxide monitoring were established. The central line was introduced under ultrasound guidance in the right internal jugular vein, using an in-plane approach.Cefuroxime 1.5 g or vancomycin 15 mg/kg in patients with known beta lactam allergy secured surgical antimicrobial prophylaxis. Stress ulcer prophylaxis was ensured with intravenous omeprazole 40 mg. Both groups received intravenous paracetamol 1 g after induction.Before and after CPB, anesthesia was maintained with sevoflurane (1.5–2.5%) in a mixture of oxygen and air (50:50). During CPB, maintenance of anesthesia was achieved with propofol infusion. BIS values of between 40 and 60 were targeted until the end of the surgery. Intermittent doses of rocuronium were given to sustain the neuromuscular blockade.−1·h−1, and the following rules were applied to achieve NAN balance in both groups: (1) if the NOL index is >25 for more than 60 s, increase the infusion by 0.5 µg·kg−1·h−1 and bolus 1 µg/kg; (2) if the NOL index is <10 for more than 60 s, decrease the infusion by 0.5 µg·kg−1·h−1; (3) after a dose change, allow an observation window of three minutes; and (4) stop the fentanyl infusion when it reaches 0.5 µg·kg−1·h−1 with the NOL index ≤ 25 for more than ten minutes.For both groups, optimum nociception-antinociception (NAN) balance was defined as a NOL index between 10 and 25 on a scale from 0 to 100, where 0 indicates a lack of nociception and 100 indicates extreme nociception. Because the NOL index is, in part, a time-variable photoplethysmographic output, it is inoperative during CPB and may be altered after coming off CPB by cardiac pacing. Hence, NAN balance monitoring was available only until CPB initiation. After induction, fentanyl was started at 2 µg·kgFrom CPB initiation until the end of the surgery, fentanyl administration was conducted in both groups to ensure hemodynamic stability within 15% of the mean arterial blood pressure (MAP) recorded during optimum NAN balance. MAP is highly non-specific; hence, integrating MAP determinants  was endorsed consistently to help differentiate noxious from non-noxious stimuli in the absence of objective NAN monitoring.Transesophageal echocardiography was standard to monitor and guide the administration of cardiovascular drugs  and fluids.Standard monitoring was resumed after intensive care unit (ICU) admission, and transthoracic echocardiography was applied to guide hemodynamic management.−1·min−1; (3) mean arterial pressure ≥60 mmHg; (4) no surgical bleeding and normal coagulation profile; (5) adequate gas exchange defined as an arterial partial pressure of oxygen to fraction of inspired oxygen ratio ≥250 mmHg with a positive end-expiratory pressure < 7 mbar; (6) sustained respiratory effort  with pressure support ≤ 7 mbar; and (7) wakefulness and adequate cough reflex.Extubation was performed when a minimum set of weaning criteria were met: (1) normothermia (temperature > 36 °C); (2) adequate perfusion with dobutamine ≤ 5 µg·kgThe nursing staff was instructed to administer all patients’ intravenous paracetamol 1 g every 6 h starting with ICU admission. Pain assessment was initiated immediately after extubation. NRS scores with in-bed mobilization were recorded at 2 h intervals for the first 12 h and then at 6 h intervals until 48 h of ICU stay or discharge, whichever came first. Intravenous morphine 0.03 mg/kg was prescribed for NRS scores ≥4 by attending anesthetists.The primary endpoint was total intraoperative fentanyl consumption. The secondary endpoints were: (1) pre-CPB intraoperative fentanyl consumption ; (2) cumulative morphine consumption 48 h after ICU admission; (3) the number of morphine-free patients 48 h after ICU admission; (4) the time to the first dose of morphine; (5) quality of analgesia assessed with NRS scores at 0, 6, 12, 24, and 48 h after extubation and 1 h after drain removal; (6) the time to extubation after ICU admission; (7) the number of extubated patients 2 h after ICU admission; (8) norepinephrine (NE) consumption during the intervention and 12 h after ICU admission; (9) the time to wean off NE after ICU admission; (10) the number of NE-free patients 2 h after ICU admission; and (11) ICU and hospital LOS. ESPB-related complications and the number of ESPB patients requiring fentanyl rescue before CPB initiation were recorded. Additionally, the incidence of opioid-related events  and postoperative atrial fibrillation (POAF) was dichotomously  assessed in both groups until ICU discharge or 72 h after ICU admission, whichever came first. Respiratory depression was defined as a respiratory rate of fewer than 10 breaths per minute following extubation, regardless of underlying gas exchange parameters.−1·h−1. The null hypothesis was that ESPB and NDGA combined are not superior to NDGA in total intraoperative fentanyl consumption by more than a superiority margin (δ) of 30%. For a study power of 90%, the sample size was estimated at 23 patients per group . To account for potential patient loss to follow-up of 20%, the minimum number of patients required per group was 28.There is a dearth of literature examining the role of nociception-directed anesthesia in cardiac surgery. One recent study demonstrated decreased intraoperative sufentanil consumption with the pupillary pain index (PPI) . ContrarAll statistical analyses were performed using NCSS 2022 Statistical Software . Visual inspection and the Shapiro–Wilk test were used to check normality for quantitative variables. Quantitative data were expressed as mean (±SD) if normally distributed or median  if non-normally distributed. Categorical variables were presented as absolute numbers and percentages.−1·h−1) was applied. Nonparametric quantitative secondary outcomes were evaluated with the Mann–Whitney U test. Binary secondary outcomes and adverse events shared by the two study groups were compared using the chi-squared test or Fisher’s exact test, depending on the number of observations.For primary outcome analysis, the Mann–Whitney U test for superiority by a margin  and the Mantel–Cox log-rank test to compare the curves between the two groups of patients. For all comparisons, a two-tailed The CONSORT flow diagram shows the patient distribution and allocation . From DeThere were no differences in demographics, surgery characteristics, surgical procedures, medical history, preoperative risk heart function, and intraoperative monitoring parameters .−1·h−1 in the Control group (p < 0.001 for δ = 1.4 µg·kg−1·h−1)  fentanyl dose of 1.2 (1.1–1.5) vs. 4.5 (3.8–5.5) µg·kgg−1·h−1) .−1·h−1 (p < 0.001).Compared to the Control group, ESPB significantly decreased pre-CPB fentanyl consumption, with a median (IQR) of 3.3 (2.7–4.5) vs. 7.2 (5.7–9) µg·kgp < 0.001).Cumulative morphine consumption 48 h after ICU admission was significantly reduced by the addition of ESPB to NDGA. The median (IQR) doses were 22.1 (0–40.4) vs. 60.6 (40–95.7) µg/kg (p < 0.001). Additionally, the first time to receive morphine was significantly extended by ESPB, with a median (IQR) from 345 (67.5–795) to 540 (285–1110) minutes (p = 0.008). Finally, the Cox proportional hazard ratio for 48 h morphine was 0.3 for the ESPB-to-Control group   a.p = 0.261).NRS scores were compared at six time points: 0, 6, 12, 24, and 48 h after extubation, and 1 h after drain removal. Compared to the Control group, ESPB significantly reduced NRS scores at the first five time-points, with the largest absolute median difference at 6 and 12 h  . There wp < 0.001). At 2 h after ICU admission, 87.5% of patients given the block were extubated compared to 0% within the Control group (p < 0.001). The Cox proportional hazard ratio for extubation in the ESPB-to-Control group was 5.24   from 360 (285–510) to 90 (60–105) minutes (< 0.001) b.p < 0.001). The time to wean off NE was significantly shorter with than without ESPB, with a median (IQR) of 0 (0–60) vs. 240 (0–720) minutes (p < 0.001). Correspondingly, more ESPB patients were free of vasopressor support 2 h after ICU admission . There was no difference between the groups in total intraoperative NE consumption (p = 0.147).ESPB hastened liberation from vasopressor support after ICU admission. NE consumption, measured from ICU admission until 12 h later, was significantly less in the interventional group compared to the Control group with a median (IQR) of 0 (0–1.3) µg/kg vs. 9 (0–32.3) µg/kg (p = 0.102) and hospital LOS (p = 0.598) (There were no group differences for ICU LOS (= 0.598) .Following skin incision, two ESPB patients exhibited a NOL index higher than 25 and required rescue fentanyl according to the intraoperative analgesic protocol. Following sternotomy, one of these and two other patients needed rescue fentanyl, as indicated by the NOL index. Eventually, all three achieved a NOL index of less than 25 before CPB initiation. All the other ESPB patients showed adequate nociception control throughout the incision and sternotomy and did not require rescue fentanyl after induction.This prospective randomized study demonstrates that the Nociception Level index-directed bilateral single-shot ESPB compared to no block is superior in reducing total intraoperative fentanyl consumption in adult patients undergoing open heart surgery. In addition, ESPB reduced the 48 h morphine and 12 h NE consumption, hastened liberation from vasopressors, enabled fast-tracking, and improved pain scores, but, in this patient population, it did not affect ICU or hospital LOS.There are several considerations to make concerning the authors’ results. Firstly, this study adds to the growing clinical evidence that bilateral ESPB is an efficient analgesic adjunct for cardiac surgery with midline sternotomy, consistent with the blockade of dorsal and ventral rami of spinal nerves ,9,10,18.−1·h−1, similar to the authors [This leads to the second consideration that continuous objective monitoring of intraoperative NAN balance is fundamentally important. It is the first study to apply the NOL index from the induction of anesthesia to CPB initiation. The pre-CPB period represents a critical window to evaluate the block’s success and tailor opioid administration to its effectiveness. Indeed, it is before CPB initiation that substantial noxious stimuli, including skin incision, sternotomy, and chest retraction, amass and provide a full-scale nociceptive challenge that the block is yet to meet. The NOL index-directed ESPB decreased pre-CPB fentanyl consumption by 54.2%, attesting to the block effectiveness. By the end of the surgery, this difference had reached 73.3%. Previous studies demonstrated the intraoperative opioid-sparing effect of ESPB. Macaire et al., found a 75% reduction in median intraoperative sufentanil . After aThirdly, postoperative pain scores and morphine consumption likely depend on whether a catheter or a single-shot technique is chosen. In less complex surgeries, single-shot blocks meet two essential ERAS requirements: simplicity and benefit. Compared to catheters, single-shots are less time-consuming, easier to perform, and do not run the risk of displacement. Nevertheless, these advantages come at the cost of a shorter duration of analgesia. Macaire et al. found that catheters can eliminate morphine consumption, improving postoperative rehabilitation and pain scores . In thisFourthly, the ESPB group met extubation criteria sooner, achieving a significantly shorter duration of mechanical ventilation. The authors attribute this finding to a fentanyl-sparing effect elicited by ESPB. Other studies that used fentanyl corroborate our findings ,10. ConvFifthly, in the ESPB group, the authors observed a significant norepinephrine-sparing effect postoperatively but not intraoperatively. This may indicate improved postoperative hemodynamic control in patients with less residual sedation who are closer to being weaned off the ventilator or are already extubated.Finally, no difference was observed for ICU and hospital LOS. This observation is at variance with Krishna et al.  and StatThis study had a few limitations. Firstly, it was a single-centered study that mainly included uncomplicated surgeries, hence the use of a single-shot ESPB approach. Secondly, a sham block design would have been ideal to ensure blinding but was not used since this has remained a controversial issue . ThirdlyThis study demonstrated that, within a NOL index-directed anesthetic protocol, bilateral single-shot ESPB benefits adult patients undergoing open heart surgery in perioperative opioid usage. ESPB reduced the total intraoperative fentanyl and cumulative 48 h morphine requirements by 73.3% and 63.5%, respectively. Other gains included faster extubation and liberation from vasopressor support, and better quality of analgesia up to 48 h after surgery.


Our results showed that the activities of β–glucosidase (βG), N–acetylglucosaminidase (NAG) and acid phosphatase (APH) were improved in RTS+MN. The bacterial and fungal abundances in RTS+MN and RTS+HN were 1.27–27.51 times higher than those in other treatment groups. However, the bacterial and fungal alpha diversities were enhanced in PTS+MN and PTS+CK compared with other treatments, respectively. Proteobacteria and Basidiomycota were the predominant phylum for the respective bacterial and fungal communities. Moreover, significant interactive effects were found in the fungal community composition, but only minor impacts were observed on the bacterial community composition. Soil water content and penetration resistance contributed more to the soil enzyme activity and microbial community than other soil properties investigated, whereas there was a significant positive correlation between βG and APH activities and microbial abundance. These findings can provide new insights into tillage with straw incorporation and N fertilization on maize cultivation in northeast China.Soil enzymes and microorganisms are both important to maintaining good soil quality and are also sensitive to changes in agricultural management. The individual effects of tillage, straw incorporation and nitrogen (N) fertilization on soil enzymes and microflora have been widely acknowledged, but their interactive effect remains largely unknown. In a 5–year in–situ field study, effects of rotary (RTS) and plow tillage (PTS) practices with straw incorporation combined with three N fertilization levels (0 kg N ha Soil microorganisms are important participants in assessing agricultural soil quality and ecosystem function through their integral and unique roles in mediating the decomposition of soil organic matter and the cycling of nutrients . Soil enReturning crop straw to fields is a sustainable approach that provides nutrients for crop growth and a carbon (C) source for soil microbe reproduction in agricultural systems . This meThe application of nitrogen (N) fertilizer to agroecosystems usually increases soil organic matter, available nutrients, and thus improves crop yields . HoweverNortheast China is one of the major areas of agricultural production, with the region’s total maize yield accounting for approximately 30% of the nation’s total maize production . Maize sin–situ field experiment was done in 2015 in Tieling City , Liaoning Province, China. Maize is the dominant crop in this area and is harvested once per year. The climate in this area is subtropical arid with a mean annual temperature of 20.9 °C and average precipitation of 543.0 mm during the spring maize growth period (from the beginning of May to the end of September). Before the field experiment started in April 2015, the basic soil chemical properties of the top 0–20 cm soil layer were: 15.7 g kg–1 of soil organic matter (SOM), 1.2 g kg–1 of total nitrogen (TN), 25.7 mg kg–1 of available phosphorus (AP), 109.3 mg kg–1 of available potassium (AK) and pH of 5.64.This –1 , 187 kg N ha–1  and 337 kg N ha–1  and were applied to the subplots. We designed five different nitrogen fertilizer gradients including 0, 112, 187, 262 and 337 kg N ha−1, it has been found in previous studies that maize grain yield would not increase significantly when N application more than 187 kg N ha−1  and plow (PTS) tillage with straw incorporation were applied to the main plots. The three N fertilization levels were: 0 kg N hag N ha−1 . Therefo2O5 ha–1 (Superphosphate) and 90 kg K2O ha–1 (potassium chloride) was used when the maize was planted. The N fertilizer (urea) was applied as split doses: 1/3 of the total N was basal fertilizer and the remainder was jointing fertilizer. Spring maize  was manually sown in early May and harvested at the end of September. Maize was planted at a density of 67,500 ha–1 with 60 cm interspaces.After the maize harvest every year, maize straw was chopped into segments 5–10 cm in length. For RTS treatment, a rotary tiller mixed the soil twice to incorporate maize straw into the 0–15 cm soil layer. For PTS treatment, the soil was inverted with a plow tiller to bury the maize straw beneath the 30 cm soil layer. Basal fertilizer with 90 kg PSoil samples were collected from the upper 30 cm of the soil at the maize jointing stage (June 2019). Five soil cores were randomly taken from three sampling sites in each plot and mixed to obtain three representative samples for each treatment. A portion of the soil sample was air–dried for soil property analysis. Subsamples were passed through a 2 mm sieve and stored at 4 °C and –80 °C for enzyme activity and molecular analyses, respectively.3––N) and ammonium (NH4+–N) were extracted with 2 M KCl for 1 h and determined colorimetrically using a Smart Chem 200 auto discrete analyzer according to the method described by Soil bulk density (BD) was measured using the the core ring method . Soil waH4 BioTek, Winooski, VT, USA) using a microplate fluorometer with 365 nm excitation and 450 nm emission filters. After accounting for negative controls and quenching, activities were expressed in units of nmol g–1 h–1.Soil β–glucosidase (βG), N–acetylglucosaminidase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APH) activities were measured based on the method used by The soil microbial DNA of each sample was extracted from 0.5 g frozen soil using a PowerSoil DNA Isolation Kit  according to the manufacturer’s instructions. The extracted DNA was checked on 1.2% agarose gel and the DNA quality and concentration were evaluated using the NanoDrop 2000 Spectrophotometer .−1 dNTP, 1 μl of 10 μmol L−1 forward primer, 1 μl of 10 μmol L−1 reverse primer, 1 μl of DNA template, 9.75 μl of ddH2O and 0.25 μl of Q5 DNA polymerase . The PCR process consisted of a 5 min initial denaturation step at 98 °C, followed by 25 or 28 cycles of 30 s at 98 °C, 30 s of annealing at 52 °C, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min for bacteria and fungi, respectively. The PCR products were checked on 2% agarose gel and further purified using the TruSeq Nano DNA LT Library Prep Kit . Subsequently, the purified amplicons were sequenced (468 bp for bacteria and 280 bp for fungi) according to an equimolar and paired–end method with the Illumina MiSeq platform . The PCR products of the 16S rRNA and ITS1 genes were used as a template for establishing the sequencing library using the Illumina Miseq Platform.The primer pair 338F (5′–ACTCCTACGGGAGGCAGCA–3′) and 806R (5′–GGA CTACHVGGGTWTCTAAT–3′) targeted the V3–V4 region of the bacterial 16S rRNA gene . The ITSThe raw sequence data was processed using the QIIME2 bioinformatics software. The very first step applied the DADA2 pipeline for de-noising and de-replicating of the paired-end sequences including chimera removal at the end . The gen2 value was 0.991, 0.994 for the bacterial and fungal communities, respectively.Real–time PCR amplification of the bacterial 16S rRNA and fungal ITS1 genes were performed on the CFX96  using the AceQ qPCR SYBR Green Master Mix . The reaction mixtures contained 7.5 μl SYBR Green Mix (2×) for each primer and 1 μl of DNA extract as the template. The amplification profile was 95 °C (5 min), followed by 40 cycles of 95 °C (10 s), 55 °C (15 s), 75 °C (30 s), and 1 cycle of 95 °C (15 s), 60 °C (1 min), 95 °C (45 s) for bacteria and fungi. Cycle threshold (Ct) values were determined with the Bio–Rad CFX Manager software . Standard curves were generated by serially diluting plasmids, as described by p value < 0.05 was considered statistically significant.The differences between the effects of tillage with straw incorporation or N levels and their interaction on soil properties, enzyme activities, alpha diversity indices and abundance of bacterial and fungal were tested using two factor analysis of variance (ANOVA) and Duncan’s multiple range Test with SPSS 23.0 . A Spearman’s correlation analysis was performed to reveal the relationships between soil properties, enzyme activities, gene abundance and the bacterial and fungal alpha diversity indices. The clustering of different samples and the microbial community structure was illustrated by nonmetric multidimensional scaling (NMDS) as microbial beta diversity. Correlations among the soil properties and microbial compositions were determined using a redundancy analysis (RDA) in CANOCO 4.5. The figures were drawn in Origin 9.0. A 3––N and AP contents under RTS treatment increased with N addition, and NH4+–N, AK and TP contents under PTS treatment were also greater with increasing N levels. However, SOC and TN contents decreased in PTS treatment (3––N and NH4+–N contents under RTS were lower compared to PTS (p < 0.05).Tillage with straw incorporation (Ts), N fertilization (N) and their interaction (Ts × N) greatly affected soil properties and enzyme activities . The addreatment . Averagep < 0.05). Furthermore, the activities of βG, NAG, LAP and APH were positively related to soil AP and AK contents, and negatively related to pH (p < 0.05).As for soil enzyme activity, the activities of βG, NAG and APH were significantly enhanced by N addition in RTS treatments . Howevered to pH . Meanwhi5 to 2.5 × 106 copies g−1 soil) were detected in RTS+MN treatment and PTS+MN treatment, respectively. The highest fungal gene copy number (1.5 × 106 copies per g−1 of soil) was in RTS+HN treatment and the lowest (4.1 × 104 copies per g−1 of soil) was in PTS+MN treatment.Ts and its interaction effect with N had significant effects on the bacterial and fungal gene abundance of the soil . N addit4+–N, NO3––N, βG, NAG and APH (p < 0.05). The fungal gene copy number was significantly related to soil pH, SWC, BD, PR, SOC, TN, NH4+–N, βG and APH (p < 0.05).The Pearson’s correlation coefficients between soil properties and enzyme activities, as well as microbial abundance, showed that the bacterial gene copy number was significantly related to soil pH, SWC, BD, PR, SOC, TN, TP, TK, NHp < 0.05), but greater variations were obviously observed from the fungal diversity indices compared to the response of bacteria on treatments . Similarly, the bacterial Shannon index was significantly related to pH, SWC, BD, PR, SOC, TN, NH4+–N, AP, AK, βG, NAG and APH (p < 0.05). However, the fungal Chao 1 index was significantly related to SWC, PR and TN (p < 0.05). Similarly, the fungal Shannon index was significantly associated with pH, SWC, PR and TN (p < 0.05).The results of the Pearson’s correlation coefficient tests showed that the bacterial Chao 1 index was significantly related to SWC, PR, TP, TK, NHA nonmetric multidimensional scaling analysis (NMDS) reflecting microbial beta diversity indicated that there was no significant changes in the number of bacteria observed . With reProteobacteria, Actinobacteria, Acidobacteria, unclassified_Bacteria, Bacteroidetes, Gemmatimonadetes, Chloroflexi and Patescibacteria, with contributions of 36.65%, 27.48%, 12.09%, 7.74%, 4.45%, 4.43%, 2.90% and 1.46%, respectively  across all treatments were ectively . Among t as well  and S3. ectively . Among tposition  and S4.Proteobacteria, Actinobacteria, Acidobacteria, unclassified_Bacteri and Chloroflexi for bacterial phyla . This suggests that tillage practices changed the location of straw incorporation into soil and affected soil structure, further regulating soil enzyme activities. In addition, there was a significant negative correlation between soil enzyme activities and soil pH, which was consistent with Soil enzymes played a vital role in the cycling of soil nutrients in agroecosystems, and soil enzyme activity could be used as an index of soil microbial activity . Previou2 emissions , which are adapted to break down the straw into simpler compounds in the early stages of decomposition  and orders  are oligotrophic microorganisms (K–strategists), involved in the degradation of complex substrates  abundance and decreased alpha diversity compared to plow tillage with straw incorporation. Increasing N input was not beneficial for fungal diversity, but a medium N application amount could improve soil enzyme activities. Moreover, the fungal community composition varied significantly relative to the community composition of bacteria in response to the combined effects of tillage with straw incorporation and applied N levels. Additionally, soil water content and penetration resistance played important roles in driving the soil enzyme activity, microbial abundance and community composition. Overall, these results suggest that rotary tillage with straw incorporation and medium N fertilizer application were helpful for the short–term improvement of soil properties and soil microbes in northeast China.10.7717/peerj.13462/supp-1Supplemental Information 1–1 applied. The values are mean ± standard deviation (n = 3). The groups accounting for 1% are shown, whereas those accounting for <1% are combined (Others).Rotary tillage with straw incorporation (RTS), Plow tillage with straw incorporation (PTS), 0 (CK), 187 (MN) and 337 (HN) kg N haClick here for additional data file.10.7717/peerj.13462/supp-2Supplemental Information 2–1 applied. The values are mean ± standard deviation (n = 3). The groups accounting for 1% are shown, whereas those accounting for <1% are combined (Others).Rotary tillage with straw incorporation (RTS), Plow tillage with straw incorporation (PTS), 0 (CK), 187 (MN) and 337 (HN) kg N haClick here for additional data file.10.7717/peerj.13462/supp-3Supplemental Information 3–1 applied. The values are mean ± standard deviation (n = 3). The groups accounting for 1% are shown, whereas those accounting for <1% are combined (Others).Rotary tillage with straw incorporation (RTS), Plow tillage with straw incorporation (PTS), 0 (CK), 187 (MN) and 337 (HN) kg N haClick here for additional data file.10.7717/peerj.13462/supp-4Supplemental Information 4–1 applied. The values are mean ± standard deviation (n = 3). The groups accounting for 1% are shown, whereas those accounting for <1% are combined (Others).Rotary tillage with straw incorporation (RTS), Plow tillage with straw incorporation (PTS), 0 (CK), 187 (MN) and 337 (HN) kg N haClick here for additional data file.