Escherichia coli, strain ECOR57. Phage-specific growth curves were observed where infections by SU10 resulted in a moderate production of phages and infections by SU57 resulted in a fast and extensive production of phage progeny. Sequentially adding SU10 followed by SU57 did not produce a significant change in growth rates, whereas adding SU57 followed by SU10 resulted in a decrease in SU10 titer The efficiency of the plating assays showed that ECOR57 exhibited a resistance spectrum after infection by both the single and combined phages. Phage-resistant bacteria exhibited four different morphotypes . The normal and edgy morphotypes had a high frequency of developing resistance. Bacterial growth and biofilm assays indicated that the edgy and pointy morphotypes reached a stationary phase faster and produced more biofilm compared to the wild type. These findings suggest that the dynamic structure of phage–bacteria communities dictate resistance evolution and development. Understanding when and how resistances arise and phage(s)–hosts interactions could aid in the design of phage therapy treatments.In this study, we looked at the population dynamics of a two phages-one host system using phages vB_EcoP_SU10 (SU10) and vB_EcoD_SU57 (SU57) and the bacteria  A substantial fraction of the human microbiome is located within the gastrointestinal tract, which has a delicate balance that is maintained by a variety of microorganisms such as bacteria and viruses . Abnorma10 per gram of the gut microbiome, phages are species specific and can play a role in bacterial genetic exchange through transduction [Viruses infecting bacteria, also known as bacteriophages (phages), maintain gut bacteria diversification and homeostasis ,4. Composduction ,5,6,7. Psduction . Infectisduction . Phages sduction ,12. Oftesduction . Under ssduction ,15,16. Vsduction ,17. The sduction . Bacteria can reduce adsorption and phage infection by employing phenotypic resistance mechanisms to prevent phages binding to their cell surface receptors. Phenotypic resistance can be divided into three categories: induced where exposure to phage-lysed bacteria triggers a gene expression change in uninfected bacteria, intrinsic where a physiological state or gene expression conferring resistance exists in a subset of bacteria population prior to phage introduction, and dynamic where phage proteins released when bacterial lysis degrades or blocks bacterial receptors . ExampleBacteria have a high rate of developing mutations in their anti-phage defense mechanisms against single phages, which can rapidly spread throughout the bacterial population ,28,29. DEmploying anti-phage defense mechanisms, such as those previously described, are highly dependent on the type and infection kinetics of the infecting phage. Virulent phages ideally would have high adsorption rates, short latent periods, and large burst sizes . HoweverEscherichia coli reference collection [Podoviridae C3 morphotype phage vB_EcoP_SU10 (SU10) and Drexlerviridae B1 morphotype phage vB_EcoD_SU57 (SU57) [In this study, the population dynamics of a two phages–one host system was investigated. Strain 57 from the llection  was infe7 (SU57) ,45, in c9 and 1010 plaque forming units (PFUs) or colony forming units (CFUs)/mL. A steady state was reached approximately after 48 h, with phage titers of approximately 1010 for SU10 and 107 PFU/mL for SU57, and bacterial titers of approximately 109 CFU/mL. Phages and bacteria coexisted for the entirety of the experiment, 120 h after infection. SU10 alone resulted in a moderate production of phages in ECOR57, where phage and bacterial titers eventually stabilized   B. The SUduration . In geneduration .Following phage exposure, ECOR57 bacteria in each chemostat were collected at 16 different time points  and analyzed for resistance/susceptibility against phages SU10 and SU57 using efficiency of plating (EOP). The number of colonies analyzed at each time point were randomly selected. A trend whereby ECOR57 was sensitive to phage adsorption during early experimentation and developed resistance during later experiment time points was observed . A mix oUpon sampling ECOR57 bacteria from each chemostat following phage exposure, four distinct colony morphotypes  were observed . Those tWhen comparing the growth curves of wild-type ECOR57 and the various morphotypes over 24 h, the following was observed: Those with the normal phenotype had the shortest lag time similar to the wild type (approximately 347 vs. 341 min), those with the edgy and slimy morphologies had a similar lag time with the longest duration (approximately 391 vs. 392 min), and those with the pointy morphology had a lag time of approximately 379 min. Bacteria presenting the edgy morphotype reached a stationary phase earlier, approximately 10 h after starting the growth experiment, compared to the other morphotypes and wild-type ECOR57 bacteria .The EOP of phages were performed in order to determine whether these various morphotypes provided resistance against phage infection. Bacteria with the normal and edgy morphotypes showed the highest frequency or resistance  to infection by phages SU10 and/or SU57. Those with the slimy phenotype had the second highest resistance frequency. For those with the pointy morphotype, resistance and susceptibility frequencies were inconclusive . p ≤ 0.001 using one-way ANOVA with post hoc Tukey; n = 5) amounts of biofilm, with the edgy generating higher amounts than the pointy morphology (p ≤ 0.001) differences in the amount of biofilm formed, except between the normal and slimy morphologies. Using antibiotic disk diffusion assays, the correlation between the varying ECOR57 bacterial morphotypes, including those presenting double resistance, and their susceptibility to cell-wall targeting antibiotics, such as ampicillin, imipenem, and ceftazidime, proved negative  (Podoviridae family [Drexlerviridae (formerly Siphoviridae) family [–10 mL × min–1 [Chemostat experiments revealed that both SU10 and SU57 produced phage-specific growth curves. Infection by SU10 resulted in a moderate production of phages, where both phage and ECOR57 bacterial titers were stable. One possible explanation for this lies with SU10’s infection efficacy. When infecting ECOR57, SU10 had an adsorption rate of 1.43 × 10 mL/min) . SU57 one family ,49, whil) family . Compare × min–1 . Like it × min–1 ,51, SU57 × min–1 ,49. With × min–1 ,52. It i × min–1 . This woPrevious studies have shown that the effect of a combinational phage infection depends on the order and timing of the addition of phages to a bacterial system . Phage-sOne of the properties that makes phages an interesting candidate to treat antibiotic-resistant infections is auto-dosing ,55. Unde“Leaky resistance”, on the other hand, could be an explanation for the phage survival observed . Phage-rIn addition to the development of single resistances and cross-resistance mechanisms, the ECOR57 bacteria underwent phenotypic changes under pressure from phage infection. Infected ECOR57 bacteria underwent phenotypic changes that bred true following multiple re-streaks . PreviouE. coli strain K12 [Neisseria gonorrhoeae has been shown to affect the shape of early biofilm formation and as well as the standing variation of bacterial populations that affect bacterial positioning and competition dynamics within a bacterial colony [The various colony morphotypes observed could be the result of phase variation . Phase vrain K12 . Large, rain K12 . It is prain K12 ,69. Modul colony . This cocsgBA  [adrA (encoding for cellulose biosynthesis) [The mucoid shiny appearance of bacteria can be attributed to the overproduction of exopolysaccharide, the main EPS component found in many biofilms ,72. Thisimbriae) ,78 and anthesis) ,79. CsgDnthesis) , wherebynthesis) ,76. It inthesis) . This alAll materials utilized and experiments performed were treated and carried out using standard microbiological laboratory practices in order to reduce any contamination risks.E. coli [600) reached 0.6. The bacterial strains used for this paper were ECOR10, ECOR57, and ECOR63 from the ECOR standard reference collection of E. coli , which wPodoviridae with C3 morphotype) and SU57 (T1-like Drexlerviridae with B1 morphotype), previously isolated from Käppala water treatment plant 15 km East of Stockholm, Sweden [g for 10 min and the supernatant was filtered through 0.45 µm sterile syringe filter . Phages were precipitated by adding solid NaCl and PEG8000  to the partially purified suspension to have a final concentration of 1 M NaCl and 10% (w/v) PEG8000 and stored at 4 °C for two weeks. Phages were recovered through centrifugation at 11,000× g for one hour at 4 °C. The phage pellet was re-suspended in 50 mL phosphate buffered saline (PBS) and stored at 4 °C until experimentation. Phage quantifications, measured as PFU/mL, were determined using the overlay agar (OA) method consisting of 65% (w/v) (22.775 g/L) TYA [8 CFU/mL) and 100 µL of serially diluted phage in PBS. The OA was poured over pre-prepared TYA plates and incubated at 37 °C for at least 18 h. The stock titer for SU10 was 4.1 × 1010 PFU/mL and 7.5 × 1011 PFU/mL for SU57.Phages SU10  TYA ,83. In s−1. Homemade chemostats  were conECOR57 bacteria were inoculated into the chemostats and was left to grow for 24 h in a 37 °C water bath until they reached steady state. Phages SU10 and/or SU57 were added at different time points and different MOIs in order to investigate changes in population dynamics between phages and bacteria. Five hundred microliter aliquots of the phage(s)–bacteria mixture were removed at 16 specific time points  for quantification and characterization (as described below). Throughout experimentation, the following conditions were kept: Chemostat A contained ECOR57 and SU10, Chemostat B contained ECOR57 and SU57, Chemostat C contained ECOR57 which was first infected with SU10 and then with SU57, and Chemostat D contained ECOR57 which was first infected by SU57 and then by SU10. The MOIs tested for SU10 were 0.33, 0.42, 0.43, 0.50, 0.55, 0.65, 0.83, 1.00, 1.10, 1.50, 2.30, and 1300. The MOIs tested for SU57 were 0.13, 0.12, 0.16, 1.04, 1.14, 1.20, 3.40, 3.60, 4.50, 10.4, 25.9, and 46.9. ECOR57 bacteria were quantified using the drop count methodology . SU10 an600 0.6). Once OD600 0.6 was reached, bacterial suspension was removed; the final volume of ECOR10 bacterial suspension used for experimentation was 44.991 mL and the final volume of ECOR57 was 44.9933 mL. A total of 9 µL of SU10 phage stock (2.85 × 1010 PFU/mL) was added to mid-log phase ECOR10 bacteria (approximately 3.95 × 107 CFU/mL) at a multiplicity of infection (MOI) of 0.144 and mixed by swirling (T = 0). One microliter of aliquots, withdrawn every five minutes for an experimental duration of 25 min, were centrifuged at 6000× g for one minute. Using the supernatant, 1:10 serial dilutions in PBS were done to determine the phage titer at each time point. All experiments were done in triplicates. This procedure was repeated for ECOR57 bacteria whereby, 6.7 µL of SU10 phage stock was added to mid-log phase ECOR57 bacteria (approximately 3.70 × 107 CFU/mL) at a MOI of 0.107. The adsorption rate constants for both ECOR10/SU10 and ECOR57/SU10 infection were determined using the following formula, where N is the bacterial density, Po and P are the starting and ending phage titers, k is the adsorption constant, and t is the time in minutes over which adsorption occurs:The adsorption rate constant of SU10 on bacteria ECOR10 and ECOR57 were determined using the modified one-step growth curve protocol used by Koonjan et al., (2020) . In summIt should be noted that the adsorption rate constants were determined using only two time points (T = 0 and T = 5).−4 for SU10 and 10−6 for SU57. As such, these dilutions were used to test the frequency of bacterial resistance development. One hundred microliters of overnight bacterial culture was used to inoculate OA containing 100 µL of either 10−4 SU10 dilution or 10−6 SU57 dilutions. LA plates containing the OA were incubated overnight at 37 °C. Phage resistance was based on the number of plaques observed rather than based on plaque morphologies. Resistance was determined as a lawn of bacteria with no phage plaques observed. Susceptibility was determined by having clear phage plaques covering the plate without having complete bacterial lawn lysis. Partial resistance was assessed as having between 1 to approximately 1000 phage plaques present. The frequency of phage resistance incidence was determined using EOP plaque assay method. Previous experiments showed optimal phage plaques when the phage stocks were diluted to 10E. coli  and three other E. coli strains  were performed using the E. coli housekeeping gene rpos, which encodes for the RNA polymerase sigma factor RpoS [In order to confirm that the various morphotypes observed were indeed ECOR57 and not contamination, preliminary genomic analyses were carried out. The genomic DNA of wild type ECOR57 and all its variants were extracted using DNeasy Blood & Tissue Kit  according to the manufacturer’s protocol specific for bacterial DNA extraction. The genomes were sequenced using Illumina Miseq™ sequencing system at Stockholm University, The Wenner–Gren Institute, Stockholm, Sweden. The raw reads were trimmed using Trimmomatic version 0.36 and assembled using SPAdes ,87. Opentor RpoS . Alignmetor RpoS ,93. A ne600 0.6) with 37 °C shaking incubation. Optimal bacteria concentration used for the BioscreenC analyses was assessed by the drop count method [−4 to 10−7 were plated in duplicates and incubated at 37 °C. The dilution which gave between 3–30 individual colonies was used. Using this dilution for all bacterial samples (for this experiment it was 10−6), 100 µL was used to inoculate 900 µL fresh LB, of which 150 µL was loaded onto the 100 well BioscreenC honeycomb plate. All samples were run as technical quadruplets and experiments were performed in triplicates. Each run included PBS negative controls and LB blanks. The BioscreenC EZ Experiment Software had the following settings: 37 °C incubation temperature, 600 nm filter, three-day experiment duration, and 10-min measurement interval with continuous shaking and medium amplitude. Growth curves and growth rate calculations were carried out using R [The growth curves for ECOR57 wild type bacteria and the different morphologies observed for phage resistance were determined using BioscreenC . LB was inoculated with 100 uL of overnight bacterial culture and allowed to reach mid-log phage  from which 200 µL was dispensed per well (40 wells per phage). 10 µL of bacteria diluted to have 106 CFU/mL were added to each well. Using the SpectraMax i3x system, the plate was incubated at 37 °C with shaking for 20 h and the bacterial growth was measured using OD600. The mutation rate (µr) was calculated using the P0 method:P0 is the proportion of wells that were sensitive (wells that exhibited no growth), N is the number of cells inoculated in each well, and N0 is the number of cells used to start cultures (approximately 103 CFU). Fluctuation experiments were replicated in triplicate. In order to quantify the mutational rates of ECOR57 resistance against SU10 and SU57, Luria-Delbrück fluctuation tests were performed. Modified from Borin et al., (2021) Lang (2018), and Luria and Delbrück (1943), the following was performed: An overnight culture of ECOR57 (initiated from a single colony) was diluted such that each well of a 96 well plate contained 200 µL of approximately 10600 was approximately 0.06  and plated on Mueller Hinton broth II agar plates . Antibiotic resistance/susceptibility were determined using the European committee on antimicrobial susceptibility testing (EUCAST) antimicrobial disk diffusion zone diameter breakpoints for the following antibiotics: 10 µg imipenem, 10 µg ampicillin, and 10 µg ceftazidime . Plates were incubated at 37 °C overnight, after which zones of inhibition were measured. Samples showing ampicillin resistance were further tested on Mueller Hinton broth II plates containing different concentrations of ampicillin . Phage-resistant ECOR57 bacteria exhibiting different morphologies and phage-resistance patterns were re-suspended in PBS until ODn = 5 was performed using GraphPad Prism version 5.0.Biofilm production for the various morphologies of ECOR57 was determined using a modified protocol from O’Toole (2011) . OvernigWithin the human gut microbiome, interactions between phages and bacteria are inevitable. In a two phages–one host system, the types of phages, their individual phage kinetics, and MOI used for infections influenced their infection efficacy and growth on a single bacterial host strain. Additionally, over time and under pressure from phage infection, bacteria were prone to developing a range of resistances  and underwent phenotypic changes most likely due to the phage tolerance, “leaky” resistance, and phase variation. While these experiments provide insight into the mechanics of a two phages–one host system, the pervasiveness of these interactions within the human gut microbiome remains largely unknown. The two phages–one bacteria interactions in the gut could ultimately dictate whether it will be possible to use solely phage cocktails therapeutically against the same bacterium or if, instead, complementary to antibiotics ,98,99. FThis study provides the foundation and a way to observe and quantify different phage(s)–bacteria combinations. Phages intended for therapeutic usage must be further researched and characterized in such a manner in order to elucidate their infection kinetics in combination with other phages, the coevolution of resistance mechanisms by both phages and exposed bacteria, as well as the dosing to reach the site of infection and produce an active infection.


Cytotoxicity measurements revealed high potency of CC-loaded NGs for various cancer cell lines with low IC50 values (up to 30 μg·mL−1), whereas free polymer was well tolerated up to a concentration of 1.50 mg·mL−1. Confocal laser scanning microscopy (CLSM) was used to monitor internalization of free and CC-loaded NGs and demonstrate the protein cargo’s release into the cytosol.The sensitivity of therapeutic proteins is a challenge for their use in biomedical applications, as they are prone to degradation and opsonization, thus limiting their potential. This demands for the development of drug delivery systems shielding proteins and releasing them at the site of action. Here, we describe the synthesis of novel polyglycerol-based redox-responsive nanogels and report on their potential as nanocarrier systems for the delivery of cytochrome C (CC). This system is based on an encapsulation protocol of the therapeutic protein into the polymer network. NGs were formed via inverse nanoprecipitation using inverse electron-demand Diels–Alder cyclizations (iEDDA) between methyl tetrazines and norbornenes. Coprecipitation of CC led to high encapsulation efficiencies. Applying physiological reductive conditions of  Therapeutic proteins have attracted growing interest due to their increased specificity and versatility of use in biological processes. Compared to small organic molecules, proteins do not have solubility problems for medical applications, but they suffer from proteolytic aggregation at surfaces, as well as physical unfolding under physiological conditions ,2. This NGs are highly water-swollen, crosslinked, three-dimensional networks of hydrophilic or amphiphilic polymers in the size range of 10–1000 nm ,14. Due Based on their reaction kinetics, click-type reactions such as the thiol-Michael addition ,30, stra−1, whereas the CC-loaded NGs exhibited high potency against all three cell lines, with low IC50 values. Overall, these NGs show excellent biocompatibility, biodegradability, high tunability with a narrow size distribution, and good stability under acidic, neutral, and basic conditions. This makes our system a promising candidate for a delivery platform of therapeutic proteins in possible future biomedical applications.In this work, we present the synthesis of new dPG-based redox-responsive NGs for the intracellular delivery of cytochrome C (CC), a protein that is apoptotic when released into the cytosol. We obtained NGs via iEDDA-mediated inverse nanoprecipitation. Redox responsiveness was introduced by using a cystamine-based linker functionalized with norbornene on both sites. Release of CC under reductive conditions showed excellent protein activity. As a proof of concept, we investigated the cell viability of the loaded compared to the free NGs and their internalization for different cell lines. Unloaded NGs showed no cytotoxic effects at concentrations of up to 1.50 mg·mLH-1,2,3-triazolpyridinium-3-oxid-hexafluoro-phosphate (HATU) was purchased from TCI . 4-(Dimethylamino)pyridin (DMAP), bicyclo[2.2.1]hept-5-en-2-ylmethanol, 4-nitrophenyl chloroformate, and cystamine hydrochloride were purchased from Sigma-Aldrich. Dichloromethane (DCM) and ethyl acetate were purchased in high-performance liquid chromatography (HPLC) grade from Sigma-Aldrich and used without any further purification. l-glutathione (GSH) and fluorescein isothiocyanate (FITC) were purchased from TCI. Cyclohexane was obtained in technical pure grade from Sigma-Aldrich and used without any further purification. Dry DCM, N,N-dimethylformamide (DMF), pyridine, and methanol (MeOH) were purchased from Sigma-Aldrich. Water was used from a Milli-Q station from Millipore . All reactions were performed under an argon atmosphere using standard Schlenck techniques and oil pump vacuum.Acetamidine hydrochloride, zinc triflate, 4-cyanobenzoic acid, and hydrazine were purchased from Sigma-Aldrich . 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid-hydrochlorid(EDC·HCl) was purchased from Carl Roth . 1-[bis(Dimethylamin)methylen]-1Qualitative thin-layer chromatography (TLC) was carried out on silica-coated aluminum plates (silica gel 60 F254) that were obtained from Macherey Nagel . Analysis of the TLC was done by fluorescence quenching under UV-light (254 nm) or by a stain of potassium-permanganate- , 25.0 mL anisaldehyde, 25.0 mL conc. sulfuric acid, 8.00 mL acetic acid) purchased from Sigma-Aldrich and heat as developing agents.2 columns and cartridges were obtained from TELEDYNE. As eluants, cyclohexane  and ethyl acetate (HPLC grade) were used. All dialyses were performed in regenerative cellulose (RC) dialysis tubes with a molecular weight cutoff (MWCO) of 1 kDa and 100 kDa purchased from Sigma-Aldrich and Fisher Scientific .Medium-pressure liquid chromatography (MPLC) was performed with a TELEDYNE ISCO Combi-Flash FR 200 from TELEDYNE . Prepacked SiO1H-NMR spectra were recorded at 300 K on a Joel ECX 400 MHz  or a Bruker Avance III 700 MHz  in the reported deuterated solvents. Chemical shifts δ are indicated in parts per million (ppm) with reference to the residual solvent peaks. To designate multiplicities, the following abbreviations were used: s = singlet. In case that no multiplicity could be identified, they were stated as m = multiplet and their chemical shift range of signal was given. 13C-NMR spectra were recorded at 300 K on a Bruker Avance III (176 MHz). Chemical shifts δ are indicated in parts per million (ppm) with reference to the residual solvent peaks. The spectra were decoupled from proton broadband.−1.Fourier transform infrared spectroscopy (FTIR) was performed on a JASCO FT/IR spectrometer  via attenuated total reflectance (ATR). Wave numbers High-resolution mass spectra (HRMS) were obtained from an AGILENT 6210 ESI-TOF spectrometer .Dynamic light scattering (DLS) and zeta potential were measured on a Malvern zeta-sizer nano ZS ZEN 3600 ) using an He–Ne laser (λ = 532 nm) at 173° backscatter and automated attenuation at, unless stated otherwise, 25 °C. All sample measurements were performed in triplicate, yielding a mean size value plus standard deviation. All measurements were performed in triplicate.−1 sample concentration using a pullulan standard, 0.1 M NaNO3 solution, as an eluant, and a PSS Suprema column  10 μm with a flow rate of 1 mL·min−1. The signals were detected with an RI detector from PSS Polymer Standards.Gel permeation chromatography (GPC) was measured on an AGILENT 1100 at 5 mg·mLUV/Vis measurements were conducted on an AGILENT Cary 8454 UV–visible spectrophotometer, using half-micro quartz cuvettes.For the preparation of cryo-sample droplets, 4 µL of the sample solution was placed on hydrophilized holey carbon filmed grids  at room temperature. The excess fluid was blotted off using filter paper to generate an ultrathin layer of the solution  spanning the holes in the carbon film. The grids were vitrified in liquid ethane using an automated vitrification robot (FEI Vitrobot Mark III from Fisher Scientific) and stored in liquid nitrogen prior to the measurement.Cryogenic transmission electron microscopy (cryo-TEM) was carried out using a Talos Arctica transmission electron microscope . The vitrified grids were stabilized by a copper autogrid and fixed with a spring clamp under liquid nitrogen. These autogrids were transferred under liquid nitrogen into the transmission electron microscope using the microscope’s autoloader transfer routine.Micrographs were recorded using the microscopes low-dose protocol at a primary magnification of 28,000× and an acceleration voltage of 200 kV. Images were recorded by a Falcon 3CE direct electron detector  at full size (4k) from Thermo Fisher. The defocus was chosen to be 5 µm in all cases to create sufficient phase contrast.The reported spectra can be found in the Both compounds were synthesized according to the literature ,34.4) was performed according to a reported procedure +: 453.1876, found: 453.1834; calc. for C22H33N2O4S2Na+ [M + Na]+: 475.1695, found: 475.1679; calc. for C22H33N2O4S2K+ [M + K]+: 491.1435, found: 491.1412.1M (dPG-metTet) to 2M (Norb-Cys-Norb) was set to 1:1.5. The standard batch size was set to 5 mg·mL−1. Acetone was used as nonsolvent. As an example, a general procedure for the preparation a NG is described below.NGs were synthesized via inverse nanoprecipitation. The ratio of the precursor 1M was stored in a stock solution (150 mg·mL−1) in water and 2M was stored as a stock solution (50 mg·mL−1) in acetone. An aliquot was taken and separately diluted with water to a final volume of 1 mL. For this, 30 μL of 1M was diluted with 470 μL of water, and 10 μL of 2M was diluted with 490 μL of acetone. Both solutions were cooled in an ice bath to 4 °C. 2M was added quickly to the solution of 1M via a syringe, briefly vortexed for 5 s, and injected quickly via a syringe into a 30 mL glass vial, containing a solution of 20 mL of acetone under vigorous stirring (1100 rpm). The suspension was stirred for another s and then kept still. After 30 min, 10 mL of phosphate buffer solution  and 20 μL of 2-(vinyloxy)-ethan-1-ol were added under stirring (1100 rpm.). The solution was transferred into a 50 mL round-bottom flask, acetone was evaporated, and the NGs were obtained as stable dispersions in an aqueous medium with an average yield of 95%. Characterization was performed by means of DLS, NMR, and cryo-TEM measurements.M1  was dissolved in dry DMF (10 mL). FITC  was added, and the solution was stirred for 48 h at 60 °C. The solution was cooled to room temperature and dialyzed against deionized water for 48 h to obtain the FITC–dPG-metTet .2O): λmax (ϵ): 500 nm (1 mg·mL−1).UV/Vis  against PB (10 mM) for 24 h. All encapsulation experiments were performed in triplicate. Protein loading content and protein loading efficiency were determined via UV/Vis, using the absorbance of CC at 410 nm.CC was stored in a 5 mg·mL−1, 1 mL in PB ), GSH (10 mM) was added and the pH was adjusted to 7.4. The resulting solution was incubated for 2 days at 37 °C. As a control, a NG sample without GSH was prepared accordingly. DLS measurements were performed after 0, 1, and 2 days.To a solution of the NGs  or PB  with 10 mM GSH in triplicates.−1) were placed in a dialysis tube (MWCO = 100 kDa) with 20 mL of release medium. At each time stamp, 5 mL of medium was withdrawn, and 5 mL of fresh release medium was refilled. The released amount of CC was quantified via UV/Vis spectroscopy using the protein absorbance at 410 nm and calculated on the basis of a calibration curve of the protein.First, 1 mL of dPG-NGs loaded with CC (CC@NGs)  and 100 μL of 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) with a concentration of 1.00 mg·mL−1 were added. Then, the absorbance at 405 nm of the oxidized product was monitored via UV/Vis spectroscopy every 30 s for 180 s. As a control, native CC (4 μg·mL−1) and native CC (4 μg·mL−1) incubated with GSH (10 mM) were used and treated in the same manner.For this, CC was taken from the release medium described in v/v) FBS, 100 U·mL−1 penicillin, and 100 µg·mL−1 streptomycin . Cells were regularly subcultured at least twice a week when they reached 70% to 90% confluency. For the cytotoxicity assay, 90 µL of a cell suspension in DMEM containing 5 × 104 cells per mL was seeded in each inner well of a 96-well plate and incubated overnight at 37 °C and 5% CO2. In the outer wells, 90 µL DMEM without cells were added. On the next day, serial dilutions of all the samples were prepared, and 10 µL of each was added to the cells in triplicate, in addition to one outer well for background correction. SDS (1%) and nontreated cells served as controls. After another 72 h at 37 °C and 5% CO2, the CCK-8 solution was added (10 µL/well), and absorbance at a measurement wavelength of 450 nm and a reference wavelength of 650 nm was measured after approximately 3 h incubation using a Tecan plate reader . Measurements were performed in triplicate and repeated three times. The cell viability was calculated by setting the nontreated control to 100% after subtracting the background using the Excel software. All cell experiments were conducted according to German genetic engineering laws and German biosafety guidelines in the laboratory (safety level 1).The effect of the dPG-NGs (NG) and the NG@CC, as well as free CC, on three cancer cell lines, A549, Hela, and MCF7, was determined using the cell viability assay Cell Counting Kit 8 (CCK-8) from Sigma-Aldrich Chemie GmbH  according to the manufacturer’s instructions. A549 (DSMZ no.: ACC 107), Hela (DSMZ no.: ACC 57), and MCF7 (DSMZ no.: ACC 115) cells were obtained from Leibniz-Institute DSMZ  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (50) was calculated with GraphPad Prism 6.01  using the log (inhibitor) vs. normalized response variable slope equation.The half-maximal inhibitory concentration (IC−1), washed twice with PBS, and covered with fresh cell culture medium (DMEM). Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8  with a 63×/1.4 HC PL APO CS2 oil immersion objective using the manufacturer-given LAS X software in sequential mode with the following channel settings: transmission Ch , excitation laser line 405 nm, detection of transmitted light (photomultiplier); Ch1 (Hoechst 33342): excitation laser line 405 nm, detection range 410 nm–484 nm (hybrid detector); Ch2 (FITC): excitation laser line 488 nm, detection range 493 nm–712 nm (hybrid detector).The uptake of FITC-labeled dPG-NGs with and without CC in A549, Hela, and MCF7 cells was analyzed using confocal laser scanning microscopy (CLSM). The cells were propagated as described above. For CLSM, HeLa cells were seeded in eight-well ibidi slides (ibidi treat) in 270 µL of DMEM. After cell attachment for 4 h to 24 h, 30 µL of a post-seeding solution containing compound was added, and the cells were further incubated overnight. Before imaging, the cells were stained with Hoechst 33342 (1 µg·mL1) hept-5-en-2-ylmethanol (5) into its reactive carbonate derivative (6) by using 4-nitrophenyl chloroformate. This allowed carbamate coupling of norbornene to the amines of cystamine under mild conditions, yielding Norb–Cys–Norb (2M) with an overall yield of 35%. Both precursors were further used for NG formation via inverse nanoprecipitation (Starting with dPG (1) , dPG–NH2nditions. dPG–metTpitation .1M, dissolved in an aqueous solution and 2M dissolved in acetone. The resulting mixture was injected into acetone, and then water was added to obtain NGs as a stable dispersion in water  from 0.25 to 2.00 equivalents. The obtained NGs were analyzed by DLS. This procedure yielded NGs in a range of 171 nm to 257 nm with a narrow dispersity (PDI > 0.11) and a slightly positive surface charge that derived from the remaining amine groups of dPG–metTet  and protein loading efficiency (PLE), loading experiments with different protein loading contents ranging from 5–20 wt.% were performed. A maximum PLC of 8.7%, with an efficiency of 87%, was obtained using 10 wt.% CC. Increasing the amount of CC led to similar PLC values, indicating protein saturation of the system at 9 wt.% PLC. For the saturated CC@NGs, a narrow PDI of >0.2 with a neutral surface charge was obtained . DLS meaIn tumor cells, an increased concentration of GSH (2–10 mmol) can be found . Therefo−1, as well as for free CC up to a concentration of 0.24 mg·mL−1. CC-loaded NGs in the same concentration caused a significant decrease in cell viability in all three cell lines at a loaded CC concentration of 30 μg·mL−1. The results indicate cellular uptake of the loaded NGs and subsequent release of CC to the cytosol, a finding reinforced by the obtained low IC50 from these measurements . For the CLSM study, 50 values (<30 μg·mL−1) for the CC-loaded NGs, as well as low toxicity of their unloaded analogues, indicating release of the protein cargo to the desired area. The obtained cell viability data, along with the observed internalization of our NGs into different cell lines, demonstrate the viability of our concept for a redox-responsive NG-based protein delivery platform.This research demonstrated the synthesis of dPG-based methyl tetrazines and cystamine-based norbornenes. We were able to reproducibly obtain NGs with dispersity indices of 0.11 and below. The prepared NGs retained their size under acidic, neutral, and basic conditions, indicating high stability of the hydrogel network under physiological conditions. Coprecipitation of CC revealed high encapsulation efficiencies of more than 80%, with up to 10 wt.% protein feed ratios. Applying reductive conditions led to degradation of the NGs and release of 80% of loaded CC within 48 h. ABTS assays revealed full functionality of the protein cargo after loading and in vitro release. Cell viability studies on McF7, HeLa, and A549 cell lines revealed low IC


Furthermore, E1RK1/2 phosphorylation was reduced during cryopreservation. Six hours after transfection with piR-121380 mimic and inhibitor, the phosphorylation of ERK2 was significantly increased and decreased (p < 0.01), respectively. Furthermore, the highest and lowest total sperm motility, forward motility, and capacitation rate were observed after piR-121380 mimic and inhibitor treatments, respectively. The concentration of intracellular calcium ([Ca2+]i) showed no significant difference after transfection with either piR-121380 mimic or inhibitor at 1, 3, and 6 h. In conclusion, we demonstrated that piR-121380 modulates ERK2 phosphorylation by targeting PTPN7, which induces sperm cryo-capacitation, and eventually affects the motility and fertility potential of post-thawed sperm.Cryopreservation induces capacitation-like (cryo-capacitation) changes, similar to natural capacitation, and affects the fertility potential of post-thawed sperm. The molecular mechanism of sperm cryo-capacitation during cryopreservation remains unknown. PIWI-interacting RNAs (piRNAs) have been reported to be involved in cryo-capacitation of post-thawed sperm and regulation of sperm motility, capacitation, and chemotaxis. In this study, protein tyrosine phosphatase nonreceptor type 7 (PTPN7) was positively targeted by piR-121380 after a dual luciferase assay. The mRNA expression of PTPN7 and piR-121380 was significantly decreased ( Howeverrylation , similarrylation .Sperm undergoes physiological and biochemical changes and then acquires fertilizing ability after capacitation . ProteinThe level of protein tyrosine phosphorylation is strictly regulated by downstream protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), which links the cAMP pathway that affects protein tyrosine phosphorylation. Additionally, PTKs and PTPs are essential for regulation of human sperm acrosome reaction . SeligmaIt is well known that PIWI-interacting RNAs (piRNAs) are widely involved in physiological processes including gametes development, germ stem cell differentiation, embryonic development, and sex determination . Our preAll procedures starting from semen collection to treatments were implemented strictly considering the Regulation of the Administration of Affairs Concerning Experimental Animals  approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, China, under permit No: 2019202012.n = 10) were collected by glove-handed technique from healthy and mature Yorkshire boars. Immediately, the fresh semen was diluted with semen extender  in the ratio of 1:3. After stirring the mixture, the semen was placed in a temperature-controlled container (17°C) and transported to the laboratory. All the fresh ejaculates were confirmed to exhibit normal morphology, more than 80% viability, 90% motility, and over 1 × 108 ml−1 density for subsequent experiments.Fresh ejaculates  for 1, 3, and 6 h at 37°C. The capacitation group was incubated with 40 ng/ml heparin as described previously  The first aliquot was directly frozen in a liquid nitrogen (LN) tank and then stored at −80°C for RNA and protein extraction; (2) the second aliquot was cryopreserved as described in the next section; (3) the third aliquot was divided into two groups: (1) negative control group (NC) and (2) capacitation group (capa-). The negative control group was centrifuged  and then incubated in BTS medium  with 20 nM of piR-121380 mimic, inhibitor, mimic NC, inhibitor NC, and negative control (NC) ; pulse conditions were adjusted as 4 × 300 V for 100 µs. The mixture was incubated at 37°C within 12 h, and after 12 h at 17°C after transfection. The relative expression of piR-121380 and PTPN7 was assessed at 17°C from 12 to 60 h. Western blot was performed at 1, 3, and 6 h after transfection. Similarly, capacitation rate and intracellular calcium were determined at 1, 3, and 6 h after transfection (37°C).Electro-transfection was performed based on a previously standardized method . Initial−∆∆CT method  as described previously . RNA witT method . InternaT method .Potential target genes of key piRNAs were predicted by miRanda , and the5 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a humid CO2 incubator (5% CO2). Transfection was performed until cell confluency was above 70%. The transfection groups were prepared as follows: pWT-PTPN7-mimic, pWT-PTPN7-mimic NC, pMT-PTPN7-mimic, and pMT-PTPN7-mimic NC, which were individually co-transfected into Hela cells using FuGENE HD Transfection Reagent . After 6 h, luciferase activity was measured with the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. The final luciferase activity was normalized with Renilla luciferase activity.Hela cells were cultured in 24-well plates at 10Fresh ejaculates, after transfection, were subjected to 12–60 h of incubation under liquid preservation conditions. Prior to measuring sperm motility, the sperm counting chamber  was pre-heated on 37°C hot-stage and the CASA detection system  was debugged until the camera image was clear . The folAfter transfection for 1, 3, and 6 h, sperm was washed and then fixed with paraformaldehyde for 10 min. Peanut agglutinin (PNA) was used to measure the acrosomal status according to the method used by 2+]i was determined by the following equation: ΔF/F0 (%) = (F − F0)/F0 × 100%. F indicates the fluorescence intensity of each group, and F0 indicates the fluorescence intensity of sperm suspension without treatment.The concentration of intracellular calcium was measured with Fluo 4, AM . After transfection and incubation with 40 ng/ml heparin, sperm were washed once and re-suspended in HBSS buffer with 4 µM Fluo 4, AM . The sperm suspension was incubated at 37°C for 30 min and then washed three times. After that, another incubation was performed at 37°C for 20 min to ensure complete de-esterification of AM groups in sperm. The fluorescence of sperm was detected using a microplate reader  at 494 nm excitation/516 nm emission. The i. Since the change of calcium ions is fast, we detected the intracellular concentration of Ca2+ at 0, 1, 3 and 6 h after transfection (p < 0.01) when there was no extracellular Ca2+  in the mimic group as compared to the mimic NC group at 6 h (p < 0.01) in the inhibitor group as compared to the inhibitor NC group at 6 h. Similarly, the protein level of PTPN7 was significantly increased (p < 0.01) in frozen–thawed sperm, which showed no significant difference between the NC group and capacitation group (p < 0.01) in frozen–thawed and capacitation groups (p < 0.01) and decreased (p < 0.01) in the mimic group and inhibitor group at 6 h, respectively. The protein bands of phosphorylated ERK1/2 and PTPN7 in transfected sperm at 0 and 3 h are shown in To observe the effect of piR-121380 on ERK1/2 phosphorylation, and the difference in pattern of PTPN7 and ERK1/2 phosphorylation between cryopreservation and capacitation, we detected the protein levels of PTPN7, ERK1/2, and phosphorylated ERK1/2 in transfected, frozen–thawed, and capacitated sperm. Results showed that the protein level of PTPN7 was significantly decreased , and non-coding RNAs . Althougp < 0.01) in frozen–thawed sperm, but no differences were observed between fresh and capacitated sperm , all of which are involved in sperm motility and capacitation . In thised sperm , implyinectively , consistectively . Normallectively . In thisNotably, after transfection with piR-121380 mimic, the decreased mRNA expression of PTPN7 was inconsistent with the amount of PTPN7 protein, which was similar with the changes during cryopreservation , 7B. Com2+ is one of the decisive events to regulate sperm motility, acrosomal reaction, and chemotaxis /cAMP/PKA (p < 0.01) in the absence of extracellular Ca2+ in the culture medium. Therefore, we believe that piR-121380, downstream of calcium influx, regulates PTPN7 to affect sperm motility.Phosphorylation of ERK1/2 has been demonstrated to be tightly coupled with intracellular calcium concentration . Influx emotaxis . The infcAMP/PKA . In thisIn addition to the roles in sperm motility, piR-121380 also plays an important role in acrosomal reaction in sperm. In our study, increased and decreased capacitation rates were observed in mimic and inhibitor groups, respectively. Defective capacitation and failure to bind and penetrate the zona pellucida have been reported in pachytene piRNA mutant sperm . Choi etvia targeting PTPN7. We explored the role of piRNA in the process of sperm cryo-capacitation, which helps to further understand the molecular mechanism of cryo-capacitation and improve the quality of frozen–thawed boar sperm.In this study, we demonstrated that the mRNA expression of piR-121380 was significantly decreased in post-thawed boar sperm. Further studies showed that piR-121380 regulates boar sperm motility and is involved in sperm cryo-capacitation through phosphorylation of ERK2


The highest growth parameters  were observed at the 20/15 °C growth conditions in both types of grass. Conversely, the lowest growth parameters were observed at 10/5 °C. However, the low growth temperature of 10/5 °C resulted in increased levels of bioactive compounds , antioxidant activities ), and antioxidant enzymes  in both types of grass. Therefore, proper temperature growth conditions of wheat and barley grasses may be a convenient and efficient method to increase bioactive compounds and antioxidant potential in our diet to exploit the related health benefits.Wheat and barley grasses are freshly sprouted leaves of wheat and barley seeds, and are rich sources of phytochemicals. This study was conducted to investigate the effects of day and night temperatures on the growth, bioactive compounds, and antioxidant potential of wheat and barley grasses. Briefly, each grass was cropped in an organic growing medium at 10/5 °C, 20/15 °C, and 30/25 °C (day/night temperature) in a growth chamber by maintaining specific light (12/12 h light/dark; light intensity 150 µmol photons m Germination and plant growth are generally influenced by various growth factors, including temperature, time, duration, humidity, light intensity, light wavelength, darkness, carbon dioxide, treatment, ozone, and water supply ,2,3. TheMeanwhile, in a field, sprout growth mainly depends on the seed’s moisture, soaking, priming, soil moisture content, atmospheric temperature, rainfall, frost, field environment, sowing date, fertilizer, treatment, and water supply ,8. In a Triticum aestivum L.) and barley (Hordeum vulgare L.) are major cereal crops that are consumed worldwide. Various researchers have investigated barley and wheat seeds under different growth conditions. In particular, different soil and atmospheric temperatures (10–20 °C) have been tested to grow barley [Wheat  and barley  seeds collected from the National Institute of Crop Science, Korea were used to conduct this experiment. Seeds weighing 50 g were soaked in distilled water (dH2O) (seed: dH2O ratio of 1:1) for 24 h in a plant growth chamber at 20 °C. Five replicates were conducted for each treatment. Then, the seeds were relocated to the organic growing system in the plant growth chamber . The organic growing system contained 35% coco peat, 25% granite soil, 25% mushroom culture, 7.4% perlite, 5% zeolite, 2.5% vermiculite, and 0.1% guano.Wheat (−2 s−1 with 10 W quantum dot light-emitting diodes), at a relative humidity of 60%. After 8 d (13 d after sowing), the 6–15 cm height young grass leaves were collected to measure the bioactive compounds and antioxidant capacities. The enzymes and analysis reagents have been acquired from Sigma-Aldrich, Korea.The temperature was maintained at 20/15 °C (day/night) during the germination period (5 d). Then, during the young grass growth period, the growth chamber temperatures (day/night) were adjusted for low (10/5 °C), medium (20/15 °C), and high (30/25 °C) with light  for 24 h. The absorbance of the supernatant was measured with an ultraviolet-visible spectrophotometer  at 647 nm for chlorophyll b and 664 nm for chlorophyll a, as described by Moran [The chlorophyll contents of the fresh young wheat and barley grasses (0.10 g) were examined using 5 mL of by Moran , using aw/v, grass: dH2O) at 20 °C. Approximately 10 g (1 cm length) of each raw grass (separately) was ground with a pestle in 40 mL of dH2O. The filtered liquid samples were stored at 4 °C until further analysis.First, the young wheat and barley grasses were extracted  were then mixed with 5 mL of acetone, and the mixtures were incubated under dark conditions at 4 °C for 24 h. The absorbance of the final supernatant was measured using a spectrophotometer at 510 nm.The wheat and barley grass extracts (1 mL) were each mixed with 1 N Folin–Ciocalteu reagent (0.4 mL) and 7% sodium carbonate (2 mL) , before 2O (2.8 mL), and were then incubated and vortexed for 45 min at 20 °C [2O was used in lieu of 0.1 mL of aluminum chloride.The wheat and barley grass extracts (2 mL) were mixed with 10% aluminum chloride (0.1 mL), 1 M potassium acetate (0.1 mL), and dHat 20 °C  to measuw/v) metaphosphoric acid (5 mL) for 30 min at 4 °C, and the absorbances were measured at 525 nm.The wheat and barley grass extracts (2 mL) were each vortexed with 5% (2O2 solution (12.3 mM) were added and mixed. The absorbance was then measured at 436 nm. The guaiacol dehydrogenation product (GDHP) formation was measured as a guaiacol peroxidase activity (POD) (GDHP unit/min/mL), in which one unit of enzyme activity is expressed as the amount of enzyme required to oxidize 1.0 µM of GDHP/min/mL:The guaiacol peroxidase activity was measured as described by Putter , with a −1cm−1.Unit activity (unit/min/mL) = /(extraction coefficient × volume of sample taken in mL), where the extraction coefficient is 25 mM2O2 solution (150 mM), and 500 µL of enzyme extract were added to a test tube and mixed. The absorbance of the supernatant was measured at 240 nm. Note that one unit of enzyme activity is expressed as the amount of enzyme required to oxidize 1.0 µM of H2O2/min/mL:The catalase activity (CAT) was measured as described by Aebi , with a −3 mM−1cm−1.Unit activity (unit/min/mL) = /(extraction coefficient × volume of sample taken in mL), where the extraction coefficient is 6.93 × 10The glutathione reductase (GR) was measured according to the method described by Carlberg and Mannervik , with a −1cm−1.Unit activity (units/min/mL) = /(extraction coefficient × volume of sample taken in mL), where the extraction coefficient is 6.22 mM2O (0.2 mL) was used in place of the leaf extract.The wheat and barley grass extracts (0.2 mL) were each mixed with 50 mM Tris-HCl buffer (pH 8.5) (3.0 mL) and pyrogallol (0.2 mL), incubated, and shaken for 30 min under dark conditions for 10 min at 20 °C. Then, the mixtures were reacted with 1 N HCl (1 mL) to terminate the reaction. The absorbances of the supernatants were measured at 420 nm. For the control, dH50) equivalent ABTS capacity (µg/mL) was measured, as per the method described by Ozgen et al. [+•)) solution was prepared (7 mM ABTS in water) with potassium persulfate (2.45 mM), and incubated at 20 °C for 12–18 h under dark conditions to achieve a stable oxidative state. Then, the ABTS solution (3 mL) was mixed with the standard (200 µL) or test extract (200 µL), and stored in the dark for 2 h at 20 °C before measuring the absorbance of the supernatant at 734 nm. The absorbance of the ABTS+• control was also measured. α-Tocopherol was used as a positive control.The half-maximal inhibitory concentration  equivalent capacity (µg/mL) of DPPH was calculated. α-Tocopherol was used as a reference.The wheat and barley grass extracts 1.8 mL) were mixed with 0.4 mM methanol containing 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals (1.8 mL), vigorously shaken, and incubated for 10 min under dark conditions before measuring the absorbance at 525 nm [ mL were Using SPSS V.25 , a one-way analysis of variance (ANOVA) followed by a Tukey test were conducted for the statistical analyses. In addition, the Pearson’s correlation was performed using SPSS software.The growth parameters of the wheat and barley grasses grown at different temperatures are listed in The chlorophyll and carotenoid contents of the wheat and barley grass extracts are shown in Compared with chlorophyll, the carotenoid exhibited the opposite result in both the wheat and barley grasses. At a growth temperature of 10/5 °C, both grass extracts had the highest carotenoid levels (yellow to orange color). A good carotenoid level is desirable because it can contribute to scavenging reactive oxygen species (ROS) , which mThe bioactive compounds in the wheat and barley grasses grown at different temperatures are listed in de novo synthesis [Meanwhile, the vitamin C content probably increased in the germinated wheat and barley as a result of ynthesis ,10, and,ynthesis . At 20/1The antioxidant enzyme contents in the wheat and barley grasses grown at different temperatures are listed in p ≤ 0.01) and barley  grass extracts, and the antioxidant activity may increase because of the increase in phenolic content in the extracts. Further, wheat grass showed higher levels of antioxidant activity than barley grass in the ABTS and DPPH assays, which may be due to crop variation.The antioxidant activities of the wheat and barley grass extracts are summarized in This study was conducted to investigate the effects of day and night growth temperatures  in the organic growing medium on the bioactive compound concentrations, antioxidant enzyme concentrations, and antioxidant activities of wheat and barley grass extracts. Both the low and high growth temperatures caused some level of damage in the wheat and barley grass growth parameters, but triggered some defense mechanisms by increasing the amount of bioactive compounds, antioxidant enzymes, and antioxidant activity. Based on these findings, the proper control of day and night growth temperatures when producing wheat and barley grasses may be a convenient and efficient way to improve the bioactive compound contents and related health benefits.


Methylobacteriaceae. The abundance of Methylobacteriaceae was low in the roots of field-grown soybean without symptoms of nitrogen deficiency. Although Methylobacteriaceae isolated from soybean roots under nitrogen deficiency did not promote growth or nodulation when inoculated into soybean roots, these results indicate that the enrichment of Methylobacteriaceae in soybean roots is triggered by nitrogen-deficiency stress.Nitrogen deficiency affects soybean growth and physiology, such as symbiosis with rhizobia; however, its effects on the bacterial composition of the soybean root microbiota remain unclear. A bacterial community analysis by 16S rRNA gene amplicon sequencing showed nitrogen deficiency-induced bacterial community shifts in soybean roots with the marked enrichment of  NRT1.1B, which encodes a rice nitrogen transporter and sensor, is a key gene that affects the community composition of the microbiota of field-grown rice  is one of the most important legume crops and establishes a symbiotic relationship with certain rhizobia, which are predominantly influenced by the supply of nitrogen. A heavy nitrogen supply suppresses nodule formation  photoperiod and fertilized weekly with 50 mL 1/2 nitrogen-free medium   under nitrogen-sufficient and -deficient conditions. Soybean seeds were surface-sterilized with 70% ethanol and 1% sodium hypochlorite (NaClO) and germinated on sterile paper. After 5 days, seedlings were transferred to plastic pots (7.0 cm diameter×9.0 cm height) filled with vermiculite. Approximately 3.5 g soil, collected from a soybean field of gray lowland soil without pesticide application at the Kyoto University of Advanced Science, Kameoka, Kyoto, Japan , was placed on the surface of vermiculite as a bacterial inoculation source (hereafter designated as ‘soil’). Soil chemical properties were analyzed at the Tokachi Federation of Agricultural Cooperatives: pH of 7.1, 0.27% total N, 3.4% total C, 5.8% humin, 12.3 ppm NOe medium  supplemectively) A. Plant ctively) . HarvestBacterial community profiling was performed by 16S rRNA gene amplicon sequencing, as previously described . Detailed methods for the isolation, identification, phylogenetic analysis, and whole-genome sequencing of bacterial isolates are available in Supplemental Methods.Root-inhabiting bacterial strains were isolated from soybean roots grown under nitrogen-deficient conditions, corresponding to the roots subjected to community profiling experiments B. Indivi7 for a single inoculation and 2×107 CFU mL–1 for a dual inoculation. A one-milliliter bacterial suspension was inoculated into plants  in a greenhouse set at a minimum of 20°C. After harvesting, the fresh weight of shoots and leaf chlorophyll contents were measured  filled with sterilized vermiculite and watered with 1/2 nitrogen-free media. Each bacterial strain was cultured in growth media  until sto plants C. The coon 4.0.1 .P=2.5×10–2 and 2.6×10–2, respectively; Proteobacteria, includingenrichment of the bacterial phylum  bacteria orders Betaproteobacteriales, Rhizobiales, and Sphingomonadales, was significantly greater in soybean roots than in soil  and Methylorubrum extorquens (classified into Group II), respectively  and Methylorubrum (Group I), respectively  Nitrogen Deficiency-induced Bacterial Community Shifts in Soybean Roots. https://doi.org/10.1264/jsme2.ME21004Supplementary Material 1Supplementary Material 2


Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of  Therefore, it is critical to have stringent quality control mechanisms in place to sequester and dispose of damaged mitochondria.Mitochondrial quality control (MitoQC), consisting of mitochondrial dynamics and mitophagy, has been a major area of focus in the context of many disease conditions including cerebral ischemia/reperfusion (I/R) injury. Mitochondrial damage and subsequent dysfunction have been well characterized as precursors to cell death following I/R9. Moreover, these stressors, and the accompanying increase in mitochondrial fission, are also reportedly associated with an upregulation in mitophagy11. However, despite the importance of these insights, two major gaps in knowledge remain. First, it is unknown whether fission is necessary for the upregulation of mitophagy and subsequent disposal of damaged mitochondria following I/R injury. Second, there is no agreement on whether the upregulation of mitophagy observed in the setting of I/R is beneficial or detrimental to neuronal viability17. Indeed, past studies underscore a complex and seemingly disparate relationship between mitochondrial dynamics and mitophagy, i.e., inhibiting fission during ischemia may attenuate cell death19, or, conversely, may worsen outcomes by compromising the clearance of damaged mitochondria that accumulate during reperfusion. To further complicate the roles and relationships between fission and dynamics in the context of I/R, mitophagy is a dynamic process, and it has been technically challenging to reliably quantify overall mitophagic flux in an unbiased manner17.Multiple stressors  have been demonstrated to initiate a transition to a fragmented mitochondrial phenotype, and there is a consensus that stress-induced activation of mitochondrial fission is directly linked to cell death21. For example, previous studies have demonstrated that overexpression of Drp1 in HeLa cells resulted in a 70% decrease in mitochondrial mass10, suggesting a direct connection between induction of fission, activation of mitophagy, and degradation of mitochondria. Further supporting this connection between fission and mitophagy, Endophilin B1, a Drp1-dependent mediator of fission, was found to colocalize with autophagic markers LC3, Atg5, and Atg9 in response to nutrient starvation20. Finally, overexpression of Parkin in N2a neuroblastoma cells purportedly protects against I/R injury through ubiquitination and proteasomal degradation of Drp121. These connections suggest a potential mechanism where Drp1-dependent fission would aid in recovery from I/R injury by: (i) inducing fragmentation of damaged/dysfunctional mitochondrial segments, and (ii) facilitating mitophagic degradation.Dynamin-related protein 1 (Drp1) is considered the “master regulator” of mitochondrial fission. However, the role of Drp1 may extend beyond the regulation of fission per se; Drp1 has also been implicated as a component of the mitophagic process, responsible for sequestering damaged portions of the mitochondrial network for recycling through mitophagy/biogenesis22, with and without conditional knockout (KO) of Drp1, to quantify changes in: (i) mitochondrial dynamics, and (ii) mitophagic flux  in an in vitro model of cerebral I/R injury. Specifically, we hypothesized that simulated I/R  followed by reoxygenation (OGD/R)) would induce mitochondrial fragmentation and activation of PINK1/Parkin-mediated mitophagy. Moreover, we posited that Drp1KO would prevent mitochondrial fragmentation and swelling and limit mitophagy.To provide a robust test of this as-yet unproven concept, we utilized MitoQC reporter mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J)16. To establish whether mitochondria are undergoing active degradation during I/R, we utilized primary cortical neurons isolated from MitoQC mice22. Neurons were subjected to OGD/R and analyzed for the accumulation of mCherry fluorescence in the absence of mitochondrial GFP fluorescence (mCherry puncta) as a marker for mitochondria undergoing mitophagy  neurons were immunolabeled with PINK1 or Parkin (red) and co-labeled for the β-subunit of ATP synthase (ATPB—green) as a marker of mitochondria Fig. . ColocalIn support of the concept that OGD/R-induced mitophagy is mediated through the PINK1/Parkin pathway, western blot analysis revealed an increase of PINK1 and Parkin in mitochondrial fractions Fig. . The pre39. To investigate this possibility, Parkin activity was analyzed by ubiquitination of mitochondrial proteins via western blot  identification of dysfunctional mitochondria, (ii) sequestration through either the phagophore or early endosomes, respectively, and (iii) degradation of mitochondria via fusion of the autophagosome or late endosome with the lysosomeion Fig. . Followiion Fig. , while c43. The classification system was based on four distinct mitochondrial morphologies: networks, unbranched, swollen, and punctate. Because mitochondrial dysfunction, characterized by mitochondrial fragmentation and mitochondrial swelling, is well accepted as the driving force of neuronal death following IR injury46, we investigated whether swollen mitochondria were preferentially cleared following OGD/R. Therefore, we developed a model to classify distinct mitochondrial morphologies in our MitoQC mice  their qualitative/semi-quantitative measurement of mitochondrial dynamics; and (ii) the use of pharmacological agents that are recognized to have off-target effects47. Accordingly, we utilized our machine learning classification system as well as Lentiviral-Cre KO of Drp1fl/fl/QCKi/+ primary cortical neurons to investigate the mechanistic role of Drp1KO in mitochondrial dynamics and cell death during OGD/R  and LV-EF1a-empty . Morphological classification in lentiviral-control neurons revealed significant reductions in networks and unbranched mitochondrial objects in cells subjected to OGD/R vs control Fig. . Drp1KO ria Fig. , and a sD/R Fig. . Althougrol Fig. . This suons Fig. .Drp1KO on the total number and area occupied by punctate and swollen mitochondrial morphologies. Interestingly, there was no increase in the number of punctate objects in lentiviral-control neurons in response to OGD/R; however, there was a significant increase in the total area of mitochondria in a punctate state—an increase that was attenuated with Drp1KO  with no difference between control and Drp1KO groups  underwent mitophagy, which may have contributed to their accumulation during reoxygenation. It is unclear why swollen mitochondria evade elimination; however, their larger size relative to punctate mitochondria may limit their inclusion in autophagosomes. Another possible explanation is that the increase in mitochondrial membrane permeability, the underlying cause of swelling, may disrupt identification by the PINK/Parkin pathway and endosomal sorting complexes required for transport.Consistent with the evidence in the field, we demonstrate that mitophagy and fission are increased in neurons during reoxygenation. As expected, punctate mitochondria were the preferred morphology for mitophagy. Previous studies visualizing mitochondria inside autophagosomes by confocal and electron microscopy demonstrated that these sequestered mitochondria are generally small (<1 μm in diameter)61. Moreover, lysosomes are ~1.2 μm in diameter62, suggesting that mCherry objects present in lysosomes represent individual mitochondria.The MitoQC dual-fluorophore that provides the foundation for our analysis is designed to allow identification of mitochondria inside lysosomes by neutralizing pH-sensitive GFP. A potential limitation of this technique is that it is difficult to distinguish with certainty whether the mCherry puncta within lysosomes are, as we propose, mitochondria with mCherry-only fluorescence, or whether the lysosomes are filled with mCherry that has been freed from degraded mitochondria. However, our estimations of the size of punctate mitochondria as determined by mCherry fluorescence are comparable to those reported in previous studies64. However, recent studies have challenged the causal role of fission and Drp1 in cell death47. Findings in support of this paradigm have often utilized mdivi1, a chemical inhibitor of Drp1 translocation, and interpretation of these data may be confounded by evidence that mdivi1 is not selective for Drp1. Rather, mdivi1 purportedly acts as an inhibitor of NADH dehydrogenase and indirectly suppresses fission by inhibiting complex I-mediated ROS generation47.The large dynamin-related GTPase, Drp1, is the primary regulator of fission and has been implicated as a driver of I/R-induced fission and cell death65. This may be explained by a possible role of Drp1 in mitochondrial outer membrane permeabilization through the association and recruitment of pro-apoptotic proteins such as Bax and Bak—a process that triggers mitochondrial swelling, the release of cytochrome c, and cell death69.Our current study, using a genetic (rather than pharmacologic) strategy to modify Drp1, provides robust mechanistic support for the concept that increased fission and cell death following OGD is, indeed, Drp1 dependent. Furthermore, we report that Drp1 expression plays a causal role in mitochondrial swelling observed during reoxygenation—results that are consistent with those of a previous study, showing that Drp1 KO in muscle cells caused a swollen mitochondrial phenotypeDrp1 KO did not alter the morphology of the mitochondrial network under baseline conditions . These data suggest that, under normoxic conditions, the mitochondrial network at 7 days following conditional KO of Drp1 is in a state of equilibrium, and the consequences of Drp1 KO only manifest after exposure to the stress of OGD/R.Interestingly, in contrast to previous studies, 70, while fusion and elongation rescues mitochondria from mitophagy48. Moreover, on a molecular level, fission is associated with mitochondrial depolarization10 , while Parkin rapidly ubiquitinates Mitofusins 1 and 2, preventing fusion and thus promoting mitophagy.The systems that regulate mitochondrial maintenance are likely integrated. On a physical level, only fragmented mitochondria will fit inside autophagosomes15, the concept that Drp1 plays a direct mechanistic role in regulating mitophagy is controversial71. Evidence obtained in yeast72 and mammalian cells10 suggest that Drp1  is required for mitophagy while, in contrast, others have reported that Drp1 KO had no effect on mitophagy73 or hypoxia-induced upregulation of mitophagy74, but may protect mitochondria from PINK/Parkin-mediated “wholesale mitophagy”11. Our results, showing no effect of Drp1 KO on induction of mitophagy or mitophagic flux, are consistent with these latter observations and argue against a direct role of Drp1 as a molecular mediator of mitophagy in our neuronal cell model.Despite evidence that fission regulates mitophagyDrp1 KO, we make the novel observation that changes in mitophagy are independent of Drp1. Our results are consistent with the paradigm that dysregulation of mitochondrial maintenance drives neurological I/R injury and suggests that mitophagy serves as a protective mechanism to eliminate dysfunctional mitochondria in this disease process.In this study, we provide evidence that mitochondrial fragmentation, mitochondrial swelling, and mitophagy are upregulated in primary neuronal cells subjected to OGD/R. While fragmentation and swelling are attenuated by 22. Drp1 floxed (Drp1fl/fl) mice (Dnm1ltm1.1Hise) were provided by Dr Hiromi Sesaki, Johns Hopkins, Baltimore, MD75. WT mice (B57BL/6Crl) were purchased from Charles River Laboratories.All experimental procedures were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee under protocol #IACUC-18-08-0767 at Wayne State University and under protocol #PRO00009531 at the University of Michigan. MitoQC reporter mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) were provided by Dr Ian Ganley, University of Dundee, Scotland, UK−4 N NaOH , 10 ng/ml APV , 50 µl of Papain ) and incubated at 37 °C for 30 min. After 30 min, the tissue was washed with Dulbecco’s phosphate buffered saline twice and replaced with Hibernate complete medium, and the tissue was dissociated by pipetting up and down 25 times to generate a cell suspension. The suspension was passed through a 40-micron filter to separate tissue debris. Cells were seeded at 190,000 cells/cm2 and incubated for 30 min at 37 °C in 5% CO2. After 30 min, media was replaced with neurobasal complete medium , 1% B27 plus , 0.5 mM Glutamax Supplement , and 1% Penn/Strep ) and cultured at 37 °C in 5% CO2. Half-media changes occurred every 3–4 days with neurobasal complete medium. Cultures were randomly assigned for lentiviral treatment as well as different time points of OGD/R.Brains were isolated on postnatal day 0–2 (P0–P2), and cortical neurons were seeded separately for individually biological replicates. Tissue was digested with enzyme digestion solution , 1.4 × 102 Control InVitro Glove Box (Coy Lab Products). The hypoxic chamber was maintained at 0.1% O2 and 5% CO2. OGD media, composed of 0.20 g/l CaCl2 , 0.4 g/l KCl , 0.097 g/l MgSO4 , 6.8 g/l NaCl , 2.2 NaHCO3 , 0.14 g/l NaH2PO4-H2O , was bubbled with 95% N2/5% CO2 inside of the of the hypoxic chamber for 60 min. Cells were transferred into the hypoxic chamber, washed 2× with OGD media, and then incubated with OGD media inside the hypoxic chamber for 150 min. After OGD, cells were removed from the hypoxic chamber, media was replaced with Neurobasal medium without antioxidants ), and incubated at 37 °C in 5% CO2 for 6 h.OGD was achieved utilizing an OCells were infected with lentivirus on DIV 7. Experiments were performed 7 days post transduction (DIV 14). Lentiviral plasmids and lentivirus were generated by the vector core at the University of Michigan : Lenti-EF1a-Cre-VSVG, Lenti-EF1a-VSVG, and Lenti-EF1a-GFP-VSVG. The EF1-alpha promoter, possessing a broad host range, demonstrated robust, constitutive, and long-term expression of both Cre-recombinase and GFP in our primary cortical neuron cultures. Vesicular stomatitis Indiana virus G-protein (VSVG) envelope was used to enhance viral entry. Viruses were made in a concentrated 10× form in Neurobasal medium. Titration curves were performed with Lenti-EF1a-VSVG and Lenti-EF1a-GFP-VSVG to evaluate toxicity and transduction efficiency, respectively. Toxicity was tested at 0.5×, 1×, 2×, 4×, and 5× concentrations. Transduction efficiency was tested at 0.5× and 1× concentrations.Viability was assessed after 2.5 h of OGD followed by 6 h of reoxygenation using Thiazolyl Blue Tetrazolium Bromide (MTT) assay. Following the 2 h incubation with MTT, media was removed and replaced with MTT solubilization solution  in isopropanol ) and placed on a shaker at room temperature for 15 min. Then, 100 µl of each sample was then pipetted into a 96-well plate and analyzed for absorbance at 570 nm utilizing a PerkinElmer Multimode Plate Reader containing Enspire interface software.TM protease and phosphatase inhibitor cocktail ) and homogenized utilizing a Teflon Potter-Elvehjem homogenizer (10 strokes at 300 rpm) . Cell homogenates were centrifuged at 1000 × g for 10 min at 4 °C. The supernatant (containing mitochondria and cytosol) was centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected as the cytosolic fraction. Mitochondrial pellets were washed once and resuspended with fresh homogenization-A buffer and sonicated. Whole cell pellets were saved and sonicated to be used as whole cell lysate samples.To obtain mitochondria, primary cortical neurons were collected in homogenization-A buffer (10 mM HEPES (pH7.5) , 1 mM EDTA , 1 mM EGTA , 100 mM KCl , 210 mM mannitol , 70 mM sucrose , and 1× HaltDynamin-like protein 1 (Dlp1/Drp1) (Clone 8) mouse antibody , mono- and polyubiquitinylated conjugated mouse monoclonal antibody clone FK2 , PINK1 rabbit polyclonal antibody , Parkin (Prk8) mouse monoclonal antibody , Rab5 (C8B1) rabbit monoclonal antibody , VDAC (D73D12) rabbit monoclonal antibody , GAPDH (14C10) rabbit monoclonal antibody , and LC3 A/B rabbit antibody .Cells were scraped in homogenization-A buffer (with MG132 proteasome inhibitor when probing for ubiquitination), sonicated, and the protein concentration was measured using the Bradford Plus Assay Reagent . Polyacrylamide gels , 375 mM Tris pH 8.8 , 0.1% sodium dodecyl sulfate , 0.1% ammonium persulfate , and 0.1% TEMED ) were loaded with 10 μg of whole cell lysates and 5 μg of mitochondrial samples and transferred to nitrocellulose membranes. Membranes were incubated with indicated primary antibodies at 4 °C overnight. Membranes were then washed with Tris-buffered saline and 0.1% Tween  and incubated in secondary antibodies for 60 min at room temperature. Membranes were washed 3× with TBST, for 5 min. The membranes were then incubated in SuperSignal West Pico Plus Chemiluminescent Substrate , imaged utilizing a BioRad ChemiDoc XRS+ imager and quantified by densitometry using ImageJ software.MAP2 chicken polyclonal antibody , Parkin rabbit polyclonal antibody , PINK1 rabbit polyclonal antibody , Lamp1 (1D4B) rat antibody , and PINK1 rabbit polyclonal antibody . Secondary antibodies were donkey anti-mouse or donkey anti-rabbit HRP , Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 633 goat anti-rat (Life Technologies).9. Briefly, cells were fixed with 3.7% PFA  in 200 mM HEPES  for 15 min at 37 °C. Coverslips were washed 3× with PBS and replaced with blocking solution  and 0.3% Triton-X100  in PBS) for 60 min. Blocking solution was replaced with primary antibodies (with 1% BSA and 0.3% Triton-X100 in PBS) at 4 °C overnight. After primary incubation, coverslips were washed 3× in PBS and incubated with indicated secondary antibodies for 60 min at room temperature, shielded from light. Coverslips were subsequently washed and mounted using Fluoroshield with DAPI .Proteins were visualized with immunofluorescence as previously described76. The following was performed using Fiji’s (ImageJ) batch processing feature in three separate phases: processing, segmentation, and red puncta analysis.For each coverslip containing MitoQC neurons, 12 random images were acquired utilizing a Zeiss Axio Observer.Z1 inverted microscope at 63× oil immersion. Each image was deconvolved using the regularized inverse filter method on Zeiss Zen Pro software. Images were further processed in Fiji ImageJ to segment and count mCherry-only punctaProcess > Subtract Background Process > Filters > Unsharp Mask 77Process > Enhance Local Contrast Process > Filters > Median (radius = 2.0 pixels)(1) Processing:(2) Segmentation:78. The program was trained to segment “mitophagy” particles (red-only), “healthy mitochondria” (green/yellow), and “background” (“black”). Once the classifier was trained, photos were segmented using the category “probabilities” as an output.Trainable Weka Segmentation was employed to segment red particles. Trainable Weka Segmentation is a plugin in ImageJ that uses a combination of visualization tools and machine learning algorithms to produce a pixel-based segmentationImage > Stacks > Stacks to ImageClose “Background” and “Healthy mitochondria” windowsSelect window titled “Mitophagy”31)Image > Adjust > Threshold (3) Red Puncta Analysis:Results of red puncta counts were tabulated into an excel sheet for eacg image and normalized to the number of cells analyzed.43. In brief, classification was performed in R computing language using the R Caret package79. Fiji ImageJ was used for image processing and mitochondrial object measurement collection76. R studio was used for all computation. The workflow was performed in two separate phases: Feature Extraction utilizing ImageJ and Random Forest Classification utilizing R studio.Machine learning-based classification of mitochondrial objects was performed as described in (1) Feature Extraction:Image > Type > 8-bitImage > Type > 16-bitImage > Type > 8-bitProcess > Binary > Make BinaryAnalyze > Set Scale (9.7 pixels/micron)Analyze > Measure (if mean is ≤ 0.75 then invert (Edit > Invert), otherwise continue)BioVoxxel_Toolbox (ImageJ plugin) > Extended particle analyzer .The green channel of the MitoQC images was used for mitochondrial morphology analysis. Processing was done in the same workflow as above. Trainable WEKA segmentation was employed to segment green mitochondrial signal using “Labels” as an output. Segmented images were then analyzed to obtain measurements of each mitochondrial object. Workflow is listed below:80. A total of 32 measurements were recorded for each object: area, perimeter, circularity, feret, FeretAngle, Min Feret, aspect ratio (AR), Round, Solidity, Feret AR, Compactness, Extent, CircToEllipse Tilt, AR_Box, AR_Feret, Round_Feret, Compact_Feret, Elongation, Thinnes Ratio, Angle_0-90, Feret_Angle_0-90, Convexity, Roundness corrected to AR, area equivalent diameter, perimeter equivalent diameter, spherical equivalent diameter, interfacial density, Hexagonal Side, Hexagonal Perimeter, Hexagonal Shape Factor, Hexagonal Shape Factor Ratio, and Hexagonality.Measurements were then expanded using “extended descriptors” macro from the Applied Superconductivity Center(2) Random Forest Classifier:n = 2342) containing the 32 measurements (predictors) listed above were imported into R Studio as the train/test set. These objects were labeled with one of four different classifications: networks (n = 236), unbranched (n = 851), swollen (n = 458), or punctate (n = 797). The data were then split based on their classifications into the training (80%) and the test (20%) sets using the createDataPatition function from the caret package79. A random forest algorithm (rf) from the caret package was trained using the training set through 25 iterations utilizing 32 predictors. The final model consisted of 500 trees with an mtry = 2, which refers to the number of randomly selected predictors considered at each split within the tree.Hand-classified mitochondrial objects (t-test was used to detect differences between two groups. Differences between conditions across time (Lenti-cre vs Lenti-empty) were analyzed using a two-way ANOVA with Dunnett’s post-hoc analysis for differences across time points compared to control. Multiple comparisons to detect differences between conditions were computed using Sidak’s post-hoc test. Differences were considered significant when p < 0.05.Statistical analysis was performed using GraphPad Prism. Principal component analysis was done in R using the built-in function prcomp. Data are presented as means ± SEM. All experiments were independently repeated and data were collected from four to eight biological replicates with two to three technical replicates for each experiment. Based on variability, power analysis was conducted to determine appropriate sample size. Differences between three or more groups were analyzed using one-way ANOVA with either Tukey post-hoc test  or Dunnett’s post-hoc test (for comparisons to control). Student’s Supplementary Material