DOI: 10.5061/dryad.6hdr7sr78
title: Data from: DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress

description: Environmental factors, infection, or injury can cause oxidative stress in
 diverse tissues and loss of tissue homeostasis. Effective stress response
 cascades, conserved from invertebrates to mammals, ensure reestablishment
 of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like
 cells, rapidly respond to oxidative stress by immune activation. However,
 the precise signals how they sense oxidative stress and integrate these
 signals to modulate and balance the response to oxidative stress in the
 adult fly are ill-defined. Furthermore, hemocyte diversification was not
 explored yet on oxidative stress. Here, we employed high throughput single
 nuclei RNA-sequencing to explore hemocytes and other cell types, such as
 fat body, during oxidative stress in the adult fly. We identified distinct
 cellular responder states in plasmatocytes, the Drosophila macrophages,
 associated with immune response and metabolic activation upon oxidative
 stress. We further define oxidative stress-induced DNA damage signaling as
 a key sensor and a rate-limiting step in immune-activated plasmatocytes
 controlling JNK-mediated release of the pro-inflammatory cytokine
 unpaired-3. We subsequently tested the role of this specific immune
 activated cell stage during oxidative stress and found that inhibition of
 DNA damage signaling in plasmatocytes, as well as JNK or upd3
 overactivation, result in a higher susceptibility to oxidative stress. Our
 findings uncover that a balanced composition and response of hemocyte
 subclusters is essential for the survival of adult Drosophila on oxidative
 stress by regulating systemic cytokine levels and cross-talk to other
 organs, such as the fat body, to control energy mobilization.
description: <em>Drosophila melanogaster stocks:</em>
 Flies were reared on a high yeast food containing 10% brewer’s
 yeast, 8% fructose, 2% polenta and 0.8% Agar. Propionic acid and nipagin
 were added to prevent bacterial or fungal growth. All crosses (except
 those containing the
 <em>tub-Gal80<sup>ts</sup></em> construct or
 otherwise noted) were performed at 25°C with a 12 hours dark/light cycle.
 The crosses with <em>tub-Gal80<sup>ts</sup></em>
 were performed at 18°C to ensure the inhibition of the Gal4-protein during
 developmental stages of the fly. The experimental male F1 flies were
 transferred to 29°C as soon as they hatched and were aged for six days.
 All transgenic lines used in this study are listed below:
 
 List of Drosophila lines used in this
 study Source 
 w<sup>1118</sup>;Hml∆ -Gal4,UAS-2xeGFP
 Gift from Marc S. Dionne 
 w<sup>1118</sup>;UAS-tefu-IR
 Vienna Drosophila Research Center 
 w<sup>1118</sup>;UAS-mei41-IR
 Vienna Drosophila Research Center 
 w<sup>1118</sup>;UAS-nbs-IR
 Vienna Drosophila Research Center 
 
 w<sup>1118</sup>;UAS-mei41-IR;UAS-tefu-IR
 made for this study 
 w<sup>1118</sup>;UAS-bsk-DN
 Vienna Drosophila Research Center 
 
 w<sup>1118</sup>;;HmlΔ-DsRed.nuc
 Gift from Katie J. Woodcock 
 w<sup>1118</sup>;UAS-upd3-IR
 Vienna Drosophila Research Center 
 w<sup>1118</sup>;Hml∆
 -Gal4,UAS-2xeGFP;tubGal80/tm6csb´ Gift from Katie J.
 Woodcock 
 w<sup>1118</sup>;UAS-hep[act]/CyO;
 Dr/tm6csb' Gift from Anne-Kathrin
 Classen 
 w<sup>1118</sup>;UAS-upd3/Sm6a
 Gift from Katie J. Woodcock 
 w[*] upd3[Δ] Bloomington Drosophila Stock
 Center w´upd3Δ;
 UAS-upd3/Sm6a made for this study
 
 w<sup>1118</sup>;tub-Gal80<sup>ts</sup>/Sm6a;crq-Gal4/TM6 c, Sb' Gift from Marc S. Dionne w1118;;UAS-rpr/TM6 c, Sb1 Bloomington Drosophila Stock Center w1118;UAS-CD8-mCherry Bloomington Drosophila Stock Center <em>Lifespan/Survival assays:</em> Male flies were collected after eclosion and groups of 20 age-matched flies per genotype were housed in a food vial. The survival experiments were performed at 29°C. The vials were placed horizontal to avoid that flies fall into the food and become stuck. Dead flies were counted in a daily manner. The flies were transferred into a fresh food vial twice per week without CO<sub>2</sub> anesthesia.  <em>Paraquat treatment:</em> Flies were maintained at 29°C for six days prior to treatment. On day six they were starved for 6 hours. A filter paper soaked in 5% sucrose solution with or without 15mM Paraquat (PQ, Methyl viologen hydrate, Acros Organics) was added after starvation. Flies were fed for 18 hours on the PQ containing food in groups of ten. The PQ treatment was performed at 29°C in the dark<sup>20</sup>. To assess the survival rate the dead flies per vial were counted and the percentage was calculated. The living flies were further analyzed in other assays used in this study. <em>Starvation experiments:</em> 10-20 age-matched male flies were kept in a vial containing 1% agar supplemented with 2% 1xPBS. The starvation experiments were performed at 25°C. Dead flies were counted every hour. Several individual experiments were performed and started on different daytimes to exclude diurnal derived artifacts. The data of the individual experiments were pooled and analyzed with GraphPad Prism 9.2.0. <em>Paraffin sections and stainings:</em> Anaesthetized flies were washed in 75% ethanol for 5-10 seconds and transferred into 4% PFA for 30 minutes at room temperature. The flies were washed for 1 hour in PBS on a shaker. Subsequently the flies were transferred into tissue cassettes, dehydrated and embedded in paraffin. The flies were cut in 7µm thick sections de-paraffinated and stained on slides with Hematoxylin-Gill (II) for five minutes. Subsequently the sections were blued in running water for ten minutes. It followed a 0.5% Eosin treatment for five minutes and a final ethanol treatment with increasing concentrations until 100%. The sections were transferred into Xylol and finally covered with synthetic resin and a cover slip. Cryo sections and Oil Red O staining: Anaesthetized flies were washed in 75% ethanol for 5-10 seconds and transferred into 4% PFA for 30 minutes at room temperature. Subsequently, the flies were dehydrated for 1h at 37°C in 30% sucrose solution. The dehydrated flies were placed in a Tissue-Tek cryo-mold and embedded in Tissue-Tek® O.C.T.™ Compound. The cryo-mold was placed on dry-ice which was embedded in a 100% ethanol bath until the whole block was completely frozen. The flies were cut in 10µm sections and stained on the slide. The slides were put on RT and dried. Subsequently, they were submerged in Oil red O solution for 30 minutes. The slides were rinsed two times in deionized water and subsequently stained for two minutes with hematoxylin to stain the nuclei. Finally, the slides were washed with water for 5 minutes and covered with a cover slip and glycerin. <em>Semi-quantitative Real-Time PCR:</em> Three flies were pooled and smashed in 100µl TRIzol to stabilize and isolate the RNA. Chloroform was used to extract the RNA followed by an isopropanol precipitation step. The RNA was cleaned with 70% ethanol and solubilized in water. A DNAse treatment was performed to digest potential genomic DNA contaminations. The purified and DNAse treated RNA was written into cDNA using RevertAid Reverse Transcriptase (Thermo Scientific) at 37°C for one hour. The reaction was stopped by incubating for 10 minutes at 70°C. The subsequent RTqPCR was done in SensiMix SYBR Green no-ROX (Meridian Bioscience) and was performed on a LightCycler 480 (Roche). The qPCR cycling started with a 10-minute 95°C step followed by 50 cycles with the following times and temperatures: 15s at 95°C, 15s at 60°C and 15s at 72°C. The gene expression levels were normalized to the value of the measured loading control gene Rpl1. <em>Confocal microscopy:</em> The flies were anesthetized with CO2, glued on a cover slip and imaged immediately. Confocal microscopy was done with a Leica SP8 microscope (Leica) and a 10x/NA0.4 Leica air objective. The images were acquired in a resolution of 512x512 with a scan speed of 600 Hz. Z-Stacks with a step size of 5µm and tile scans (2x2 or 2x3 images per fly) were acquired to quantify the hemocytes in whole flies. The tiled images were merged with the LAS-X software (Leica) and maximum projections were created using Fiji/ImageJ. The hemocytes were counted in the maximum projection images of whole flies. <em>Smurf Assay:</em> Smurf assays were performed to check the feeding behavior as well as the gut integrity of 7 days old, male flies upon PQ treatment. Brilliant Blue dye (FCF) was added into 5% sucrose solution in a concentration of 1% (w/v). PQ was added in concentrations of 2mM, 15mM and 30mM. Control food was 5% sucrose and 1% Brilliant Blue without PQ. The flies were fed with blue food according to the PQ-treatment protocol, as described above. Flies were analyzed after 18h of feeding on brilliant-blue food. Three kinds of flies were distinguished. Flies that did not eat at all were excluded. Flies with a blue gut or crop were classified as “non-smurf”. Flies which turned completely blue or showed distribution of blue dye outside the gut were classified as “smurf”. <em>Thin Layer Chromatography (TLC):</em> Each sample in the TLC analysis contains 10 flies of the respective genotype. The flies were starved for one hour before they were anesthetized with CO2 and transferred into 100µl chloroform:methanol (3:1) on ice. The flies were centrifuged for 5 minutes at 15,000g at 4°C and subsequently homogenized with a pestle. The sample was centrifuged again with the same settings. Lard was dissolved in chloroform:methanol (3:1) and served as triglyceride control (standard 1). Standard 1 was used to produce a standard curve with decreasing triglyceride concentrations. The samples and the standard curve were loaded on a glass silica gel plate (Millipore). The mobile phase in the TLC chamber was made with hexane:diethylether (4:1). The plate was placed in the chamber until the solvent front reached the upper end of the plate. The plate was taken out, air dried and stained with ceric ammonium heptamolybdate (CAM). Subsequently the plate was incubated at 80°C for 2 hours to visualize the stained triglyceride bands. Images were taken with a gel documentation system (gelONE). Triglyceride density was measured and the concentrations were calculated according the pre-determined concentrations of the standard curve. Image analysis and calculations were done using ImageJ. <em>Glucose Assay:</em> Seven days old male flies were used for the analysis. They were treated for 18h with PQ or sucrose respectively. Subsequently the flies were starved for one hour to bring all flies into the same metabolic state. Three flies were pooled as one sample and manually homogenized in 75µl TE + 0.1% Triton X-100 (Sigma Aldrich). The samples were incubated for 20min at 75°C to inactivate any intrinsic enzymatic activity. 5µl of the samples were loaded into a flat-bottom 96-well plate. Each sample was measured four times to measure glucose, trehalose, glycogen and the fly background respectively. The fly background was determined by diluting the fly sample with water. Glucose reagent (Sentinel Diagnostics) was added to the fly sample to measure the glucose levels. Trehalase (Sigma Aldrich) or amyloglucosidase (Sigma Aldrich) was added to the glucose reagent to measure trehalose or glycogen respectively. Plates were incubated for 24h at 37°C before the colorimetric measurement was performed at a wavelength of 492nm on a Tecan® Spark plate reader. The concentrations were calculated according to standards loaded on the same plate. <em>S2 Cell Culture, CellROX staining and flow cytometry:</em> Drosophila S2 cells (Thermo Scientific, R96007) were cultured in Schneider’s Drosophila medium (Gibco™) at 26°C and atmospheric oxygen and carbon dioxide conditions. The medium contained 10% FCS and 1% penicillin/streptomycin. PQ was added into the medium in concentrations of 15mM or 30mM to investigate the influence on the production of reactive oxygen species (ROS). Control cells remained untreated. To visualize ROS and oxidative stress, the cells were stained with CellROX™ Deep Red Reagent (Invitrogen) for 30 minutes directly in the medium in a concentration of 1:500 at 26°C. The cells were washed twice with 1xPBS containing 2mM EDTA. In order to stain dead cells DAPI was added to the samples in a concentration of 1:1000. Samples were acquired using a BD LSRFortessa™ (BD Biosciences) and analyzed with FlowJo analysis software. <em>COMET assays:</em> Comet assays were performed to analyze the amount of DNA damage in S2 cells treated with PQ. S2 cells were treated with 15mM or 30mM PQ for 24h. The cells were brought into a concentration of 1.8x10<sup>5</sup> cells/ml. Low melting agarose (LMA) was boiled at 90°C and subsequently cooled down to 37°C. The cells were diluted in low melting agarose (LMA) at a ratio of 1:10 (cells:LMA) and transferred onto a CometSlide™ (Trevigen). To ensure proper attachment of the LMA, the slides were cooled for 30 minutes at 4°C. The lysis was performed for one hour at 4°C. Therefore, the slides were submerged in CometAssay® Lysis Solution (Trevigen). Subsequently, the cells were submerged in alkaline unwinding solution (200mM NaOH, 1mM EDTA, pH 13) for 1h at 4°C. The electrophoresis was performed under alkaline conditions (pH13) for 30 minutes with a current of 20V (1V/cm). The slides were neutralized with ddH<sub>2</sub>O, dehydrated with 37% ethanol and dried properly at 37°C before they were stained with SYBR™Gold (Invitrogen) for 30 minutes. Comets were imaged with an Olympus BX61 fluorescence microscope. Comet data was analyzed via TriTrek CometScore 2.0.0.38 software.
description: # Drosophila macrophage response to oxidative stress
 The data deposited here
 includes survival data, imaging analysis, glucose/glycogen measurements,
 qPCR assays as well as TLC to measure triglyerides. The precise methods
 and analyses are described in the method section of this dataset. ##
 Description of the data and file structure The data files deposited here
 include the following datasets: Fly survivals were performed as indicated
 in the method section. Genotypes and treatment are included in the raw
 counting files below. Dead flies are indicated as 1 in the table living
 flies are indicated as 0. *Survival data (Lifespan, Paraquat, Starvation)*
 Paraquat survival: Fig.1_S1A_PQ survival_2mM_15mM_30mM_raw data.xml:
 Fig.1I_crq-reaper 15mM PQ survival raw data.txt
 Fig.5_S1E_Hml;tubGal80_DDR-IR_survival15mMPQ_raw data.xml Fig.5A_15mM PQ
 survival Hml-Gal4_DDR-IR.txt Fig.6A_Hml-upd3 and Hml-upd3-IR 15mM PQ
 survival_raw data.xml Fig.6C_Hml-upd3 and Hml-upd3-IR 15mM PQ hemocyte
 quantification_raw data.xml Fig.6D_upd3 null rescues survival on 15mM
 PQ_raw data.xml Fig.6E_JNK-signaling in Hemocytes_15mMPQ_survival_raw
 data.xml Lifespan analysis: Fig.5_S1B_Hml-Gal4_DDR-IR_lifespan_raw
 data.xml Fig.5_S1D_Hml;tubGal80_DDR-IR_lifespan_raw data.xml
 Fig.5_S1G_lifespan_Hml-upd3 and Hml-upd3-IR.xml Fig.5_S1H_lifespan of
 Hml-hep and Hml-bsk-DN.xml Starvation:
 Fig.5_S1C_Hml-Gal4_DDR-IR_starvation survival_raw data.xml *Smurf assays*
 Includes the counting of flies, which were identified as smurf (blue dye
 leaked out of the gut into the abdomen) or non-smurf (blue dye is only
 seen in the gut) on 30mM Paraquat. The following n-number of adult flies
 was analyzed per condition: 2mM PQ (n=79), 15mM PQ (n=90), 30mM PQ (n=80)
 and controls (n=80) Fig.1_S1B_Smurf assay_PQ 0-30mM_raw data.xml *Raw data
 files for semiquantitative qPCR* The following txt files include raw data
 sets obtained for gene expression analysis in whole flies after 15mM PQ
 treatment compared to control food. Three flies are included in one
 analyzed sample. Fig.1_S1C_qPCR_DNA-Damage_AttA.txt
 Fig.1_S1C_qPCR_DNA-Damage_defensin.txt
 Fig.1_S1C_qPCR_DNA-Damage_diptericin.txt Fig.1_S1C_qPCR_DNA-Damage_Dro.txt
 Fig.1_S1C_qPCR_DNA-Damage_Drs.txt Fig.1_S1C_qPCR_DNA-Damage_Mtk.txt
 Fig.1_S1D_qPCR_DNA-Damage_daw.txt Fig.1_S1D_qPCR_DNA-Damage_dpp.txt
 Fig.1_S1D_qPCR_DNA-Damage_eiger.txt The following txt files include the
 raw data obtained by gene expression measurement in whole flies with
 different genotypes after 15mM PQ treatment compared to control food.
 Three flies are included in one analyzed sample. The depicted values
 represent the Cp value as well as the predicted concentration of the gene
 product. Standard curve for semiquantitative concentration measurement was
 performed for each gene product. The measured gene products are indicated
 in the file names. Genotypes of flies are indicated.
 Fig.1+5_qPCR_DNA-Damage_daw.txt Fig.1+5_qPCR_DNA-Damage_dilp-2.txt
 Fig.1+5_qPCR_DNA-Damage_dilp-3.txt Fig.1+5_qPCR_DNA-Damage_dilp-5.txt
 Fig.1+5_qPCR_DNA-Damage_InR.txt Fig.1+5_qPCR_DNA-Damage_InR (2).txt
 Fig.1+5_qPCR_DNA-Damage_pepck.txt Fig.1+5_qPCR_DNA-Damage_puc.txt
 Fig.1+5_qPCR_DNA-Damage_rpl1.txt Fig.1+5_qPCR_DNA-Damage_socs36e.txt
 Fig.1+5_qPCR_DNA-Damage_stat92e.txt Fig.1+5_qPCR_DNA-Damage_Thor.txt
 Fig.1+5_qPCR_DNA-Damage_TotA.txt Fig.1+5_qPCR_DNA-Damage_upd1.txt
 Fig.1+5_qPCR_DNA-Damage_upd2.txt Fig.1+5_qPCR_DNA-Damage_upd3.txt
 Fig.6B_qPCR_Hml-upd3 and Hml-upd-IR gene expression_raw data.xml
 Fig.6F_qPCR_JNK_rpl-1.txt Fig.6F_qPCR_JNK_upd3.txt
 Fig.6F_upd3_expression_in_Hml-hep_and_Hml-bsk-DN_raw_data.xml The
 following txt files include the raw data obtained by gene expression
 measurement of S2 cells after PQ treatment compared to control . . The
 depicted values represent the Cp value as well as the predicted
 concentration of the gene product. Standard curve for semiquantitative
 concentration measurement was performed for each gene product. The
 measured gene products are indicated in the file names.
 Fig.3_S1_qPCR_S2_daw.txt Fig.3_S1_qPCR_S2_dpp.txt Fig.3_S1_qPCR_S2_dro.txt
 Fig.3_S1_qPCR_S2_drs.txt Fig.3_S1_qPCR_S2_InR.txt Fig.3_S1_qPCR_S2_mtk.txt
 Fig.3_S1_qPCR_S2_pepck.txt Fig.3_S1_qPCR_S2_puc.txt
 Fig.3_S1_qPCR_S2_rpl-1.txt Fig.3_S1_qPCR_S2_socs36e.txt
 Fig.3_S1_qPCR_S2_thor.txt Fig.3_S1_qPCR_S2_TotA.txt
 Fig.3_S1_qPCR_S2_upd1.txt Fig.3_S1_qPCR_S2_upd2.txt
 Fig.3_S1_qPCR_S2_upd3.txt *Triglyceride measurement via thinlayer
 chromatography (TLC)* The following files include the original images of
 the TLC plates and the quantifications thereof. Genotypes are indicated in
 the xlsx files. 10 flies were inlcuded in one individual sample.
 Qunatification was performed according to the standard row applied on each
 plate. Representative Images: Fig.1F_Hml-control_TLC image.tif
 Fig.1F_Hml-control_TLC image.tif Fig.1J_TLC_image_crq-reaper.tif
 Fig.5F_TLC_ddr1,2.tiff Fig.5F_TLC_ddr2,2.tiff Fig.5F_TLC_ddr3,3.tiff
 Fig.5F_TLC_ddr4,2.tiff Quantifications: Fig.1F_Analysis_TLC_Hml-control
 PQ.xlsx Fig.1J_analysis TLC crq_reaper PQ.xlsx Fig.5F_TLC_analysis TLC 5
 DNA damage genotypes plate1_2 .xlsx Fig.5F_TLC_analysis TLC 5 DNA damage
 genotypes plate2_1 .xlsx Fig.5F_TLC_analysis TLC 5 DNA damage genotypes
 plate3_1 .xlsx Fig.5F_TLC_analysis TLC 5 DNA damage genotypes plate4_1
 .xlsx *Quantification of hemocytes (Imaging)* The data presented here
 include the original images (tif or lif files) quantified for different
 genotypes and treatments. Fig.1G_Hml-control_NF.tif
 Fig.1G_Hml-control_PQ.tif Fig.5B_Hemocyte Quantification
 ra_Hml-mei41-IR_tefu-IR_NF.lif Fig.5B_Hemocyte Quantification
 ra_Hml-mei41-IR_tefu-IR_PQ.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-control_NF.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-control_PQ.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-mei41-IR_NF.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-mei41-IR_PQ.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-nbs-IR_NF.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-nbs-IR_PQ.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-tefu-IR_NF.lif Fig.5B_Hemocyte Quantification raw
 data_Hml-tefu-IR_PQ.lif Fig.5B_DDR Hemocyte quantification raw data.xml
 Fig.6C_Hemocyte Quantification_Hml-control_NF.lif Fig.6C_Hemocyte
 Quantification_Hml-control_PQ.lif Fig.6C_Hemocyte
 Quantification_Hml-control_PQ.lif Fig.6C_Hemocyte
 Quantification_Hml-upd3_NF.lif Fig.6C_Hemocyte
 Quantification_Hml-upd3_PQ.lif Fig.6C_Hemocyte
 Quantification_Hml-upd3-IR_NF.lif Fig.6C_Hemocyte
 Quantification_Hml-upd3-IR_PQ.lif *Glucose and gycogen measurement
 (calorimetric assays)* The xlsx file include the quntification fo glycogen
 and glucose in calorimetric assays. Amount of gluocse and glycogen
 measured was determined according to standard rows measured on each
 individual plate. Genotypes and treatments are indicated in the xlsx file.
 Fig.1K+L_Glucose analysis 24h_crq reaper.xlsx *CellRox assays (FACS
 analysis of S2 cells)* The files include the raw fcs files of S2 cells
 treated with different concentrations of PQ (0mM, 15mM, and 30mM) after 6
 hours and 24 hours. Acquisition was performed with an LSR Fortessa flow
 cytometer. Dead cells were excluded and CellROX assays were eprformed as
 described in the method section. Fig.3_S1_CellROX_6h_15mM 1_004.fcs
 Fig.3_S1_CellROX_6h_15mM2_005.fcs Fig.3_S1_CellROX_6h_15mM3_006.fcs
 Fig.3_S1_CellROX_6h_30mM1_007.fcs Fig.3_S1_CellROX_6h_30mM2_008.fcs
 Fig.3_S1_CellROX_6h_30mM3_009.fcs Fig.3_S1_CellROX_6h_C1_001.fcs
 Fig.3_S1_CellROX_6h_C2_002.fcs Fig.3_S1_CellROX_6h_C3_003.fcs
 Fig.3_S1_CellROX_24h_15mM 1_004.fcs Fig.3_S1_CellROX_24h_15mM2_005.fcs
 Fig.3_S1_CellROX_24h_15mM3_006.fcs Fig.3_S1_CellROX_24h_30mM1_007.fcs
 Fig.3_S1_CellROX_24h_30mM2_008.fcs Fig.3_S1_CellROX_24h_30mM3_009.fcs
 Fig.3_S1_CellROX_24h_C1_001.fcs Fig.3_S1_CellROX_24h_C2_002.fcs
 Fig.3_S1_CellROX_24h_C3_003.fcs *Comet Assays (DNA damage)* The files
 included here are the measurements of comet assays performed on S2 cells
 upon PQ treatement or on isolated hemocytes from transgenic flies. Method
 details are provided in the method section. Fig.3_S1_S2-cells_olive tail
 moment_PQ treatment_raw data.xml Fig.5C_Olive Tail Moment.xml ##
 Sharing/Access information Not applicable ## Code/Software No new codes
 were generated for the analysis of the deposited datasets.