DECR1, encoding the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown selectively inhibited β-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation and metastasis in mouse xenograft models. Mechanistically, targeting of DECR1 caused cellular accumulation of PUFAs, enhanced mitochondrial oxidative stress and lipid peroxidation, and induced ferroptosis. These findings implicate PUFA oxidation via DECR1 as an unexplored facet of FAO that promotes survival of PCa cells.Fatty acid β-oxidation (FAO) is the main bioenergetic pathway in human prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify  Prostate cancer (PCa) is the most prevalent male cancer and the second leading cause of cancer deaths in men in Western societies . For patIncreasingly, targeting cancer cell metabolism is a focus of research efforts . While fUntil recently, most attention has focused on the therapeutic targeting of de novo FA synthesis and, most recently, uptake of FAs in PCa to limit their availability as a source of energy and cell membrane phospholipids . HoweverIn addition to our recent report of enhanced FAO in PCa cells , an accumeta effect size scores in PCa malignant versus matched normal tissues  in four clinical datasets with malignant and matched normal RNA sequencing data . Genes w tissues . The met tissues , despiteDECR1 mRNA expression was significantly higher in malignant compared to benign prostate tissues in ten independent expression datasets of PCa tissues versus non-malignant tissues , suggestive of a relationship between DECR1 expression and hormone signaling  sequencing data. In VCaP cells, AR bound strongly to the DECR1 promoter in response to DHT treatment, but not when co-treated with AR antagonists  signaling and lipid metabolic genes is well established. Many studies have reported a marked stimulatory effect of AR on key lipid metabolism pathways either directly or indirectly through activation of a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) . We therpression ; GSE5901ecreased . To dete tissues . Collectacetyl-CoA, which enters the tricarboxylic acid cycle (TCA) and produces ATP and NADH as energy for the cell. Unlike saturated FAs, all unsaturated FAs with double bonds originating at even-numbered positions, and some unsaturated FAs with double bonds originating at odd-numbered positions, require three auxiliary enzymes to generate intermediates that are harmonious with the standard β-oxidation pathway (Enoyl CoA isomerase (ECI1), 2,4 Dienoyl-CoA reductase (DECR1) and Dienoyl CoA isomerase (ECH1) (FAs are metabolized mainly in mitochondria through the β-oxidation process to generate  pathway : Enoyl Ce (ECH1) . DECR1 ce (ECH1) . Given te (ECH1) . DECR1 ke (ECH1)  as well e (ECH1) , an intee (ECH1) . Howevere (ECH1) , indicatNext, we employed extracellular flux analysis to determine the intrinsic rate and capacity of PCa cells to oxidise PUFAs in conditions where other exogenous substrates were limiting. Exogenous linoleic acid stimulated basal oxygen consumption rates (OCR), as a measure of mitochondrial oxidative phosphorylation, and maximal respiration, ATP production, and mitochondrial spare capacity  as deterThe consistently increased expression of DECR1 in PCa tissue and its association with shorter-relapse times and survival rates  and 2, tDECR1 knockdown resulted in inhibition of PUFA β-oxidation and led to accumulation of PUFAs in phospholipids . InspectCD36 deletion slowed cancer progression in prostate-specific -/-PTEN mice. However, it is increasingly evident that PCa exhibits plasticity in attaining FAs and that crosstalk between de novo synthesis and FA uptake requires dual targeting of the two pathways to achieve maximal efficacy , acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) are all overexpressed in PCa compared to benign tissue . While mefficacy , an apprWe showed previously , as have-/-Decr1 mice and DECR1-deficient patients  versus low fat diet and reported that the former promotes AKT and ERK activity, tumor growth, tumor incidence in genetically engineered/transgenic mouse models, tumor progression to CRPC and metastasis , whilst in contrast DECR1 is specific for PUFA. Homozygous  in mice , but Decc stress . The marc stress , and thuIn summary, herein we strengthen the evidence base for the critical importance of FAO and, specifically, PUFA oxidation in PCa, thereby identifying a promising new therapeutic candidate, DECR1.eBayes pipeline  datasets composed of matched normal versus tumor prostate cancer patient tissue samples were acquired and are listed as follows:  The Cancpipeline .Effect sTranscriptomic data was downloaded from The Cancer Genome Atlas (TCGA) data portal, cBioPortal , and GEO2 in a humidified atmosphere at 37°C. Prior to androgen treatment, cells were seeded in medium supplemented with 5% dextran charcoal coated FBS (DCC-FBS) and after 24 hr, 1 nM or 10 nM of dihydrotestosterone (DHT) was added. For anti-androgen treatment, cells were cultured in growth medium supplemented with 2.5 µM, 5 µM, 7.5 µM or 10 µM Enzalutamide . The sources and experimental conditions for primary antibodies used in this study are listed in the Key Resources Table. Primers were obtained from Sigma-Aldrich and their sequences are detailed in the Key Resources Table.The human normal prostate epithelial cell lines PNT1 and PNT2 were obtained from the European Collection of Authenticated Cell Cultures (ECACC), and prostate carcinoma cells LNCaP, VCaP, and 22RV1 were obtained from the American Type Culture Collection . Castrate resistant V16D and enzalutamide resistant MR49F cells were derived through serial xenograft passage of LNCaP cells  were a k3 pieces and cultured on Gelfoam sponges (80 × 125 mm Pfizer 1205147) in 24-well plates pre-soaked in 500 µl RPMI‐1640 with 10% FBS, antibiotic/antimycotic solution. Etomoxir (100 µM) or Enzalutamide (10 µM) was added into each well and the tissues were cultured in 5% CO2 in a humidified atmosphere at 37°C for 48 hr, then snap frozen in liquid nitrogen and stored at −80°C, or formalin-fixed and paraffin-embedded.Patient derived-explant culture was carried out according to techniques established in our laboratory and as described previously . 6 mm/8 Paraffin-embedded tissue sections (2–4 µm) were deparaffinized in xylene, rehydrated through graded ethanol, and blocked for endogenous peroxidase before being subjected to heat-induced epitope retrieval . IHC staCell Fractionation. Protein lysates from each subcellular fraction  were obtained from PNT1 and LNCaP cells using the cell fractionation kit  according to the manufacturer’s protocol.Protein lysates were collected in RIPA lysis buffer . Western blotting on whole cell protein lysates were performed as previously described . Cell FrGUSB and L19 for prostate cancer cells and LNCaP-derived tumors, or TUBA1B, PPIA and GAPDH for PDEs.RNA was extracted from cells using the RNeasy RNA extraction kit (Qiagen), followed by the iScript cDNA Synthesis kit . qPCR was performed with a 1:10 dilution of cDNA using SYBR Green (Bio-Rad) on a CFX384 Real-Time System (Bio-Rad). Relative gene expression was calculated using the comparative Ct method and normalized to the internal control genes AR ChIP-seq data from published external datasets, GSE56288   and GSE56 cells/plate in 15 cm plates in RPMI-1640 medium containing 10% DCC-FBS for 3 days, then treated for 4 hr with 10 nM DHT or Vehicle (ethanol). AR ChIP was performed as described previously  and control siRNA (D-001810-01-20 ON-TARGET plus Non-targeting siRNA #1) were purchased from Millennium Science . Four siRNA were tested and the two most effective were selected for our experimentation: siDECR1-1 (J-009642-05-0002) and siDECR1-2 (J-009642-06-0002). The siRNAs at a concentration of 5 nM were reverse transfected using Lipofectamine RNAiMAX transfection reagent  according to the manufacturer’s protocol. DECR1 downregulation (>80%) was confirmed on mRNA and protein levels.LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc  according to the manufacturer’s protocol .4–6.0 × 104 cells/well and reverse transfected with siRNA overnight. Cells were manually counted using a hemocytometer 96 hr post-siRNA knockdown and viability was assessed by Trypan Blue exclusion as described previously  at 40X magnification. The number of migrated cells were counted manually and presented as percentages relative to control cells ± SEM.Transwell migration assays were performed using 24-well polycarbonate Transwell inserts . LNCaP, 22RV1 and MR49F cells transfected overnight with siRNA were seeded into the upper chamber of the Transwell at a density of 8 × 102 for 48 hr, non-invading cells as well as the Matrigel from the interior of the inserts were gently removed using a cotton-tipped swab. The inserts were fixed in 4% paraformaldehyde for 20 min and stained with 1% crystal violet for 30 min. The images of invaded cells were captured using the Axio Scope A1 Fluorescent Microscope (Zeiss) at 40X magnification. The number of invaded cells were counted manually and presented as percentages relative to control cells ± SEM.Cell invasion were assessed using 24-well-plate BD Biocoat Matrigel Invasion Chambers  according to the supplier instructions. After 48 hr of siRNA transfection, 650 µl of medium containing 10% FBS was added to the bottom chamber, and equal number of cells within 1% FBS-contained medium were transferred to the upper chamber. After incubation at 37°C, 5% CODECR1 stable knockdown cells (shDECR1) or negative control cells (shControl) were prepared in a single-cell suspension before being plated in 6-well plates (500 cells/well). Cells were incubated for 2 weeks at 37°C and medium was replenished every 3–7 days. After 3 weeks, cells were washed with PBS, fixed with 4% paraformaldehyde and stained with 1% crystal violet for 30 min. Colonies were counted manually, and results were reported as number of colonies ± SEM.4 cells/ml. Cell suspensions (1500 cells in 20 µl) were pipetted onto the inside of a petri dish lid, and 15 ml of PBS was added to the dish to prevent the drops from drying. The petri dishes were reassembled and incubated at 37°C for 5 days. Photos of the formed spheres were captured, and the sphere volume was determined using ReViSP software . After baseline OCR is stabilized in FAO Assay Medium, 200 µM of linoleic-acid (LA) or palmitic acid (PA) were added before initializing measurements. Extracellular flux analysis was performed using the Seahorse XF Cell Mitochondrial Stress Test kit (Seahorse Bioscience) according to the manufacturer’s protocol. Extracellular flux experiments were performed on a Seahorse XF96 Analyzer and results were analysed using Seahorse Wave software for XF analyzers. The OCR values were normalized to cell numbers in each well.Cells were plated on XF96 well cell culture microplates  at equal densities in substrate-limited medium  and incubated overnight. One hour before the beginning of OCR measurement, the cells were changed into FAO Assay Medium  extraction solvent containing the internal standards (0.5 µl/samples) was added onto each well and allowed to incubate for 10 min. Cells were collected into 1.5 ml Eppendorf tubes, incubated on ice for 5 min, and centrifuged at 4°C for 5 min at 16,100 g. The supernatant was then transferred into a fresh 1.5 ml Eppendorf tube and allowed to dry in a Speedvac. Dried samples were derivatised with 20 µl methoxyamine  and 20 µl N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% Trimethylchlorosilane (TMCS). The derivatised samples were analysed using GC QQQ targeted metabolomics as described in LNCaP cells were transfected with siRNA for 96 hr in no glucose medium (containing 10% FBS) supplemented with 2.5 mM glucose in 6-well plates. Cells were washed with 1 ml of 37°C Milli-Q water on the shaker for 2 s. The plate was placed in sufficient volumes of liquid nitrogen, enough to cover the surface of the plate and was briefly stored on dry ice. 600 µl of ice cold 90% 9:1 methanol:chloroform  was mixed with 800 μl 1 N HCl:CH3OH 1:8 (v/v), 900 μl CHCl3 and 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol . 3 μl of SPLASH LIPIDOMIX Mass Spec Standard  was spiked into the extract mix. The organic fraction was evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature and the remaining lipid pellet was stored at - 20°C under argon.Lipid pellets were reconstituted in 100% ethanol. Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer . Chromatographic separation was performed on a XBridge amide column  maintained at 35°C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient:  at a flow rate of 0.7 mL/min which was increased to 1.5 mL/min from 13 min onwards. SM, CE, CER, DCER, HCER, LCER were measured in positive ion mode with a precursor scan of 184.1, 369.4, 264.4, 266.4, 264.4 and 264.4 respectively. TAG, DAG and MAG were measured in positive ion mode with a neutral loss scan for one of the fatty acyl moieties. PC, LPC, PE, LPE, PG, LPG, PI, LPI, PS and LPS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V and −4,500 V; Temperature = 550°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V and −80 V; Entrance Potential = 10 V and −10 V; Collision Cell Exit Potential = 15 V and −15 V. The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6 except for TGs which considered: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:2, 22:3, 22:4, 22:5, 22:6.Peak integration was performed with the MultiQuant software version 3.0.3. Lipid species signals were corrected for isotopic contributions  and were normalized to internal standard signals. Unpaired T-test p-values and FDR corrected p-values (using the Benjamini/Hochberg procedure) were calculated in Python StatsModels version 0.10.1.LNCaP cells were transfected with siRNA for 96 hr in 6-well plates. Cells were collected into fluorescence-activated cell sorting (FACS) tubes and stained with 2.5 µM of MitoSOX Red stain  for 30 min in a 37°C water bath. Cells were centrifuged at 1,500 rpm for 5 min, washed twice with 500 µl of PBS, and resuspended in 500 µl of pre-warmed PBS before the samples are read on a BD FACSymphony flow cytometer.Total lipids were extracted from cells using two-phase extraction with methyl-tert-butyl-ether (MTBE)/methanol/water  . Cell pe m/z). The ten most abundant ions in each cycle were subjected to fragmentation . An exclusion list of background ions was used based on a solvent blank. TraceFinder v5.0 (Thermo Fisher Scientific) was used for peak detection and integration, based on exact precursor ion mass (m/z tolerance four ppm) and m/z 85.0 acylcarnitine product ion. Peak areas were normalised to the D3-palmitoylcarnitine internal standard.Acylcarnitines were quantified by liquid chromatography-tandem mass spectrometry using a Q-Exactive HF-X mass spectrometer with heated electrospray ionization and a Vantage HPLC system (ThermoFisher Scientific). Extracts were resolved on a 2.1 × 100 mm Waters Acquity C18 UPLC column (1.7 µm pore size), using an 18 min binary gradient at 0.28 mL/min flow rate, as follows: 0 min, 80:20 A/B; 3 min, 80:20 A/B; 6 min, 57:43 A/B; 8 min, 35:65 A/B; 9 min, 0:100 A/B; 14 min, 0:100 A/B; 14.5 min, 80:20 A/B; 18 min, 80:20 A/B. Solvent A: 10 mM ammonium formate, 0.1% formic acid in acetonitrile:water (60:40); Solvent B: 10 mM ammonium formate, 0.1% formic acid in isopropanol:acetonitrile (90:10). Data was acquired in positive ion mode with data-dependent acquisition  and incubated with 5 µM BODIPY-581/591 C11 stain (Thermo Fisher Scientific). Cells were washed and fixed with 4% paraformaldehyde (PFA), and mounted with Prolong Gold anti-fade solution with DAPI (Thermo Fisher Scientific). Cells were imaged at 60 X magnification using a Olympus FV3000 Confocal Microscope. Quantification of BODIPY-C11 stain was performed using ImageJ analysis software.For imaging, LNCaP cells following DECR1 knockdown were plated at 5 × 106 cells in 50 μL 10% FBS/RPMI 1640 medium) were co-injected subcutaneously with 50 μL Matrigel in 6-week-old NOD Scid Gamma male mice . When tumors reached ~200 mm3, mice were randomized in different therapy groups. One group was left untreated (n = 5), one group was treated with vehicle control , one group was treated with enzalutamide (10 mg/kg MDV3100 in 10% DMSO/PBS) and one group was castrated by surgical castration under isofluorane anesthesia (n = 9). Five of the ten castrated mice were then treated daily with enzalutamide (10 mg/kg MDV3100 in 10%DMSO/PBS) by oral gavage for 7 days. Enzalutamide therapy of castrated mice started five days after surgery.LNCaP cells (5 × 106 cells in 50 μL 10% FBS/RPMI 1640 medium) were co-injected subcutaneously with 50 μL matrigel in 6-week-old NOD Scid Gamma male mice. Tumors were measured using callipers and their volumes were calculated using the formula length × width2/2.DECR1 stable knockdown cells (shDECR1) or negative control cells (shControl) (5 × 106 DECR1 stable knockdown cells (shDECR1) or negative control cells (shControl) were -injected intraprostatically in 8 week old NOD/SCID male mice. Whole-body imaging to monitor luciferase-expressing LNCaP cells was performed at day 3 of the injection and once weekly after that using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). D-luciferin  was dissolved in sterile deionized water (0.03 g/ml) and injected subcutaneously (3 mg/20 g of mouse body weight) before imaging. Bioluminescence is reported as the sum of detected photons per second from a constant region of interest. After the animals were sacrificed, lungs and livers were excised for ex vivo imaging using the IVIS system.Ten microliter containing 1 × 10After each study, tumors were excised and half was snap frozen for RNA extraction while the other half was formalin fixed and paraffin embedded. All animal procedures were carried out in accordance with the guidelines of the National Health and Medical Research Council of Australia, with subcutaneous xenograft studies approved by the Austin Health Animal Ethics Committee  and orthotopic xenograft studies approved by the University of Adelaide Animal Ethics Committee .Results are reported as mean ± S.E.M. Statistical analysis was performed using GraphPad Prism (V7.0 for Windows). Differences between treatment groups were compared by T-test or one-way ANOVA followed by Tukey or Dunnett post hoc test. Significance is expressed as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your work identifies DECR1 as an important regulatory component of polyunsaturated fatty acid oxidation in prostate cancer. Your evidence suggests that DECR1 expression contributes to prostate cancer progression and that blocking this enzyme may reverse metabolic processes that are critical for disease progression. Congratulations on your body of work!Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Maureen Murphy as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "DECR1 is an androgen-repressed survival factor that protects prostate tumor cells from ferroptosis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Fatty acid oxidation is important for some types of cancer. Inhibition of fatty acid oxidation has been explored as therapeutic strategy. Drugs that inhibit CPT1, the rate-limiting enzyme in fatty acid oxidation, are effective at reducing fatty acid oxidation but are associated with considerable toxicity. In this work, the authors identify DECR1, the rate-limiting enzyme for polyunsaturated fatty acid (PUFA) oxidation as a potential therapeutic target for prostate cancer. DECR1 expression is correlated with adverse tumor pathology and prognosis. DECR1 knockdown suppresses PUFA oxidation and cell growth / viability. The authors suggest that cell death induced by targeting DECR1 occurs through ferroptosis. Overall, this is a novel body of work that is interesting and well-written with significant merits. However, there are several items that should be addressed to improve the manuscript.Essential revisions:1) The conclusion that cells are dying via ferroptosis is insufficiently supported. Ferroptosis is characterized by the accumulation of phospholipid hydroperoxides which can be measured by mass spec or using the dye BODIPY C11. Are the PUFAs channeled into specific lipids (e.g. phospholipids) or do they remain as free fatty acids? Dose response curves should be performed in the presence and absence of multiple ferroptosis inducers to determine IC50s. What is the effect of deferoxamine on cell death? What are the effect of apoptosis inhibitors on the cells?2) As it stands currently, the in vivo data are not convincing. In Figure 5 the authors stated that "LNCaP cells stably depleted of DECR1 showed highly variable growth rates in vivo, but inspection of the resultant tumors revealed significantly reduced cellular proliferation compared to control cells, concomitant with reduced DECR1 expression" the author should present the data for tumor growth and tumor volume. How do the authors explain the variability in tumor growth? For IHC, the authors should show a serial section for DECR1 and KI67 expression.3) Some of the in vitro effects, e.g., cell death, are relatively modest. Are these effects intensified with stable knockdown or knockout approaches using shRNA or CRISPR/Cas? Essential revisions:1) The conclusion that cells are dying via ferroptosis is insufficiently supported. Ferroptosis is characterized by the accumulation of phospholipid hydroperoxides which can be measured by mass spec or using the dye BODIPY C11. Are the PUFAs channeled into specific lipids (e.g. phospholipids) or do they remain as free fatty acids? Dose response curves should be performed in the presence and absence of multiple ferroptosis inducers to determine IC50s. What is the effect of deferoxamine on cell death? What are the effect of apoptosis inhibitors on the cells?We have incorporated these points into our updated Figure 6 and new Figure 6—figure supplements 1 and 2 of the revised manuscript, and in the subsection “DECR1 targeting induces lipid peroxidation and cellular ferroptosis”. To summarise the changes:In our original manuscript, we showed that DECR1 down-regulation led to accumulation of the free fatty acid linoleate  and we now include new lipidomics data to show increased levels of PUFAs in the PC, π and PS classes of phospholipids . PUFAs are highly susceptible to lipid peroxidation and ferroptosis and, in response to the reviewer’s questions, we have performed multiple additional experiments to further prove that DECR1 activity regulates ferroptosis.Firstly, in the original manuscript we showed that DECR1 knockdown increased cellular levels of malondialdehyde, a marker of lipid peroxidation . As suggested by the reviewer, we used the BODIPY C11 stain and intensity quantification, which revealed significant accumulation of phospholipid hydroperoxides, a hallmark of ferroptosis . Our original data showed that DECR1 knockdown increases mitochondrial ROS levels , while ectopic overexpression of DECR1 reduces mitochondrial ROS levels . We extend these findings to show that ectopic overexpression of DECR1 also significantly reduces PUFA-induced ROS levels in the mitochondria .50 in DECR1 downregulated cells than control cells . Moreover, we now show that two ferroptosis inhibitors, Ferrostatin-1 and Deferoxamine (as suggested), completely prevent the effects of DECR1 knockdown on LNCaP cell viability and cell death .Secondly, DECR1 knockdown increased the cell sensitivity to three inducers of ferroptosis ; and we extended the dose ranges as requested to show that the three agents exhibited a lower ICFinally, we showed using flow cytometry that DECR1 knockdown-induced cell death is not characteristic of apoptosis , and the apoptosis inhibitor ZVAD did not rescue the cells from cell death . Collectively, these data  strongly support ferroptosis as a primary mechanism of cell death in response to DECR1 targeting.2) As it stands currently, the in vivo data are not convincing. In Figure 5 the authors stated that "LNCaP cells stably depleted of DECR1 showed highly variable growth rates in vivo, but inspection of the resultant tumors revealed significantly reduced cellular proliferation compared to control cells, concomitant with reduced DECR1 expression" the author should present the data for tumor growth and tumor volume. How do the authors explain the variability in tumor growth? For IHC, the authors should show a serial section for DECR1 and KI67 expression.Subcutaneous growth of LNCaP tumor xenografts is widely acknowledged as being highly variable. Nevertheless, there was a significant decrease in proliferative index in the shDECR1 tumors compared with shControl cells. We have included a serial section for DECR1and Ki67 staining of representative tumors as requested . We have also included the individual tumor growth curves as requested .In order to confirm these data as well as study the effect of DECR1 downregulation on PCa in the prostate microenvironment, we undertook a second study of orthotopically-grown LNCaP xenografts. These new data confirmed that DECR1 knockdown significantly inhibits tumor growth in the prostate , but also significantly inhibits lung metastasis , and have been added to the revised manuscript text:“In vivo, LNCaP cells stably depleted of DECR1 showed highly variable growth rates in a subcutaneous model , but inspection of the resultant tumors revealed significantly reduced cellular proliferation compared to control cells, concomitant with reduced DECR1 expression . To study the effect of DECR1 downregulation on PCa in the prostate microenvironment, we undertook a second study using LNCaP orthotopic xenografts. DECR1 knockdown significantly retarded tumor growth , and significantly inhibited lung metastasis in the orthotopic tumor model .”3) Some of the in vitro effects, e.g., cell death, are relatively modest. Are these effects intensified with stable knockdown or knockout approaches using shRNA or CRISPR/Cas?We used our shRNA stable knockdown LNCaP cells to assess this, showing a similar effect to the transient knockdown; with a ~25% decrease in cell viability and almost 2-fold increase in cell death detected .


Logistic regression models were performed to identify the impact of p16ink4a positivity.Retrospective analysis of disease-free (DFS) and disease-specific survival (DSS) of patients with invasive vulvar cancer at a single tertiary care center. Histology, HPV and p16ink4a expression of over 25%. Disease-free and disease-specific survival was longer in p16ink4a positive patients . In multivariate analysis, p16ink4a positivity was an independent parameter for DFS ), but not for DSS , in contrast to age and tumor stage.135 patients were included in the analysis. 32 (23.7%) showed a p16ink4a positive invasive vulvar cancer.Age and tumor stage negatively affect survival. However, disease-free survival is significantly longer in patients with p16 Transient HPV infections can be excluded and only tumors with HPV as the primary cause of the oncologic process remain [ink4a status may strongly influence the patient’s outcome, which is already established in oropharyngeal cancer [ink4a positivity and HPV status on the clinical outcome in IVC.Over the past two decades, the incidence rates of invasive vulvar cancer, which is responsible for about 5% of all gynecological cancers, increased continuously. Especially in women younger than 50 years of age, the incidence rates almost tripled over the past decades –3. Especnowadays . Similarnowadays . In a menowadays . In a rer in IVC . P16ink4s remain –12. We pl cancer . Age at ink4a was evaluated. A sensitive assay, using SPF10 broad spectrum primers PCR and DEIA (DNA enzyme immunoassay) was used for HPV–DNA detection. Positive samples were subsequently analyzed by SPF10 PCR/DEIA/LIPA25 , which is a reverse hybridization technique that detects 25 high-risk (HR) and low-risk (LR) HPV types . Furthermore, IVC cases were tested for cyclin-dependent kinase-4 inhibitor (p16ink4a), which is reported to be overexpressed in at least 90% of HPV-related VIN and IVC cases. Therefore, CINtec® PLUS Cytology Kit by Roche  was used to detect p16ink4a in the invasive vulvar cancer cases. A case was considered to be positive if more than 25% of invasive cancer cells showed a diffuse overexpression [Austria was one of 39 countries participating in the international collaborative study . Patientpression . The cliAll patients included were managed by gynecologic oncologists. Predefined uniform criteria for surgical procedure terminology, pathologic variables, and sites of recurrence were used. Based on the FIGO 2009 classification system, disease staging was performed . DependiFor the first 2 years, clinical examination was performed every 3 months, followed by 6 months intervals until the completed fifth year, then by annually consultations. At every follow up check, the patient received a vulvoscopy, vagino-rectal palpation and groin inspection. Furthermore, serum squamous cell carcinoma antigen (SCC) was evaluated. Biopsy and or computed tomography were performed when recurrent disease was suspected. Based on post-mortem results, patient’s death and causes of death were documented.ink4a could represent a transient infection with no role in carcinogenesis. Group 1 represented patients with HPV-positive and HPV-negative IVC with a p16ink4a expression in less than 25% (< 25% = negative) and group 2 consisted of HPV-positive and HPV-negative IVC with a concomitant overexpression of p16ink4a (≥ 25%). Values are shown as mean values with standard deviation (SD). To compare HPV-positivity with clinic-pathological parameters, t tests were performed. P values of < 0.05 were considered statistically significant. With respect to overall and disease-free survival, differences between groups were tested using the log-rank test and are presented as Kaplan–Meier survival curves. Multivariable analysis was performed using a Cox regression model including patient’s age , p16ink4a expression and tumor stage (FIGO III and IV vs. FIGO I and FIGO II) as independent variables. Furthermore, FIGO I and II adapted analysis was performed via t test and is presented as Kaplan–Meier survival curves as well. Statistical analyses were performed using SPSS 25.0 for MAC . Collected data: age at diagnosis, histopathological grading, initial therapy, HPV-subtype analysis, p16ink4a expression, FIGO classification, disease-free survival, disease-specific survival, nicotine abuse, coexistent lichen sclerosus or VIN, previous malignant disease, comorbidities.Patient’s data was divided in two different groups (group1/group2), assuming that cases in which HPV–DNA is detected without overexpression of p16ink4a status to exclude transient HPV infections (Table p < 0.001). In 30 (22.2%) of the evaluated histological specimen, HPV was detected. HPV 16 was the primarily diagnosed HPV-subtype and detected in 27 cases (77%). HPV 31 was detected in two cases (6.7%) and HPV 33 (3.3%) in one. In 20 (66.7%) of those HPV-positive cases, an overexpression (≥ 25%) of the tumor-suppressor-protein p16ink4a was detected. Contrary, only 12 (11.4%) of HPV-negative IVC presented an overexpression of p16ink4a . Further subgroup analysis, only including patients at FIGO stage I and II was performed.A coexistent lichen sclerosus was detected in 6 (4.4%) cases, all of them were HPV-negative. FIGO stages were well balanced between both groups ) but not disease-specific survival (DSS) ). Results on multivariate analysis are presented in Table 76 56.3%) patients suffered at least one relapse. Mean disease-free survival (DFS) in group 1 was 10 months (IQR: 5–36), whereas patients from group 2 had a relapse after 23 months (IQR: 4–65.5), % patientn = 80, 77.7%), still were significantly older (mean: 70.5 years (IQR: 57.4–83.6)), than patients of group 2 (mean: 63.1 years (IQR:46.0–79.5)), p = 0.028. Disease-free survival of group 2 still was significantly longer than in group 1; p = 0.011 ), but not concerning DSS , Table To have a closer look at patients with a favorable prognosis, FIGO stage I and II were analyzed separately Table . Patientp = 0.010 and p = 0.048, respectively).Furthermore, for sensitivity analysis, patients aged 80 years, or more were excluded in the subgroup analysis of FIGO stages I and II. Excluding the old ages as a possible bias, the difference between both groups for DFS and DSS remained statistically significant  [p = 0.501). Although patients with p16ink4a positive IVC were on average seven years younger than p16ink4a negative controls and the first recurrence occurred after an average of 2 years instead of one year, in multivariate analysis DSS depends on younger age and lower FIGO stage but not on p16ink4a status. Therefore, further research has to be done on data of relapse in IVC. Since there is no appropriate screening for VIN and IVC, elimination of HPV-related tumors is possible in countries with a good coverage of HPV vaccination [ink4a positive invasive vulvar cancer. There is a caveat which needs further investigation: In case of a relapse, the mortality of the initially prognostic favorable p16ink4a positive invasive vulvar cancer appears to be worse.In our dataset of 135 IVC, survival indicators, DFS and DSS, were significantly longer in p16with IVC –17. To bcination –22. In c


Several antimicrobial resistance determinants were detected for the first time within pets from Africa, including blaCTX-M-37, blaCTX-M-156, blaSHV-11, blaACT-23, blaACT25/31, blaDHA-1, and blaCMY-169. Our results revealed that pets displaying symptoms of respiratory illness are potential sources for pathogenic microbes possessing unique resistance mechanisms which could be disseminated to humans, thus leading to the development of severe untreatable infections in these hosts.Antimicrobial resistance within pets has gained worldwide attention due to pets close contact with humans. This report examined at the molecular level, the antimicrobial resistance mechanisms associated with kennel cough and cat flu. 1378 pets in total were assessed for signs of respiratory infection, and nasal and conjunctival swabs were collected across 76 diseased animals. Phenotypically, 27% of the isolates were characterized by multidrug resistance and possessed high levels of resistance rates to β-lactams. Phenotypic ESBLs/AmpCs production were identified within 40.5% and 24.3% of the isolates, respectively. Genotypically, ESBL- and AmpC-encoding genes were detected in 33.8% and 10.8% of the isolates, respectively, with  Furthermore, the emergence, widespread occurrence, and spread of antimicrobial resistance among animals, particularly within domestic pets, has raised high levels of global interest. The growing interest among AMR among animals is due to the high possibility of animal-human transmission of highly pathogenic bacteria possessing unique resistance mechanisms such as ESBL, carbapenemases, and mcr genes5. For example, human-specific pathogens have recently been isolated in dogs4. Furthermore, pathogenic strains possessing the same extended-spectrum and AmpC beta-lactamases (ESBL/AmpC) have been detected within the same households of humans and dogs5. More recently, our research group has identified highly resistant carbapenemase-producing and mcr-9 coharbouring bacteria associated with pets that had an active respiratory infection that could be transmitted between animals and humans3. Within this context, the bacteria associated with respiratory diseases in cats and dogs are of particular importance due to the widespread nature of these contagious diseases, and ease of transmission, in addition to the close proximity of pets and humans.Antimicrobial resistance (AMR) comprises a rapidly emerging growing global threat that exerts enormous economic burdens. Based on recent estimates, AMR’s economic costs varied from £3–11 billion to US $100 trillion6. Furthermore, several β-lactams are exclusively utilized in the field of veterinary medicine, including ceftiofur and cefquinome, which comprise 3rd and 4th generation cephalosporins7. Moreover, in the nation of Egypt, pets are typically treated with antibiotics manufactured for human use, including those forbidden within the veterinary practice, in order to reduce resistance rates. Hence, the heavy use of β-lactam across the veterinary and human practices has acted as a selective pressure facilitating the development of β-lactam resistance. Among the various mechanisms of β-lactam resistance, particular attention had been paid to ESBLs/AmpCs, as these especial enzymes are capable of hydrolyzing penicillins, cephalosporins, and monobactams thus facilitating resistance to multiple antimicrobial agents8. The faulty therapeutic outcomes and high mortality rates associated with ESBL/AmpC producers8, have bolstered the scientific interest to elucidate the molecular mechanism of β-lactam resistance, in particular in pets, which act as a potential consignor for human infection5.β-Lactam antibiotics comprise an important group of antibiotics that are widely utilized in human and animal fields, exhibiting strong therapeutic potential. Hence, WHO has identified several β-lactam classes, namely 3rd, 4th, and 5th generation cephalosporins, and carbapenems in addition, as being “critically important” for humans use9. Various Gram-negative bacteria have facilitated these particular challenging diseases and have been reported in several studies to be zoonotic10. Such as members of the Enterobacterales, alongside Pseudomonas aeruginosa that capable of acting as primary or secondary complicating pathogens within kennel cough and cat flu. Other Gram-negative pathogens could possibly also be involved and necessitate transtracheal wash or deep sampling to address their isolation, including Bordetella bronchiseptica, which is a commonly occurring bacterium associated with kennel cough and cat flu11. At present, a paucity of information is known and available regarding the bacterial resistance mechanisms associated with the respiratory diseases in pets. Hence, the present study was designed to determine the molecular mechanisms of resistance within Gram-negative bacteria isolated from cat flu and kennel cough cases within Egypt and intended to describe the relevant pathogen-specific signs. In our team’s present knowledge, this is the first report investigating the antimicrobial resistance mechanisms associated with respiratory diseases in pets.Respiratory diseases in cats and dogs are descriptively known as cat flu and kennel cough. These conditions are complex, with infections caused by various viruses and bacteria, and typically initiated by stress-induced immunosuppression within infected petsEnterobacter cloacae (n = 15) occurred the most commonly in isolates from infected cats. followed by Escherichia coli (n = 10), and E. fergusonii, Klebsiella oxytoca, Leclercia adecarboxylate, Pantoea septica, P. aeruginosa, and Raouletella ornithinolytica, each occurring as a singleton infection from a single animal. Symptoms and their relation to the infecting bacteria are displayed in Table Out of the 875 studied cats, 47 specimens  were collected from 44 animals with symptoms of respiratory disease in a sample fraction equal to 5% (95% CI 3.7–6.7%). Thirty-one Gram-negative bacteria were isolated from 24 cats in a percentage comprising 54.5% (95% CI 38.9–69.7%). Various species of Gram-negative bacteria were isolated from the sampled diseased cats, either as singleton or mixed infections. acae n = 5 occurreE. cloacae (n = 15) was the most commonly occurring Gram-negative bacteria isolated from dogs, followed by E. coli (n = 12), K. pneumoniae (n = 4), Citrobacter braakii, and R. ornithinolytica (n = 3 each), C. freundii (n = 2), and E. hormaechei, C. farmeri, K. oxytoca, and Serratia marcescens (n = 1 each).Out of the 503 dogs examined in the present study, 37 specimens  were harvested from 32 animals in a sampling fraction of 6.4%. Gram-negative bacteria were isolated from 27 animals . Akin to cats, P < 0.05) (see the “P < 0.08). On the other hand, there was no significant association found between the course of illness and isolate type (P > 0.05). There was no significant association either between animal species and specific type of isolates (P > 0.05) or between animal age and isolate type (P > 0.05). There was no significant association between the type of isolate and associated clinical signs except for E. cloacae which was found to be significantly associated with sneezing and ocular discharge (P < 0.05).Symptoms and their relation to the infecting bacteria are displayed in Table n = 20) (95% CI 17.4–38.6%). With the exception of carbapenems, the isolates demonstrated high resistance rates to almost all the β-lactam tested  95% CI 7.4–38.6%) 95% CI 7.4–38.6%n = 30) (95% CI 29.3–52.6%) of the isolates, and 24.3% (n = 18) (95% CI 15.1–25.7%) of the isolates were phenotypically positive to AmpC production  of the isolates; 18 isolates contained blaSHV-12; a single isolate possessed blaSHV-11. The second most prevalent ESBL-encoding gene was blaCTX-M, which was present within 17.6% (95% CI 9.7–28.2%) of the isolates, while five isolates contained blaCTX-M-15; four isolates contained blaCTX-M-14; 2 isolates contained blaCTX-M-37; 2 isolates possessed blaCTX-M-156. Nine isolates  contained blaTEM, while all isolates were confirmed to be blaTEM-1 which is not an ESBL-encoding gene. Five isolates registered as phenotypically positive for ESBL production, while genotypic screening confirmed that the isolates were either negative (n = 4) or contained blaTEM-1 (n = 1). It follows, the isolates may also possess other unchecked or rare ESBL-encoding genes.Varying ESBL-encoding genes were identified via PCR screening and DNA sequencing within 33.8% 25/74) (95% CI 23.2–45.7%) of the isolates ; two isolates contained blaACT25/31; a single isolate possessed blaCMY-2; a single isolate contained blaACT23. Two isolates were confirmed to possess blaDHA-1  (95% CI 4.8–20.2%) of the isolates contained varying AmpC-encoding genes. A-1 Fig. . Ten isoqnr gene detected within this study comprises qnrS, found in 44.6% (33/74) (95% CI 33.02–56.6%) of the tested isolates. The second most prevalent qnr is qnrA detected within 10.8% of isolates, followed by qnrB, found within 9.5% of isolates. All the isolates were negative for the qepA gene and 16S rRNA methylases.The plasmid-mediated quinolone resistance genes (PMQR) were characterized within 64.9% (48/74) 95% CI 52.9–75.6%) of the isolates 18. Furthermore, our team found it surprising that the isolates demonstrated extremely high resistance levels for different β-lactams, in particular AMP (87.7%), CRO (74.3%), AMC (68.9%), and FOX . The ampicillin resistance level is much higher than that previously reported from fecal E. coli from Portugal (51.3%)18 and Denmark (40%)19. Furthermore, β-lactams resistance was also comparable or higher than previously reported within healthy dogs in other European countries including the Netherlands, the United Kingdom, and France22. The isolates also demonstrated a high resistant rate to tetracycline (69%), which is also much higher than rates previously recorded in Portugal by 45.2%18. The widespread prevalence of β-lactam and tetracycline resistance among Gram-negative bacteria associated with respiratory diseases of pets may possibly be attributed to dependence on these drugs as a cheap and readily available remedy for the treatment of pets’ infections8. On the other hand, our results demonstrated low levels of resistance to other drugs, in particular for carbapenems, which are strictly utilized only for humans in Egypt13, and aminoglycosides and chloramphenicol, which may be due to their limited use, owing to their costs or their complications8.The results of the present study indicate a widespread prevalence of multidrug-resistant bacteria within pets inflicted with kennel cough and cat flu by 27%, but lower than that previously identified within enteric 8. Our results showed that 40.5% of them were phenotypically positive for ESBL production, with 33.8% of the isolates containing at least one ESBL-encoding gene  from pets in Egypt, the Middle East, and even the entirety of Africa. blaDHA-1 has been recently identified within pets in Italy28, and blaACT gene was identified in Germany29.The results of the present study demonstrated that 24.3% of the isolates registered as phenotypically positive for AmpC production, with 10.8% carrying AmpC-encoding genes. These results are higher than previously reported levels from pets in the UK, where 16% of the isolates were phenotypically positive, and only 4% harbored 6. Due to this fact, in recent years PMQR genes have gained special attention due to their high transmission rates between bacteria via plasmid8. Unsurprisingly, our results highlight a high prevalence of qnr genes within pets in Egypt by 64.9%, with qnrS occurring as the most prevalent (44.6%), supporting our previous reports from clinical settings and food settings in Egypt14. Our results are much higher than those previously reported within other countries, where qnr genes could not be identified in pets from France/Spain and the UK21. In a recent large-scale study within Europe, qnr genes were detected in 20% (32/160) of enrofloxacin non-wild type (resistant) Enterobacterales, which represent 3.8% of the total isolates (32/843)30. However, qnr genes induce low levels of quinolone resistance, but mediate the selection mutations leading to high quinolone resistance31. Our results are inconsistent with the previous findings, as 14.3% and 16.3% of qnr containing isolates were resistant, and intermediate to nalidixic acid, respectively, and 6.1% and 49.9% of the isolates were resistant and intermediate to ciprofloxacin (data not shown). Our findings confirm our previous studies regarding the lower role of the qepA gene and 16S rRNA methylases in the mediation of quinolones and aminoglycosides resistance, respectively8.Quinolones comprise a major group of antibiotics that are commonly used in both human and veterinary medicine and have been identified as one of the most critically important drugs for humansIn summary, the present study reveals wide variation within clinical signs, and a high prevalence of ESBL, AmpC, and PMQR genes across Gram-negative bacteria associated with cat flu and kennel cough within Egypt. Furthermore, unique resistance mechanisms and bacterial species were isolated and identified for the first time within Egypt and the entirety of Africa. Our results are of great clinical concern, as pets could possibly act as a potential reservoir for these antimicrobial resistance determinants that could further disperse them into the human environment. Taking into consideration that the majority of the resistant determinants were previously identified from clinical settings and food in Egypt, the query of animal-human transmission is still needed to be confirmed through future studies.n = 875) and dogs (n = 503) hailing from five shelters located in Giza which collect stray pets, as well as those released by their owners in lower and middle Egypt, were selected based upon information regarding outbreaks of respiratory illness. A total of 76 diseased animals (44 cats and 32 dogs) were subjected to precise clinical examination between March and April 2017, and 84 swabs—including 76 nasal swabs and 8 conjunctival swabs—were aseptically collected. All the swabs were transferred to the lab at 4 °C under aseptic conditions for further analysis. All experimental protocols were carried out according to the Kafrelsheikh University Animal Experimentation Regulations, and approved by the Committee on Animal Experiments, Faculty of Veterinary Medicine, Kafrelsheikh University.In the present study, a total of 1,378 pet animals, including cats (n = 65 isolates) and the conjunctiva (n = 9 isolates) of 51 pets, including dogs (n = 43 isolates) and cats (n = 31 isolates), that were tested in this study. All the isolates were identified using MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry). The isolates were detected to comprise Enterobacter spp. (n = 31), Escherichia spp. (n = 23), Klebsiella spp. (n = 6), Citrobacter spp. (n = 6), R. ornithinolytica (n = 4), Pseudomonas aeruginosa (n = 1), S. marcescens (n = 1), L. adecarboxylate (n = 1), and P. septica (n = 1) , AMK (30 µg), AMP (10 µg), CHL (30 µg), CRO (30 µg), CFP (75 µg), FOX (30 µg), CIP (5 µg), GEN (10 µg), IPM (10 µg), MEM (10 µg), NAL (30 µg), and TET (30 µg). All of the discs were purchased from Becton, Dickinson and Company Sparks .The Kirby–Bauer disc diffusion method was utilized in order to evaluate all of the bacterial isolates for the antimicrobial susceptibility phenotypes to different classes of antibiotics, according to the Clinical and Laboratory Standards Institute (CLSI)33. Briefly, the bacterial isolates were cultured overnight at 37 °C and a 1-μl inoculation loop was added to 2 ml of tryptic soy broth within a tube. Following 15 s of vortexing, a 10-μg MEM disc was added to the bacterial suspension. The bacterial submission containing the MEM disc was incubated at 37 °C for 4 h. Before the end of the incubation time, the indicator organism (0.5 McFarland standard E. coli ATCC 25922) was inoculated at the Mueller Hinton agar plate (MHA). After 4 h incubation of bacterial submission containing the MEM disk, the MEM disk was placed on the inoculated MHA, followed by incubation at 37 °C for 18–24 h. After the incubation period, the result was evaluated by measuring the inhibition zone around the MEM disc.Phenotypic carbapenemase detection was tested via the mCIM method as previously described34. Briefly, the bacterial isolates were cultured overnight at 37 °C on MHA plates, followed by their inoculation at a concentration of 0.5 McFarland standard on MHA plates utilizing a sterile swab. The D68C AmpC and ESBL detection kits contain four different discs, Disc A contains 10 μg of cefpodoxime, disc B 10 μg of cefpodoxime and ESBL inhibitor, disc C 10 μg of cefpodoxime and AmpC inhibitor, and disc D 10 μg of cefpodoxime and both AmpC and ESBL inhibitors. The four discs were placed on the inoculated plates, followed by incubation at 37 °C for 24 h. The final results were evaluated by measuring and contrasting the inhibition zones’ diameters around the four discs. In the phenotypic assays, E. coli ATCC 25922 was employed as a negative control and an indicator, while ESBL-, AmpC-, and carbapenemases-producers from our previous reports were employed as positive controls13.Phenotypic ESBL and AmpC detection were evaluated utilizing the D68C AmpC and ESBL detection kits  following the instruction of the producing company and as previously reported14. Briefly, a full 1-μl inoculation loop of bacteria culture incubated overnight on MHA was mixed with 100 µl of sterilized, distilled water, followed by boiling for 8 min. The bacterial suspension was thoroughly shaken then centrifuged, and the supernatant was utilized as a template for PCR experiments.The DNA was prepped by boiling lysates following the method as previously indicated40  and the AmpC-phenotypic positive isolates were tested for plasmid-mediated AmpC β-lactamase genes , and all the isolates were checked for plasmid-mediated quinolone resistance genes , qepA gene which is quinolone efflux pump determinant, and 16S rRNA methylases .Genetic characterization of the isolates was conducted by PCR experiment and sequencing. The isolates were tested for a wide range of antimicrobial resistance mechanisms via single and/or multiplex PCR as previously describedhttp://blast.ncbi.nlm.nih.gov/Blast.cgi).Target genes were confirmed via single PCR, and PCR fragments were then purified from PCR product or agarose gels using CICA GENEUS PCR & Gel Prep Kit . Following sequencing, the similarity search was performed with the sequenced data using BLAST analysis  and SAS 9.2 (Institute Inc 2008). A Supplementary Information


KTN1) variants on the putamen GMV.Selective loss of dopaminergic neurons and diminished putamen gray matter volume (GMV) represents a central feature of Parkinson’s disease (PD). Recent studies have reported specific effects of kinectin 1 gene , putamen GMV, and PD.To examine the relationship of KTN1 single nucleotide polymorphisms (SNPs) in one discovery sample , and confirmed the nominally significant associations (p < 0.05) in two replication samples . The regulatory effects of risk variants on the KTN1 mRNA expression in putamen and SNc and the putamen GMV were tested. We also quantified the expression levels of KTN1 mRNA in the putamen and/or SNc for comparison between PD and HC in five independent cohorts.We examined the associations between PD and a total of 1847 imputed KTN1-PD associations were identified (0.009 ≤ p ≤ 0.049). The major alleles of five SNPs, including rs12880292, rs8017172, rs17253792, rs945270, and rs4144657, significantly increased risk for PD (0.020 ≤ p ≤ 0.049) and putamen GMVs . The risk alleles of five SNPs, including rs8017172, rs17253792, rs945270, rs4144657, and rs1188184 also significantly increased the KTN1 mRNA expression in the putamen or SNc (0.021 ≤ p ≤ 0.046). The KTN1 mRNA was abundant in the putamen and/or SNc across five independent cohorts and differentially expressed in the SNc between PD and HC in one cohort (p = 0.047).Six replicable and two non-replicable KTN1 variants, PD risk, KTN1 mRNA expression in putamen, and putamen volumes, and a modest relation between PD risk and KTN1 mRNA expression in SNc, suggesting that KTN1 may play a functional role in the development of PD.There was a consistent, significant, replicable, and robust positive relationship among the  The nigrostriatal dopaminergic pathway connects the substantia nigra pars compacta (SNc) with the dorsal striatum, forms part of the extrapyramidal system, and plays a central role in motor control . SelectiConsistent with the loss of dopaminergic neurons, imaging studies have demonstrated altered nigrostriatal functions in PD . For exaB3GALT4 and ST3GAL2 , i.e., rs945270, demonstrated the genome-wide strongest (p = 1.1 × 10–33), replicable, and specific effects on the putamen gray matter volume (GMV) in subjects without neurodegenerative or neuropsychiatric disorders , rs8017172 (3′-UTR), and rs17253792 (3′-UTR), significantly increased the putamen GMVs too  (cis-regulate the methylation of CpG islands in the putamen (p = 4.4 × 10–6)  . Except KTN1 variants as a genetic risk factor for PD, the roles of the KTN1 variants in regulating mRNA expression in the putamen and SNc and putamen GMV, and whether KTN1 mRNA may be differentially expressed in the putamen and SNc between PD and controls. The overall design of this study is illustrated in In the present study, we aimed to examine the We examined three independent population-based Caucasian samples: “PD_ENV” (dbGaP access number: phs000196.v3.p1), “phg000022” (phs000126.v2.p1), and “lng_coriell_pd” (phs001172.v1.p2). The first sample served as the discovery sample and the other two served as the replication samples. The discovery sample included 2,000 subjects with PD  and 1,986 healthy subjects . The first replication sample, i.e., “phg000022,” included 900 subjects with PD  and 867 healthy subjects . The second replication sample, i.e., “lng_coriell_pd,” included 940 subjects with PD  and 801 healthy subjects .All cases met the UK Parkinson’s Disease Society Brain Bank clinical diagnostic criteria for PD , and werKTN1 region (Chr14:54995382-55550419) by the program IMPUTE2  (BRAINEAC dataset) (n = 170) (GTEx dataset) (cis-acting expression quantitative trait locus (cis-eQTL) analysis.After the risk dataset)  and a Eudataset)  were anan = 13,145 and 37,571, respectively) [Enhancing Neuro Imaging Genetics through Meta-Analysis (ENIGMA2) consortium – GWAS Meta-Analysis of Subcortical Volumes]1 ]  >5.17, were taken as “expressed.” mRNA expression in Cohort 2 was examined using RNA-Seq . The expression levels with RPKM values >1 were taken as “expressed” (The (UKBEC)] , 124 put(UKBEC)] ], 15 put(UKBEC)] , 9 SNc t(UKBEC)] , and 14 (UKBEC)]  extracte(UKBEC)] , 16 SNc (UKBEC)] , and 14 (UKBEC)]  extractepressed” .KTN1 mRNA in the putamen and/or SNc in Cohorts 3, 4, and 5 were compared between PD and healthy subjects using analysis of covariance (ANCOVA) with age as a covariate. A p < 0.05 indicates significantly different expressions.As the putamen volume decreases with age , the expKTN1 SNPs were analyzed in the discovery sample (“PD_ENV”), including 142 SNPs nominally associated with PD (p < 0.05). Among them, six SNPs significantly associated with PD in the discovery sample were also associated with PD in at least one replication sample (“phg000022” or “lng_coriell_pd”), including four replicable associations between “PD_ENV” and “phg000022” samples (0.009 ≤ p ≤ 0.049) and two replicable associations between “PD_ENV” and “lng_coriell_pd” samples (0.020 ≤ p ≤ 0.045). These six replicable risk SNPs, together with the other two non-replicable GMV-associated variants   (p = 0.049 for rs17253792) and/or the replication sample “phg000022” .A total of 1847 imputed s945270) , were los945270) , includictively) , were poThe eight risk variants were located in four haplotype blocks. Bioinformatic analysis showed that the variants within the same haplotype blocks were not only highly linked but also shared similar biological functions. All of the three variants in H3 were located in long intergenic non-coding RNAs (lincRNAs). The variant in H4, i.e., rs1188184, was located in an enhancer. Furthermore, two risk variants were, respectively, located on two transposons, including rs12880292 on the DNA transposon Arthur1B (163 bp), and rs8017172 on the DNA transposon MER5A (150 bp).KTN1 mRNA expression in putamen both in UK Europeans  and European-Americans . Two risk alleles of rs17253792 and rs1188184 increased mRNA expression in putamen in European-Americans too . Additionally, the risk allele of rs4144657 increased mRNA expression in SNc in European-Americans (p = 0.045). Four risk alleles of rs8017172, rs17253792, rs945270 and rs4144657 were all major alleles (f > 0.5), consistent with previous reports  and rs4144657 in H3 block  that modestly increased the putamen GMVs, and rs8017172 , rs17253792  and rs945270  in H2 block that highly significantly increased the putamen GMVs. All of these five risk alleles were major alleles (f > 0.5). These SNP-GMV associations were consistent with previous reports ], 38.70 [Transcripts Per Kilobase Million (TPM)] and 9.79 ± 0.62 [log2], respectively. KTN1 mRNA was also abundantly expressed in PD subjects in Cohort 3 (expression level = 9.86 ± 0.44), at a level higher than control subjects, although the difference was not statistically significant (p = 0.858).In three independent cohorts, Cohorts 1, 2, and 3, the KTN1 mRNA was abundantly expressed in the SNc too. The expression levels in control subjects were 5.76 ± 0.35 [log2], 55.70 [TPM], 10.04 ± 0.75 [log2], 10.76 ± 0.26 [log2], and 9.18 ± 0.49 [log2], for Cohorts 1–5, respectively. Furthermore, the expression levels in PD subjects in Cohorts 3, 4, and 5 were 10.03 ± 0.50, 10.95 ± 0.19, and 9.14 ± 0.31 [log2], respectively. The expression levels were significantly higher in PD than in control subjects in Cohort 4 (p = 0.047) but were not different for Cohorts 3 and 5 (p’s > 0.05).Across the five independent cohorts, the KTN1 variants that were associated with PD across at least two independent samples, and two other GMV-associated variants that were associated with PD in one sample. Six of these risk variants may be biologically functional, regulating either the mRNA expression in putamen or SNc or the GMVs of putamen. KTN1 mRNAs were expressed in the putamen and/or SNc across five independent cohorts, and were differentially expressed in the SNc between PD and controls in one cohort. Together, these results suggest that KTN1 plays a functional role in the development of PD, supporting previous findings of associations between KTN1 variants and PD   , substanr (ADHD) , and cogr (ADHD) . We notifor ADHD . This waKTN1 SNPs, KTN1 mRNA expression in the putamen and SNc, GMVs of putamen and PD. The findings revealed a statistical but not causal relationship. Future research to reveal the cause-effect relationships would require a prospective, functional study with an appropriate intervention, to answer whether KTN1 variants regulate the development of putamen GMVs and PD, and whether the putamen GMV alteration results in or from the development of PD. The second limitation is that these associations were not analyzed in the same samples. For example, the SNP-mRNA and SNP-GMV associations were separately analyzed only in the samples without PD; and the SNP-PD association was analyzed only in the samples without GMV data; and therefore, we were unable to know the interactive impacts of SNPs, mRNA expression, putamen GMV and PD on one another from these separate samples. Future research to know their interactive impacts would require studying them in the same sample. Finally, some associations among lncRNAs, enhancers, KTN1 mRNA, kinectin, and PD have never been studied, forming the research gaps as shown in A major limitation of the present study is that the primary analyses focused on the cross-sectional or retrospective associations among KTN1 variants and PD, the replicable associations between KTN1 variants and putamen GMVs, the potential regulatory effects of KTN1 variants on the mRNA expression in putamen and SNc and the activities of lncRNA and enhancer, the abundant KTN1 mRNA expression in putamen and SNc, the differential mRNA expression in SNc between PD patients and controls, and the reported associations between putamen GMVs and PD, suggested that KTN1 variants may underlie the putamen GMV and risk for PD.In summary, the robust associations between phs000196.v3.p1, phs000126.v2.p1, and phs001172.v1.p2.The datasets generated for this study can be found in the dbGaP: Accession The studies involving human participants were reviewed and approved by the Human Investigation Committee of Yale University. The patients/participants provided their written informed consent to participate in this study.XGL provided the resources (patients and data). QM, LF, XW, YZ, Y-CW, JJ, JX, HZ, CZ, KW, C-SL, and XGL conducted the study and performed the analysis. C-SL and XGL were responsible for the overall content as guarantors. All authors contributed to the formulation of overarching research goals and aims and the writing, reviewing, and editing of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GA3 was applied exogenously  on 15-days-old plants, and the results show that the increasing concentration of GA3 buffered the phytotoxic effects of Cu, coupled with an increase in plant growth and physiological variables. The results also showed that foliar-applied GA3 up to 100 mg·L−1 alleviated the oxidative stress, as inferred from the lower concentrations of malondialdehyde (MDA) and H2O2, which mirrored the increased activity of antioxidant enzymes, i.e., superoxide dismutase, peroxidase, and catalase. In addition, enhanced growth, physiology, and enzymatic activities were also observed in pea plants sprayed with GA3 up to 100 mg·L−1 in Cu− soil. Overall, the foliar application of GA3 boosted phytoextraction of Cu from the soil and alleviated the oxidative stress in pea plants grown in Cu-polluted soil.Phytohormones are key regulators of several stages of plant growth and development as well as provide the regulatory response against various heavy metals stresses by mediating physio-morphological responses and enzymatic activities. The current study evaluated the effects of gibberellic acid (GA3) foliar applications on the performance of pea plants grown either in Cu-contaminated (Cu+) and non-contaminated (Cu−) soil. GA3 was sprayed  on 15-days-old plants. The results showed that the increasing concentration of GA3 buffered the phytotoxic effects of Cu and enhanced plant growth, photosynthesis, and leaf chlorophyll content. Foliar-sprayed GA3 up to 100 mg·L−1 alleviated the oxidative stress, as inferred from the lower concentrations of MDA and H2O2 , and boosted the activity of superoxide dismutase (64.4 U·g−1·FW), peroxidase (122.7 U·g−1·FW), and catalase (226.3 U·g−1·FW). Interestingly, GA3 promoted Cu accumulation in different plant parts when compared to untreated plants, likely due to increased photosynthetic and transpiration rates. Overall, foliar application of GA3 promoted phytoextraction of Cu and alleviated the oxidative stress in pea plants grown in Cu+ soil.Copper (Cu) is an essential metal for plants. However, its excess in soil can adversely affect plant metabolism. The current study evaluated the effects of gibberellic acid (GA Pisum sativum L.) is an important leguminous crop, whose high protein content (up to 22–23%) is almost three times that of cereals pyrene-contaminated soil [Gibberellic acid -contaminated soil [Corchorus capsularis L. plants growing in Cu-contaminated soil [3 to enhance plant growth and development in metal-contaminated soil, however, available on other plant species, such as and Solanum nigrum L., and Tagetes patula L. [Pisum sativum L. plants due to high nutritive value and negative impacts of Cu on human health demands phytoremediation; however, sufficient information is not available regarding Cu tolerance, antioxidative defense mechanism, and Cu accumulation in different parts of this species, when grown under different concentrations of foliar applied GA3. Therefore, it is vital to explore the protective and growth promoting role of GA3 in Cu-contaminated soils. It may be hypothesized that GA3 alleviates Cu and oxidative stresses by modulating some physio-morphological and biochemical processes in pea. To test this hypothesis, pea plants were germinated on Cu-contaminated soil and then foliar sprayed with 10, 50, and 100 mg·L−1 GA3 at 15 days after germination. In addition to evaluating key stress benchmarks on plant growth , accumulation of electrolytes, H2O2 and MDA, and activities of key of radical oxygen-scavenging enzymes catalase, peroxidase, and superoxide dismutase were also quantified. Exogenous applications of GAelopment . In addid plants . Previouted soil . The posted soil . There iatula L. ,15. The −1, pH 7.5, available K 344 mg·kg−1, available P 12.21 mg·kg−1, and Cu 109 mg·kg−1. Total Cu (2000 mg·kg−1), as CuSO4·5H2O, was mixed thoroughly in soil prior to filling in pots, to simulate Cu contamination (Cu+ soil) [Pisum sativum L.) with 98% germination capacity was procured from Ayub Agricultural Research Institute, Faisalabad, Pakistan and used throughout the course of the experiment. The experiment was conducted in the glasshouse at the Research Area, Department of Agronomy, University of Agriculture, Faisalabad, from November 2018 to January 2019. Plants in the glasshouse received natural light, with day/night average temperature of 26/15 °C and day/night average humidity of 65/80%. The bi-factorial experiment was laid out under completely randomized design (CRD), with four replications (each replication contained five pots for each treatment). Ten seeds were directly sown in each pot. Each replication contained 50 plants of each experimental unit. Irrigation, weeding, and necessary agronomic practices, based on physical observations, were done when needed.The soil for the pot experiment was collected from the Research Area, Department of Agronomy, University of Agriculture, Faisalabad City, Punjab Province, Pakistan  at depth of 0–20 cm. After grinding, the soil was mixed thoroughly, air dried under shade, and sieved through a 5 mm sieve before filling in pots (10 kg soil per pot). The textural class of soil for experimentation was loam and, before the start of the experiment, had the following characteristics: Electrical conductivity (EC) 0.432 dSm3 were done 14 days after seeding. From 10:00 until 11:00 am, treatments, i.e., 0, 10, 50, 100 mg·L−1 were applied by exogenously spraying GA3 on whole seedlings until solution falls and treatments were applied only once during the whole experiment [Foliar sprays of GAFive uniform plants were randomly selected from each replication to determine the number of leaves, plant height, and plant relative fresh and dry weights at 40 days after seeding (DAS). To determine relative fresh and dry weights, plants were washed with distilled water, dried with paper and weighed for their fresh weight followed by oven drying at 70 °C until constant weight achieved. After oven drying, dry weights were recorded.2 m−2·s−1), stomatal transpiration rate (mmol H2O m−2·s−1), and CO2 concentration (µmol·mol−1) were made on fully expanded leaves from the top of the plant canopy by using an open system LCA-4 . These measurements were made from 10:00 to 11.00 a.m., with the following specifications: Ambient pressure (P) 88.76 kPa, molar flow of air per unit leaf area (Us) 201.89 mol m−2·S−1, temperature of leaf chamber (Tch) varied from 38.9 to 42.4 °C, leaf chamber molar gas flow rate (U) 236 µmol·S−1, leaf surface area 10.11 cm2, and photosynthetically active radiation (PAR) at leaf surface was maximum up to 887 µmol m−2·s−1.At 40 DAS from five uniform randomly selected plants from each replication, leaf chlorophyll content was measured with a chlorophyll SPAD meter  according to manufacturer’s instructions, and presented as SPAD values. On the same date measurements of net photosynthetic rate  [To determine malondialdehyde (MDA) content, an indicator of lipid peroxidation, 0.1 g leaves were ground with 25 mL of 50 mM phosphate buffer solution containing 1% polyethylene pyrrole with the help of pestle and mortar. After centrifugation at 12,000× ire, UK) . 2O2 concentration, leaf samples (1 g) were ground in 9 mL of normal saline solution (4.5 g NaCl added in 500 mL ddH2O) followed by centrifugation at 10,000× g for 10 min. Three tube types were prepared, namely empty, standard, and sample tubes. Briefly, reagent 1 and 2 (1.0 mL) in all tubes, H2O (0.1 mL) in empty tube, standard solution (0.1 mL) in standard tube, and sample (0.1 mL) in sample tube was added. The absorbance was taken at 405 nm with spectrophotometer according to H2O2 determination kit .To determine H1). After measuring the initial EC1, samples were autoclaved at 121 °C for 20 min followed by cooling down to 25 °C to measure the final electrical conductivity (EC2). To measure the electrolyte leakage, a pH/conductivity meter  was used, then the following equation for EL calculation was applied: To determine electrolyte leakage (EL), fully expanded leaves from the top of the plant canopy were taken, followed by cutting into minor slices (5–6 mm length), placed in sterilized test tubes having 8 mL distilled water, incubated, and transferred to water bath for 12 h prior to measuring the initial electrical conductivity . The absorbance was taken at 240 nm with the spectrophotometer. The POD reaction solution (3 mL) contained 0.1 mL enzyme extract, 50 mM sodium acetate buffer (pH 5.0), 40 mM H2O2, and 20 mM guaiacol. The absorbance was taken at 470 nm. The superoxide dismutase (SOD) reaction solution (3 mL) contained 1.3 µM riboflavin, 50 µL enzyme extract, 50 µM nitroblue tetrazolium (NBT dissolved in ethanol), 13 mM methionine, 50 mM phosphate buffer (pH 7.8), and 75 nM EDTA [To determine antioxidant activities, 0.5 g leaves were ground using a tissue grinder in 8 mL of cooled phosphate buffer  polyvinylpyrolidone) in test tubes. The homogenate was centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was used for assays of enzymes activity. The activity of catalase (CAT) and peroxidase (POD) was measured by using the method of Maehly . The rea nM EDTA . The abs3/HClO4 (4:1) solution followed by dilution of digested sample in de-ionized water up to final volume of 25 ml. The supernatant was taken and passed through a filter paper. Copper standard solution  was used as a primary calibration standard for the quantitative determination of copper in roots, leaves, and stem of pea. Readings were taken by using a Perkin-Elmer 3100 Atomic Absorption Spectrophotometer .To determine the Cu content, in root, leaves, and stems, respective parts from ten uniform randomly selected plants were taken, oven dried, and ground at 40 DAS. Briefly, 0.1 g of the respective ground sample was digested in HNO3 application) was executed to evaluate the effects of GA3 on the recorded variables both under normal  and under Cu-contaminated soil conditions. Tukey’s honestly significance difference (HSD) test was used for comparison of treatment means (p ≤ 0.05), using a statistical software package ‘Statistix 8.1’ (https://www.statistix.com/). A principal component analysis among treatments and dependent variable was executed using XLSTAT ver. 2018 (https://www.xlstat.com/) to delineate the effect of different doses of GA3 on physiological growth attributes of pea plants grown in Cu− and Cu+ soil. Clustering of observations and variables into groups were done on the basis of their highest squared cosine values corresponding to factors, F1 and F2. Correlation coefficients among variables were determined with Pearson (n) method.A two-way analysis of variance . Similarly, maximum plant relative fresh weight (34 g) was recorded in plants receiving 100 mg·L−1 GA3 grown in Cu− soil followed by the plants receiving same treatment grown in Cu+ soil, indicating that GA3 increased the above ground biomass accumulation in pea  as compared to the other treatments. Though all foliar applied GAn in pea .−1 GA3 showed the highest values in both soils (17.2 g Cu− soil and 15.2 g Cu+ soil) followed by 50 and 10 mg·L−1 GA3 (3 showed an increased number of leaves in a dose-dependent manner as compared to control. The plants treated with 100 mg·L−1 showed the maximum number of leaves in both soils (30.1 Cu− soil and 28.3 Cu+ soil). The lowest values for plant height, fresh/dry weight, and number of leaves per plant were recorded in control followed by 10 mg·L−1 GA3 application in both Cu− and Cu+ soil  followed by plants treated with 50 mg·L−1 GA3  and 10 mg·L−1 GA3  in Cu− soil. Similarly, the experimental units received foliar applied 100 mg·L−1 GA3 showed the highest chlorophyll contents, photosynthetic rate, transpiration rate, and CO2 concentration  in Cu+ soil. Whereas the untreated plants grown in Cu+ soil showed minimum values of chlorophyll contents, photosynthetic rate, transpiration rate, and CO2 concentration , showed the positive role of GA3 in improving physiological response of pea . The plants grown in Cu+ soil exhibited increased electrolyte leakage, MDA, and H2O2 contents than those were grown in Cu− soil. The exogenous application of GA3 significantly reduced electrolyte leakage, MDA, and H2O2 contents in a concentration-dependent manner. The maximum decrease in MDA, H2O2, and electrolyte leakage were observed in plants treated with 100 mg·L−1 GA3 in Cu−  and Cu+ soils  as compared to other experimental units and control  followed by the plants grown in Cu+ soil (64.39 U·g−1·FW). In similarity with the aforementioned variable, the highest POD activity (136.21 U·g−1·FW) was also observed in pea plants grown in Cu− soil treated with 100 mg·L−1 GA3. Plants receiving foliar application of 50 mg·L−1 GA3 also showed better performance in both soil conditions. In the case of CAT activity, maximum value (245 U·g−1·FW) was also recorded in plants treated with 100 mg·L−1 GA3 in Cu− soil. The reduced activity of antioxidants i.e., SOD, POD, and CAT in untreated plants grown in Cu+ soil indicates a significant effect of Cu stress on pea plants  as compared to all other treatments. The foliar application of GA3 enhanced the uptake of Cu in plants. The plants grown in Cu+ soil showed more Cu concentration in roots, leaves, and stem as compared to those grown in Cu− soil. The plants treated with 100 mg·L−1 GA3 showed maximum Cu concentration in roots, leaves, and stem of peas grown in both soils  (The GActively) . 2 index, H2O2 contents, SOD, POD, and CAT activity with 50 and 100 mg·L−1 GA3 treatment in plant grown in Cu− soil, as well as 100 mg·L−1 GA3 treatment in plant grown in Cu+ soil. Malondialdehyde, electrolyte leakage, and H2O2 contents are shown in the negative side of the F1 axis, indicating their negative correlation with 100 mg·L−1 GA3 and positive correlation with control (no GA3 treatment)    . −1 GA3 treatment on plants grown in Cu+ soil, while negative correlation with 10 mg·L−1 GA3 treatment on plants grown in Cu− soil. Thus, principal component analysis delineated the morphological, physiological, and oxidative response of pea plants under the influence of GA3 and Cu toxicity.Second factor F2, having eigenvalue 3.059 and variability of 17.99% represented Cu contents in leaves, roots, and stems. Cu contents in leaves, roots, and stem are shown in the positive half of F2 depict their positive correlation with 50 mg·L3 alleviated the toxic effect of Cu and enhanced plant height, number of leaves, and biomass accumulation in pea plants grown in both Cu+ and Cu− soil  decreased the production of MDA, H2O2 contents, and electrolyte leakage compared to control (no GA3 application). In contrast, all concentrations of foliar applied GA3 enhanced the activities of SOD, POD, and CAT, but higher values for antioxidants enzymes were linked with 100 mg·L−1 GA3 (2O2 contents [3 to ameliorate the oxidative stress response in Corchorus capsularis L. Moreover, upregulation of activity of various antioxidant enzymes such as SOD, POD, and CAT shows the higher capacity of plants to scavenge excessive ROS under Cu stress [Copper stress rigorously impeded the performance of crop plants . In addia sativa  and Brasca napus  plants u·L−1 GA3 . The enhcontents ,33,41. Tu stress , who arg3 exhibited enhanced Cu accumulation in roots, leaves, and stem under Cu stress (3 was likely the reason behind the increased Cu uptake by plants, even under phytotoxicity conditions [3 to modulate the plant physiological status depends on its concentration, application method, and plant genetics [3 application changed with change in concentration, mostly under Cu stressing conditions, overall, suggesting that GA3 promoted growth of pea plants and alleviated the Cu toxicity. To this end, foliar application of 100 mg·L−1 induced the best response in pea plants in terms morphological, physiological, and oxidative attributes. Depending on growth conditions, plants vary in their capacity of Cu uptake and accumulation. Plant roots play a central role in Cu uptake and transfer to stem and leaves through the xylem ,44. Pea u stress . The incnditions . The eff3 proved to be successful for enhancing tolerance to Cu stress in pea plants, as evidenced from higher plant growth and antioxidant activity. In this view, the foliar application of a 100 mg·L−1 GA3 solution proved to be the most effective in enhancing tolerance to Cu and related oxidative stresses in pea plants, while at the same time maximizing the Cu content in plant organs. Results also reveal that GA3 alleviated Cu-induced stress on pea plants by stimulated activities of reactive oxygen-scavenging enzymes catalase, peroxidase, and superoxide dismutase, which not only helped in reducing electrolyte leakage, but also hindered accumulation of MDA and H2O2 in Cu-stressed pea plants. Overall, this indicates the possible role of this plant hormone in sustaining the phytoextraction functions of this important, N-fixing leguminous species, when crop rotations in Cu-polluted soils are concerned. Gibberellic acid is a key regulator of several stages of plant growth, development, physiology, and morphology in plants, showing also pivotal regulatory effects against various environmental stressors. In this study, foliar application with different concentrations of GA


Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological  and pathophysiological  processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry  and Western blot analysis  were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity. Breast cancer (BC) is the most frequently diagnosed cancer in women, and the second most frequent overall . This diIn this context, so-called cancer stem/initiating cells (CS/ICs) came to the forefront of research. CS/ICs are thought to be the reason for cancer recurrences, but also for tumor invasion and resistance against chemotherapy and radiation treatment . They arCell fusion is a rare but very important process found in physiological processes, including tissue regeneration , fertiliIn previous studies, we demonstrated that hybrid cells, generated by spontaneous cell fusion of human breast epithelial M13SV1-EGFP-Neo cells and different breast cancer cell lines , exhibited individual stem cell and/or tumorigenic characteristics. M13MDA435 hybrids, for example, showed increased migratory activity and different resistance capabilities compared to the parental cell lines . In addiWithin tumor tissue, there are many different cell types, such as fibroblasts, immunocompetent cells and MSCs. To investigate if spontaneous cell fusion of MSCs and breast cancer cells could take place, in vitro studies were done. Therefore, two different human breast cancer cell lines (MDA-MB-231 Hyg and HS578T Hyg) were separately cocultured with human immortalized mesenchymal stem cells (iMSC#3). Possible hybrid clones were identified by double antibiotic selection and verified by STR-analysis . Four MDInitially, general differences between the hybrid clones and their parental cell lines were examined. In an XTT-proliferation assay, the metabolic activity, which can be correlated to the proliferation of the cells, was tested. Hybrid clones MH#7 and MM#2 showed an increased proliferation rate in comparison to both parental cell lines, while MM#3 cells seemed to grow fast in the first 72 h, but then the proliferation rate decreased as fast as it first increased a. The laSkarn et al. showed that iMSC#3 cells were able to differentiate into osteoblasts . Hence, To test the clonogenic potential of the hybrid clones, a colony formation assay was performed. Hybrid clones MH#7 and MH#9 showed a significantly increased colony formation capacity in comparison to both parental cell lines. In addition, MM#2 and MM#3 cells were able to form more colonies than iMSC#3 cells a,b.Next, the ability to form mammospheres was tested. In comparison to iMSC#3 cells and HS578T-Hyg breast cancer cells, MDA-MB-231 Hyg cells formed loose cell aggregates rather than proper mammospheres a. The hyIn summary, there seem to be correlations between the results of three in four experiments, but the constitutions differ between the single cell lines. For example, the XTT-assay, mammosphere diameter and osteogenic differentiation results demonstrate that MM#2 cells expressed these characteristics at a higher level, whereas MH#9 cells expressed them on a low level. In contrast, the CFA data show exactly the opposite result. These data indicate that hybrid cells express stem cell characteristics very individually.By the use of the AldeRed Assay, the number of expressed ALDH1 positive cells was investigated. ALDH1 has been suggested to be a marker for adult stem cells, but also for cancer stem/initiating cells . As expe+/CD24−/low cells. The three parental cell lines and the four hybrid clones were analyzed using flow cytometry. In brief, all seven cell lines exhibited more than 90% of CD44+/CD24−/low cells , HER3 and epidermal growth factor receptor (EGFR) was also analyzed. While the expression of EGFR and HER2 was detectable for all cell lines, only MM#2 expressed HER3 a.To analyze which signaling pathway may be active in the hybrid cells, cells were stimulated with epidermal growth factor (EGF) for 1, 2 or 5 min. Afterward, Western blot analysis was used to investigate if phosphorylated Akt and/or Erk1/2 is expressed b. NonphoTo test whether the hybrid clones react differently to cytostatic drugs, XTT analysis was performed using either paclitaxel or doxorubicin at concentrations of 0.1 µM/1 µM/10 µM. In brief, all cell lines died quickly after treatment with paclitaxel. Similar findings were observed after treatment with doxorubicin for iMSC#3, HS578T Hyg, and MM#2 cells. In contrast, MDA-MB-231 Hyg, MM#3, MH#7 and MH#9 cells exhibited resistance against doxorubicin at a concentration of 0.1 µM a,b.In the present study, we characterized hybrid cells obtained by the fusion of human BC cells and human MSCs. We wanted to examine whether CSC-like cells can be formed by cell fusion and if CSCs might develop certain properties during this process. We used MSCs because these cells are located in the tumor microenvironment where they are in direct contact with cancer cells and might contribute stem-like properties, i.e., inducing EMT. The tumor microenvironment affects tumor progression and cell fusion in a crucial way, enabling different cell types to come in contact and containing molecules such as cytokines, which might function as inducers for different processes such as cell fusion . Even ifWe also wanted to look for CSC properties in our hybrid cells. Therefore, we used different markers that had been described before. Dontu et al. were the first to describe the formation of mammospheres by human mammary stem/progenitor cells . They el+/CD24−/low phenotype are distinctive for stem-like cell lines [+/CD24−/low phenotype , and its reversal mesenchymal to epithelial transition (MET) might have on tumor progression. The transition of an epithelial to a mesenchymal phenotype is induced by different transcription factors such as SLUG, SNAIL, TWIST, ZEB1 and ZEB2 ,45,46, l4+ cells . Cells iIn summary, in this study, we tested the mesenchymal/cancer stem cell characteristics of four hybrid cell clones in various experiments. There were no obvious correlations between distinct properties; rather, all hybrid cells exhibited individual characteristics that were not predictable. Our results indicate that cell fusion leads to the formation of a heterogenic cell population with a variety of capabilities. These results confirm a plurality of studies describing the same results for cell fusion hybrid cells ,22,47,48In the early 2000s, cell fusion hybrids were discovered in patients who had previously had a bone marrow transplantation (BMT) ,51. GastTumor heterogeneity remains one of the biggest problems cancer therapy must face. Many studies already demonstrated the diversity of fusion partners that cancer cells must create variegated tumor tissues, making it difficult to find the right treatment for all these cell subtypes. In the future, new starting points must be considered for fighting against this leading disease.iMSC#3 cells  were culMDA-MB-231 Hyg human breast cancer cells  were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)  medium supplemented with 10% fetal calf serum , 100 U/mL penicillin, 0.1 mg/mL streptomycin  and 200 µg/mL hygromycin.HS578T Hyg human breast cancer cells  were cultured in RPMI 1640  medium supplemented with 10% fetal calf serum , 100 U/mL penicillin, 0.1 mg/mL streptomycin  and 200 µg/mL hygromycin.2 at 37 °C.MM-Hybrids were cultured in DMEM  medium supplemented with 10% fetal calf serum , 100 U/mL penicillin, 0.1 mg/mL streptomycin , 1 µg/mL puromycin and 100 µg/mL hygromycin. MH-Hybrids were cultured in RPMI 1640  medium supplemented with 10% fetal calf serum , 100 U/mL penicillin, 0.1 mg/mL streptomycin , 1 µg/mL puromycin and 100 µg/mL hygromycin. All cells were maintained in a humidified atmosphere with 5% CO6) and HS578T Hyg (1 × 106) or iMSC#3 (1 × 106) and MDA-MB-231 Hyg (1 × 106) cells were co-cultivated in media specific for the appropriate breast cancer cell line at 5% CO2 and 37 °C. After 24 h, media were replaced by media containing antibiotics ; 1 µg/mL puromycin ). Hybrid cells exhibiting double resistance were isolated and individually cultivated. Overall, 11 iMSC#3 × HS578T Hyg hybrids, named MH#X, and 2 iMSC#3 × MDA-MB-231 Hyg hybrids, namely, MM#X, were isolated (X = clone number).To generate cell fusion hybrids, either iMSC#3 (1 × 10® 16HS System DC2101 (Promega). Therefore, 100 ng genomic DNA was prepared using the NucleoSpin® tissue kit  per the manufacturer’s instructions  and sent to their laboratory. Examination of the degree of the relationship was performed using Excel.Short tandem repeat analysis was performed by the working group of W. Gerding  using the Powerplex5 cells/well were seeded into a 6-well plate and incubated at 5% CO2 and 37 °C for 1 day. On the next day, media were exchanged. Control cells received the cell-line-specific medium, while the tested cells got osteogenic differentiation medium consisting of DMEM  supplemented with 10% FCS , 100 U/mL penicillin, 0.1 mg/mL streptomycin , 100 nM dexamethasone 21-phosphate disodium salt , 10 mM β-glycerol phosphate  and 50 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate . The medium was replaced two times per week, and cells were cultured for 21 days at 5% CO2 and 37 °C. Afterward, cells were fixed in 4% paraformaldehyde  and stained with Alizarin Red S  for 30 min at RT and washed 3 times with aqua destillata (A. dest.).For osteogenic differentiation, 1 × 10On the first day, 2500 cells/well were seeded on a 96-well plate and cultured in a cell-type-specific medium. For proliferation studies, the medium was removed after 24 h and replaced by RPMI 1640 without phenol red . Then, 50 µg/well XTT and 0.25 µg/well PMS  were added, and plates were incubated for 3 h at 37 °C. After 3 h, the reaction was measured with an ELISA reader . This procedure was repeated after 48 h, 72 h and 96 h. For cytostatic studies, the medium was also removed after 24 h, and it was replaced by a medium containing either 0.1 µmol, 1 µmol or 10 µmol doxorubicin or paclitaxel . Afterward, cells were again incubated for 24 h before media were exchanged by RPMI 1640 without phenol red . Then, 50 µg/well XTT and 0.25 µg/well PMS were added, and plates were incubated 3 h at 37 °C. After 3 h, the reaction was measured with an ELISA  reader. This procedure was repeated after 48 h and 72 h.Colony formation assay:2O. The plate was air-dried at RT. Densitometric analysis was performed using ImageJ (imagej.-nih.gov/ij/).In a 6-well plate, 200 cells/well were seeded and cultured for 10 days, changing the cell-line-specific media after 2–3 days. After 10 days, the media were removed, and the cells were washed with PBS . For fixation, 4% paraformaldehyde  was used; for coloring of the cells, 0.5% crystal violet  was used. Crystal violet was incubated for 30 min at RT and afterward washed away with Hhttps://imagej.nih.gov/ij/download.html).For mammosphere assays, 96-well plates were coated with 1.2% poly(2-hydroxyethyl-methacrylate) (poly-HEMA)  in ethanol and dried for 10 days at 37 °C. Five hundred cells/well were seeded and cultured in mammosphere formation medium at 37 °C for 7 days. Mammosphere formation medium consisted of two different media (medium I and II) in a ratio of 1:4. Medium I was composed of DMEM/F12  + B27 supplement  + 20 ng/mL FGF  + 20 ng/mL EGF  + 0.39 µg/mL hydrocortisone . Medium II is composed of Methocult H4100  + DMEM  (ratio 2:3). After 7 days, images were generated with a microscope and analyzed using ImageJ 1.8.0 , which is a specific ALDH1 inhibitor; this sample served as a control. Afterward, cells were incubated for 45 min at 37 °C in the dark and centrifuged . The cell pellet was washed in 500 µL of AldeRed assay buffer. Samples were analyzed using fluorescence-activated cell sorting , and FACS data were evaluated using WinMDI 2.9.The expression of aldehyde dehydrogenase 1 (ALDH1) was examined by using the AldeRed ALDH detection assay  according to the manufacturer’s manual. First, 2 × 105 cells were stained with different antibodies in accordance with the manufacturer’s instructions and measured with a FACScalibur flow cytometer . Isotype-matched antibodies served as controls. All antibodies are listed in Flow cytometry was used to analyze CSC and EMT markers in hybrid and parental cells. In this experiment, 1.5 × 105 cells were treated with 3× Lemmli sample buffer (ratio 1:2) and lysed for 10 min at 95 °C. EGF-stimulated samples were treated with 100 ng/mL EGF  for 1, 2 or 5 min before 3× Lemmli sample buffer was added and cells were lysed for 10 min at 95 °C. For 8%, 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), the samples were separated and afterward transferred to an Immobilon polyvinyl difluoride (PVDF) nitrocellulose membrane  under semidry conditions. The membranes were then blocked with 5% (w/v) nonfat milk powder in Tris-buffered saline with 1% Tween-20 (TBS-T) for at least 1 h. All antibodies were used in accordance with the manufacturer’s instructions. Visualization of protein bands was executed by using the Pierce ECL Western blot substrate  and the Aequoria macroscopic imaging system . All images were evaluated using ImageJ (imagej.-nih.gov/ij/).For the generation of Western blot samples, 2 × 10Statistical analysis was performed using GraphPad Prism 7. In the case of a rectangular distribution of the data, a one-way ANOVA test with the following post hoc test was used. If not, a Kruskal-Wallis test and appropriate post hoc test were performed. Unless otherwise mentioned, data were generated in at least three independent experiments.