The steroid hormone progesterone accounts for immune tolerance in pregnancy. Enhanced progesterone metabolism to 6α-OH-pregnanolone occurs in complicated pregnancies such as in preeclampsia with preterm delivery or intrauterine growth restriction, and in cancer. As lymphatic endothelial cells (LECs) promote tumor immunity, we hypothesized that human LECs modify progesterone bioavailability. Primary human LECs and mice lymph nodes were incubated with progesterone and progesterone metabolism was analyzed by thin layer chromatography and liquid chromatography-mass spectrometry. Expression of steroidogenic enzymes, down-stream signal and steroid hormone receptors was assessed by Real-time PCR. The placental cell line HTR-8/SV neo was used as reference. The impact of the progesterone metabolites of interest was investigated on the immune system by fluorescence-activated cell sorting analysis. LECs metabolize progesterone to 6α-OH-pregnanolone and reactivate progesterone from a precursor. LECs highly express 17β-hydroxysteroid dehydrogenase 2 and are therefore antiandrogenic and antiestrogenic. LECs express several steroid hormone receptors and PIBF1. Progesterone and its metabolites reduced TNF-α and IFN-γ production in CD4+ and CD8+ T cells. LECs modify progesterone bioavailability and are a target of steroid hormones. Given the global area represented by LECs, they might have a critical immunomodulatory control in pregnancy and cancer. Additionally, the expression and release of the lymphangiogenic factor VEGF-C by tumors is linked to metastasis, poor prognosis and immune tolerance2. LECs are, like dendritic cells, antigen-presenting cells that mediate systemic peripheral immune tolerance4. Upon presentation of self-antigens by LECs, T-cells undergo apoptosis and LECs therefore contribute to immune tolerance. LECs also promote immune tolerance when tumorigenic antigens are presented to T-cells. T-cells, which were activated by LECs, become more rapidly apoptotic as T-cells activated by mature dendritic cells5. Though preventing autoimmune reactions and promoting immune tolerance during pregnancy, this behavior may favor tumor development. LECs in the local microenvironment of a tumor might therefore be a target for immunomodulation5.The lymphatic system, especially lymphatic endothelial cells (LECs) are thought to play a crucial role in tumor immunity. For example, tumorigenic cells travelling inside lymphatic vessels towards sentinel lymph nodes are not being recognized as foreign and an appropriate immune response is missing, leading to tumor survival7. This immunomodulatory effect of progesterone is mediated via upregulation of the lymphocyte-derived progesterone-induced blocking factor (PIBF)13. Thus, in pregnant women14 and cancer patients15, there is an increased progesterone metabolism.During pregnancy, immune tolerance is required and linked to high progesterone levels16. Horwitz et al. proposed an intracellular metabolism of progesterone into the 6α-OH-pregnanolones involving 3 different enzymes: 5α-reductase (SRD5A1-3), 3β-hydroxysteroid dehydrogenases (HSD3B1/2), and a 6α-hydroxylase17.The first step in progesterone metabolism leads to the formation of active 5α-dihydroprogesterone (5α-DHP), which is further metabolized into the 6α-OH-pregnanolones  and allopregnane-3,20-diol . The metabolism of 5α-DHP is extrahepatic, as the final step of 6α-OH-pregnanolones and allopregnane-3,20-diol formation is not catalyzed by the cytochrome P450 steroid 6α-hydroxylase expressed in the liver22, but the mechanism leading to these metabolites remains to be elucidated. Allopregnanolone, the metabolite down-stream of 5α-DHP, stimulates proliferation and migration in the ovarian cancer cell line IGROV-115 and in glioblastoma23.Several reports have demonstrated that the 5α- and 5β-DHP metabolites are biologically active in pregnancy29. The progesterone metabolite 5α-DHP produced by the enzyme 5α-reductase, is a target for prostate cancer and glioblastoma therapy33. Interestingly, HSD17B2 also exerts a high 20α-hydroxysteroid dehydrogenase activity34 and can convert the weak 20α-hydroxyprogesterone (20α-OHP) into progesterone38. Many compounds targeting HSD17B2 activity in various cancers are under investigation39.Several enzymes accounting for steroid hormone metabolism, specifically progesterone metabolism, have been involved in cancer. These are the 6α-hydroxylase, the 5α-reductase, the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 2 (HSD17B2)first, progesterone metabolism was assessed in primary human LECs isolated from lymph nodes (HLEC) and results were confirmed in LECs isolated from lymphatic skin vessels (dLEC); second, the resulting progesterone metabolite was identified and characterized by LC–MS; third, the enzymatic steps involved were clarified; fourth, human cell culture results were matched to ex-vivo mouse lymph node; fifth, the impact of the found progesterone metabolites 5α-dihydroprogesterone (5α-DHP) and 6α-OH-pregnanolone (6α-OH-Pregn) was tested on immune cells. The placental cell line HTR-8/SVneo was used as a control and reference for the experiments with lymphatic endothelial cells.The hypothesis of this investigation is that human LECs modify the bioavailability of progesterone. To prove this, 3H-cholesterol to quantify the de-novo synthesis of progesterone, corticosterone, cortisol, testosterone and estradiol (HLEC), and of progesterone, 11-deoxycortisol, corticosterone, aldosterone and cortisol (dLEC), after an incubation time of 24 h. No significant de-novo steroid hormone production could be detected out of the precursor cholesterol by thin layer chromatography (TLC) in HLEC  or 1 h/4 h/8 h/24 h (HTR-8/SVneo). Steroids were extracted from supernatants and the metabolites separated by TLC. Progesterone was time-dependently and significantly converted to one major steroid hormone, 6α-OH-pregnanolone, in all three cell lines with a production rate of 19.3% ± 3.6 (24 h) and 31.7% ± 6.3 (48 h) in HLEC, 13.6% ± 2.5 (24 h) and 23.9% ± 4.2 (48 h) in dLEC and 21.0% ± 11.5 (8 h) and 42.7% ± 17.1 (24 h) in HTR-8/SV neo  and of steroid hormone receptors in HLEC, dLEC and HTR-8/SV neo was assessed Table . All thrSteroidogenic enzymes involved in de-novo steroid hormone production were assessed thereafter Table . HLEC anAssessment of the mRNA expression of steroidogenic proteins involved in de-novo steroid hormone production Table  revealedBased on our findings by Real-time PCR, LC–MS and Thin layer chromatography, progesterone is metabolized via 5α-DHP and allopregnanolone to 6α-OH-pregnanolone in HLECs, dLECs and HTR-8/SVneo Fig. . Further−5 M and 10−6 M for HLEC and HTR-8/SV neo and 10−6 M and 10−8 M for dLEC. Supernatants were analyzed by TLC to measure the conversion of 14C-progesterone to 6α-OH-pregnanolone  and DHEA-S and of 20α-OHP to progesterone. There was a significant HSD17B2 activity, both in HLEC , were incubated for 24 h and 48 h with , were inAll assessed steroid hormones, enzymes and steroidogenic factors in this manuscript are summarized in Supplementary Fig. PBMCs of three different healthy female donors were isolated and pre-incubated with the steroid hormones progesterone, 5α-DHP, 6α-OH-pregnanolone, and with dexamethasone (Dexa) as positive control for 6 h before activation. Solvent control of all steroid hormones was EtOH. Activation of T cells was thereafter induced by PMA/Ionomycin. TNF-α and IFN-γ production was assessed 24 h later using FACS analysis. Progesterone and Dexa, strongly decreased TNF-α positive CD4+ Fig. a and CD8De-novo steroid hormone synthesis from cholesterol is absent in lymphatic tissue, which is consistent with the lack of steroidogenic acute regulatory protein (StAR) in LECs. However, FDXR, FDX1 and LRH-1, genes involved in steroidogenesis42, are expressed in LECs. Progesterone, a steroid hormone critical in regulating immune responses and pregnancy, is intensely metabolized. In primary human LECs derived either from lymph nodes or from dermal lymphatic vessels, progesterone is mainly converted to a single metabolite, which was identified as 6α-OH-pregnanolone . Enzymatic steps involved are the two isoforms of the 5α-reductase, SRD5A1 and SRD5A3, the isoform 1 of 3α-HSD (AKR1C1) and a 6α-hydroxylase. Besides converting progesterone to the down-stream metabolites 5α-dihydroprogesterone (5α-DHP), allopregnanolone and 6α-OH-pregnanolone, HLECs and dLECs were able to reactivate progesterone from the less potent metabolite 20α-OHP. Further steroidogenic activity of HLEC and dLEC included deactivation of testosterone and 17β-estradiol to androstenedione and estrone as well as androstenediol to DHEA and DHEA-S.To our knowledge, this is the first report showing detailed steroid hormone metabolism in the lymphatic system. The lymphatic endothelium could be identified as an important regulator of steroid hormone bioavailability, yet also as a target of steroid hormones by expressing the respective steroid hormone receptors. 43. The data show that progesterone is not only metabolized to the primary and active metabolite 5α-DHP which plays a critical role in pregnancy44, but is also restored from 20α-OHP.These findings are pivotal in understanding the progesterone-dependent immunomodulation observed earlier by various groupsProgesterone metabolism to 6α-OH-pregnanolone and the reactivation of progesterone from 20α-OHP by LECs serves to adapt immune tolerance and to control local and/or systemic progesterone bioavailability in high progesterone conditions as found during pregnancy. As the exact mechanism for this phenomenon is unknown and given the huge surface of the lymphatic endothelium throughout the body, it is supposable that local progesterone metabolism in LECs could be the key regulator in preventing cancer cells from being destroyed.45. Immunosuppression by progesterone is mediated via the progesterone regulated gene PIBF146, which has now also been found to be highly expressed in LECs. A reduced expression of PIBF in threatened pregnancies51 and an increased production of PIBF in cancer53 are clearly linked to a failed immune modulation. Production of TNF-α and IFN-γ in activated CD4+ and CD8+ T cells from 3 different donors was modulated upon stimulation with progesterone and its metabolites. It needs to be investigated if the interaction of LECs and the immune cells is PIBF1 regulated.The direct immunosuppressive role of progesterone in both reproduction and tumor progression was impressively shown by Szekeres-Bartho and PolgarSteroid hormone effector mechanisms such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the G protein-coupled estrogen receptor 1 (GPER1), and the progesterone receptor membrane components PGRMC1 and PGRMC2 are present in HLECs and dLECs, while no expression of nuclear progesterone receptors (PRA and PRB) was found.55. PGRMCs were overexpressed in the maternal–fetal interface and in the embryonic/fetal trophectoderm in pregnancy57 as well as in T cells during pregnancy59. Since many steroid hormone receptors are expressed by immune cells62 it is most likely that the LECs not only have an autocrine, but also a paracrine role in controlling immune responses through the action of steroid hormones via binding to a receptor.Most of the known anti-inflammatory effects of progesterone are transmitted through the GR, with progesterone binding to the GR even though progesterone can also bind to PGRMCs64 and are only sufficient to inhibit peripheral natural killer cell activity in normal pregnant women. The presence of lymphatic vessels throughout the body, together with the immunosuppressive role of the placenta, might be a much better explanation for the systemic immunosuppression.The serum concentrations of progesterone are according to the literature much too low to support the concept of a generalized immunosuppression65. High progesterone levels seem to coincide with high VEGF-C expression66. Activated macrophages and monocytes releasing VEGF-C thus enhance LEC growth and could well contribute to immune regulatory mechanisms via progesterone.The mononuclear phagocyte system stimulates VEGF-C release and regulates thereby lymphangiogenesis67. Even though HLECs and dLECs express the same enzymes, 6α-OH-pregnanolone was found instead as major metabolite of progesterone, and not 20α-hydroxyprogesterone. That LECs favour the 5α-reductase/3α-HSD pathway over the 20α-HSD pathway might be explained by the different tissues investigated and eventually the limitations of some cofactors.In 2009, Zhang et al. found, that in isolated mature adipocytes progesterone was converted to 20α-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo–keto reductases AKR1C1, AKR1C2 and AKR1C3 , respectivelyThe strength of this study is the thorough analysis of different steroid hormone metabolites using state-of-the-art technology. It shows enzyme expression on mRNA and on protein level, as well as functional assays including inhibitory experiments. All results are referred to our self-designed and validated model of a positive control, the placental cell line HTR-8/SV neo. Since fast, significant and similar progesterone metabolism was found in HTR-8/SV neo cells, they were used as a model of reference for all experiments. Taking a placental cell line as model is obvious, as the placenta is the primary organ exposed to high progesterone levels. A direct immunologic effect of the lymphatic progesterone metabolites on CD4+ and CD8+ T cells could be shown as well.Investigation of further immunomodulatory effects of the lymphatic progesterone metabolism and progesterone reactivation described herein on immune and cancer cells is the goal of future studies.Verification of our observations in an in vivo model is difficult due to the systemic presence of steroid hormones, further complicated by the delicate structure of lymphatic vessels, therefore precluding differential concentration measurements. But, the capacity of steroid hormone metabolism and reactivation in lymph nodes isolated from both sexes, including pregnancy, should be explored. Additionally, future detailed assessment in patients treated with the 5α-reductase inhibitors dutasteride or finasteride could be investigated with more accuracy.In conclusion, we identified and characterized the lymphatic system as major steroid hormone metabolizing tissue, which is additionally able to reactivate progesterone from 20α-OHP and therefore adapts progesterone availability. This is important in high progesterone conditions such as in pregnancy in order to adjust local immune tolerance. The impact of these findings on cancer and pregnancy research might be meaningful and could provide new targets for the treatment of cancer and pregnancy related diseases. Furthermore, these results could also help to elucidate disease mechanisms in autoimmunity and allergy.3H-cholesterol (ART-0255), 3H-progesterone (ART-0795) and 14C-progesterone (ARC-1398) were from American radiolabeled chemicals (ARC) Inc. . Scintillation solution Optiphase Supermix (Cat. No. 1200-439) was from Perkin Elmer Schweiz AG . The SRD5A-inhibitor dutasteride (SML 1221) was purchased from Sigma-Aldrich .Collagen I coated petridishes were from Corning . The primary HLEC  were from Sciencell  and the primary human dLEC  were from Lonza  or from PromoCell Heidelberg, Germany . Primary cells were obtained from female donors. The HTR-8/SVneo cells were a gift from Charles H. Graham . The vascular cell basal medium (PCS-100-030) and the supplementary factors (PCS-100-041) were from ATCC . The Endothelial Cell Growth Medium EGM-2 (CC-3162), including EBM-2 Basal Medium (CC-3156) and EGM-2 Supplements (CC-4176), was from Lonza . The PromoCell MV-2 medium with supplements (C-22121) was from PromoCell . Medium RPMI 1640  was from Gibco . Penicillin/streptomycin  and FBS (Cat. No. 0025) were from Sciencell . 254  and pre-coated TLC sheets Polygram SIL G/UV254  were from Macherey–Nagel GmbH, Düren, Germany. Dichloromethane (1.06050), methanol (1.06009) and choloroform (1.02445) were from Merck Millipore, Zug, Switzerland. Microbeta 2 Microplate counter 2450 was from Perkin Elmer Schweiz AG . Phosphorimager Typhoon FLA 7000 and the Storage Phosphor Screen (BAS-IP TR 2040 E Tritium Screen) were from GE Healthcare Life Sciences, Glattbrugg, Switzerland. Prime Script RT Reagent Kit Cat. RR037A was from Takara Bio Europe .Cholesterol, progesterone, and all steroid hormone standards for LC–MS and TLC analysis were purchased from Steraloids, Inc. . Pre-coated TLC plates SIL G25 UVFor LC–MS analysis, a Vanquish UHPLC  was coupled to a Q Exactive Plus Orbitrap . Separation was achieved using gradient elution over 11 min using water and methanol both supplemented with 0.1% formic acid  as mobile phases. Data analysis was performed using TraceFinder 4.1 . All steroids analyzed by LC–MS and their systematic names are listed in Table Primer pairs for SRD5A1, SRD5A2, SRD5A3, AKR1D1, AKR1C1, AKR1C2, AKR1C3, AKR1C4, HSD3B1, HSD3B2, HSD17B2, HSD11B1, FDXR, and NR5A2 , anti-CD4 PerCP (clone SK3) and anti-CD8 PE-Cy7 (clone SK1) were purchased from BD Biosciences. The fixable Live/Dead dye was obtained from Invitrogen. Anti-TNF-α AF647 (clone Mab11) and anti-IFN-γ BV421 (clone 4sB3) and their matched isotype controls were bought from Biolegend. PMA/Ionomycin was obtained from Sigma Aldrich.All methods used in this manuscript were carried out in accordance with relevant guidelines and regulations.Primary HLECs and dLECs were cultured in collagen I coated cell ware. Medium for HLECs was the vascular cell basal medium from ATCC with all supplementary factors but without cortisol (hydrocortisone). FBS concentration was 5%. Medium for dLECs was the EBM-2 Basal Medium from Lonza with all EGM-2 supplements but without cortisol. FBS concentration was 2%. Primary HLECs and dLECs were used up to passage 6.HTR-8/SV neo cells were cultured in RPMI 1640 5% FBS.In order to minimize steroid hormone contamination in our medium or FBS, all experiments were performed in cortisol-free medium containing 5% or 2% of charcoal treated (ct) FBS.PBMCs were cultured in RPMI1640 containing 10% FBS after isolation. During experiments, PBMCs were maintained in steroid-free RPMI1640.14C-progesterone or 1 μCi 3H-cholesterol were added in steroid free medium. Cell culture supernatant was collected into glass vials at the indicated time points. 5 ml ethyl acetate was added and glass vials were vortexed for exactly 1 min. Thereafter, supernatants were stored at − 20 °C until phase separation. The upper phase was transferred into new glass vials and evaporated under a nitrogen stream at 56 °C. The pellet was suspended in 30ul of cold progesterone dissolved in EtOH (10 mg/ml) for the 14C-experiments and in 30ul of the cold mixture cholesterol, progesterone, corticosterone, cortisol, testosterone and estradiol  for the 3H-cholesterol experiments in HLEC. For the 3H-cholesterol experiments in dLEC, the pellet was dissolved in 30ul of the cold mixture of cholesterol, progesterone, 11-deoxycortisol, corticosterone, aldosterone and cortisol . A 10ul aliquot of each sample was loaded on TLC glass plates or sheets. Running medium was dichloromethane, methanol, H2O (150:10:1). For Microbeta 2 measurements, spots were visualized and marked under the UV lamp. Marked spots were scratched into scintillation vials. 3.5 ml of scintillation fluid was added and vials were counted in a Microbeta 2. Individual steroid hormones were run on the same plate to allow for localization of the steroid hormones. A control without cells  was loaded as baseline/background. Counts are displayed in counts per minute (CPM = CCPM1). Conversion of 14C-progesterone to 6α-OH-pregnanolone or of 3H-cholesterol to progesterone, corticosterone, cortisol, testosterone and estradiol (HLEC), or of 3H-cholesterol to progesterone, 11-deoxycortisol, corticosterone, aldosterone and cortisol (dLEC), was calculated. For visualization by a phorphorimager, TLC sheets were exposed to a Storage Phosphor Screen. Spots were visualized by the Phosphorimager Typhoon FLA 7000. Individual steroid hormones were run on the same sheet to allow for localization of the steroid hormones. A control without cells  was loaded as baseline/background. Quantification was done using ImageJ software. All conversion rates were compared to the controls without cells. Progesterone substrate availability at timepoint 0 h (control without cells) was taken as 100% for all experiments.HLECs and dLECs were cultured in collagen I coated 6-well plates with medium containing 5% or 2% ct FBS for 24 h. After 24 h cells were washed twice with DPBS and 0.05 μCi −5 M and 10−6 M for HLECs and HTR-8/SV neo and 10−6 M and 10−8 M for dLECs. Total incubation time was 24 h. Conversion of progesterone to 6a-OH-pregnanolone was quantified by TLC.HLECs and dLECs were cultured as described above and incubated with dutasteride dissolved in DMSO and used at a final concentration of 1014C-progesterone was added in steroid free medium. Cell culture supernatant was collected into glass vials at timepoints 1 h, 4 h, 8 h and 24 h. 5 ml ethyl acetate was added and glass vials were vortexed for exactly 1 h. The rest of the procedure was done exactly as described above for the HLEC and dLEC.HTR-8/SV neo were cultured in RPMI 1640 medium containing 5% ct FBS for 24 h. After 24 h cells were washed twice with DPBS and 0.05 μCi SRD5A1, SRD5A2, SRD5A3, AKR1D1, AKR1C1, AKR1C2, AKR1C3, AKR1C4, HSD3B1, HSD3B2), of PIBF1, of the steroid hormone receptors  of the steroidogenic enzymes  and of the steroidogenic proteins StAR, FDXR, FDX1, NR5A2 in HLEC, dLEC and HTR-8/SV neo.mRNA expression of the progesterone metabolizing enzymes  and centrifuged at 800 rpm. Cell pellet was washed 2 × with DPBS and the dried pellet was lysed in 8 M UREA buffer containing a protease inhibitor cocktail. Protein amount was measured by the Qubit Protein Assay. The MS part was done by the Proteomic Mass Spectrometry Core Facility PMSCF using standard procedure.−6 M. A time-course experiment was performed with incubation times 0 h as baseline, 4 h and 24 h for HLEC and 0 h and 24 h for dLEC. Steroids were extracted from the supernatant by solid phase extraction using Oasis HLB SPE plates . Remaining substrate concentrations and metabolites were assessed using LC–MS. 20 uL of the extract were injected and separation was achieved using gradient elution over 11 min using water and methanol both supplemented with 0.1% formic acid as mobile phases. 6α-OH-pregnanolone was identified based on accurate mass measurement, comparison of MS/MS spectra and retention times between the analyte in the samples and an authentic standard. Data was normalized to condition at 0 h = 1000 nM = 10−6 M for each compound.HLECs and dLECs were cultured in collagen I coated 6-well plates with medium containing 5% ct FBS for 24 h. After 24 h cells were washed twice with DPBS and the cold testosterone, 17β-estradiol, 5α-dihydroprogesterone and androstenediol were added separately in steroid-free medium at a concentration of 1014C-progesterone for 24 h or 48 h at 37 °C. For controls, medium from wells incubated under the same conditions and incubation time but without tissue was used. After 24 h and 48 h supernatants were collected into glass vials. 5 ml ethyl acetate was added and vials were vortexed exactly for 1 min. The next steps were performed as described in the section thin layer chromatography. Products of progesterone metabolism were visualized with a Phosphorimager.Animal experimentation was approved by the Ethics Committee for Animal Experiments of the Veterinary Administration of the Canton of Berne, Switzerland and conformed to the rules of the Swiss Federal Act on Animal Protection (BE58/19). C57/Bl6 mice were bred according to the rules in the central animal facility of the university of Bern. They were maintained under 12-h dark–light cycles with unrestricted access to food and water. Males used in experiment were fed for 6–8 weeks with regular chow diet. On the day of sacrifice, lymph nodes and adrenal glands were isolated and put on ice until arrival at the lab. Thereafter, organs were washed in PBS and put into warm, steroid-free medium (PCS-100-030) containing 5% ct FBS. The fat was dissected, and the adrenal gland, and half of the lymph nodes were cut in half. The rest of the lymph nodes were uncut and contained a capsule. Every lymph node (cut or entirely), and the adrenal gland were placed in 48-well plates and incubated with 500ul of steroid-free medium containing 5% ct FBS and 0.05 µCi −3 M. After the 6 h pre-incubation period with steroid hormones, lymphocytes were activated with 15 ng/ml PMA and 1 μg/ml Ionomycin. Simultaneously, GolgiPlug was added in order to inhibit cytokine release. After a total incubation period of 24 h, PBMCs were stained for CD3, CD4, CD8, IFN-γ and TNF-α and analyzed by FACS as follows: Cells were stained with fixable Live/Dead dye for 30 min one ice. Subsequently cells were washed using PBS and fixed by adding Medium A from the Fix & Perm kit (Invitrogen) for 15 min at RT. Afterwards cells were washed and the permeabilization Medium B and the antibody cocktail containing anti-CD3, anti-CD4, anti-CD8, anti-IFN-γ and anti-TNF-α was added. Cells were incubated for 20 min, washed and resuspended in PBS containing 2% heat-inactivated FBS, 0.05% sodium azide. Data was acquired using a FACSCanto II (BD Biosciences). Matched isotype controls were used for anti-IFN-γ and anti-TNF-α Percentage of IFN-γ and TNFα positive cells was determined in alive cells, which were CD3 positive.Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy, non-pregnant female donors using Ficoll gradient centrifugation. After isolation, PBMCs were kept for 30 min in RPMI1640 medium containing 10 mM HEPES, 10% heat-inactivated FBS and 100 μg/ml Streptomycin, 100U/ml penicillin . After 30 min, PBMCs were stimulated with progesterone, 5α-DHP, 6α-OH-pregnanolone and dexamethasone for 6 h (priming). Control medium was RPMI1640 containing EtOH, the solvent of the steroid hormones. Final concentration of all steroid hormones on PBMCs was 10The % of CD4+ and CD8+ T cells owning the intracellular cytokines IFN-γ and TNF-α is shown in Fig. p < 0.05. TLC experiments were additionally controlled by CCPM1 measurements in a microbeta2 instrument. For TaqMan results ct values were calculated as mean of all experiments. All statistical analyses were performed using GraphPad PRISM version 8 .All experiments were performed at least 3 times. Pictures in figures are unprocessed. Representative blots are shown. Densitometry was performed for all phosphorimager pictures using Image J software. Conversion and production rates are displayed as mean ± SD using one-way ANOVA with Dunnetts multiple comparison test. Significance was assigned at For the phosphorimager pictures in Figs. Supplementary Information


These microsamples can provide information related to the genetic correlates of disease and can be used to quantify a range of analytes, such as proteins and small molecules. However, after routine neonatal screening, the amount of DBS sample available is limited. To optimize the use of these samples, there is a need for sensitive assays which are integrated across different analytic platforms. For example, after DNA extraction, protein extracts are available for additional analyses. We describe a sensitive and robust LC-MS/MS method for 25-hydroxyvitamin D If keptThe standards, instrumentation, chromatographic condition , tandem ® 972 or internal controls were punched  in Nunc® MicroWellTM 96 well polystyrene plates. Proteins were extracted by adding 130 µL of an in-house made extraction buffer (PBS containing 5 mL/L Tween20 and “Complete protease inhibitor cocktail with EDTA”—1 tablet dissolved per 25 mL of extraction buffer) to each well before the plates were shaken one hour at 450 rpm at room temperature.The DBS containing either the stable isotope-labelled calibrator standard or quality controls or endogenous SRM® 972 and internal control DBS punches, were subjected to the same additional extraction procedure for vitamin D quantification. Samples were wrapped in aluminum foil to avoid contact with light. First, 30 µL of each sample was transferred to a Thermo Scientific 96-well NUNC microtiter plate before 120 µL internal standard (reconstituted in acetonitrile and diluted to a working solution of 1:100 compared to the kit insert) was added. The plate was placed on an orbital shaker (450 rpm) for 10 min at room temperature to facilitate protein precipitation. Next, the precipitated proteins were removed by centrifugation for 30 min at 4000× g rpm (4 °C). Subsequently, 80 µL of the supernatants were transferred into a 96 deep-well plate already containing 400 µL ethyl acetate and 180 µL deionized water for the liquid–liquid extraction procedure. All samples were mixed well using the “pipetting-mixing” function on an electronic pipette (20 cycles of pipetting 500 µL ups and down). Two phases were separated during centrifugation at 700× g rpm for 5 min (4 °C), before approximately 200 µL of the upper organic phase (containing the purified vitamin D metabolites) was transferred to a Thermo ScientificTM WebSeal Plate+ 96-Well Glass-Coated Microplate. The samples were dried down in an Eppendorf Bench Top Concentrator PlusTM (60 °C) before the vitamin D metabolites were derivatized with 20 µL of the commercial PTAD reagent (reconstituted in ethyl acetate and diluted to a working solution of 1:12). The plate was incubated on an orbital shaker (450 rpm) at room temperature for 30 min, after which the reaction was quenched by the addition of 50 µL ethanol. Samples were dried down in a concentrator before being reconstituted in 80 µL 1:1 acetonitrile/deionized water solution. The reconstitution was carried out by mixing the plate on an orbital shaker (450 rpm) for 10 min at room temperature. The plate was centrifuged at 3000× g rpm for 10 min at 4 °C and transferred to the autosampler. Subsequently, 40 µL was injected into the LC-MS/MS system. All protein extracts, including stable isotope-labelled calibrator standards, quality controls and endogenous SRM® 972—from NIST) was converted to DBS in which the proteins were extracted and analysed according to the above approach. The performance of the assay was considered acceptable if the precision (%CV) at each concentration is <20% and the bias is ±20% of the expected reference value. Lower limit of detection (LOD) is determined when the peak height is three times the background noise. Lower limit of quantification (LOQ) was determined using dilutions of the lowest stable-isotope-labelled calibrator standard and with precision <20% and recovery within ±20%. The quantitative LC-MS/MS approach was validated in accordance with the Clinical and Laboratory Standards Institute’s approved guideline for liquid chromatography-mass spectrometry methods (C62-A). Linearity and calibration curve were evaluated using stable isotope-labelled calibration standards . Precisi2 and 25OHD3 (2H6-25OHD2 and 2H6-25OHD3) together with the different stable-isotope-labelled internal standards (2H3-25OHD2 and 2H3-25OHD3) [The assay calibration was achieved by using a combination of the commercial external stable-isotope-labelled six-point calibration standards for both 25OHD2 and 25OHD3 were Y = 2.2338 × 10−2 [25OHD2] + 4.617 × 10−3; R2 = 0.9907 and Y = 2.492 × 10−2 [25OHD3] − 3.699 × 10−3; R2 = 0.9921, respectively. The analyte responses were linear within the calibration curves for both 25OHD2 and 25OHD3. In addition, the correlation between the known nominal concentrations (nmol/L) against the ratio of the back-calculated concentrations (nmol/L) relative to the internal standard which are based on the MRM responses, were plotted too. The regression equations were Y = 0.9991 × [25OHD2] + 0.8879; R2 = 0.9988 and Y = 1.007 × [25OHD3] + 0.1279; R2 = 0.9990 25OHD2 and 25OHD3, respectively. The relative error (%), between the nominal and back-calculated concentration, fluctuated within ±6%, which is well within the CLSI limit of ±15%. The precision was investigated by quantifying the concentration of endogenous 25OHD2, 25OHD3 and total (25OHD2 + 25OHD3) in full blood concentrations in six in-house internal controls. 3 was in the range of 0.5–7.7% and the %CV for the total full-blood concentrations ranged from 1.1–8.4%, while %CV for 25OHD2 ranged between 3.9–17.1%. The suboptimal %CV for 25OHD2 mainly reflects the low concentration of endogenous 25OHD2 in the samples, which has been observed in previous DBS-related studies [Based on the commercial calibration standards with known concentrations. regression equations for 25OHD2H6-25OHD2 and 2H6-25OHD3). All three quality controls for each of the two metabolites were quantified twenty-three times, measured over a period of approximately four months. Geometric means, SD, %CV, relative error and accuracy were calculated for all measurements and are summarized in Intermediate precision was obtained by quantifying the concentration of three stable-isotope-labelled external commercial quality controls with a low, medium and high concentration of each vitamin D metabolite   . Additio2H6-25OHD2 and 2H6-25OHD3) were prepared and quantified. The concentrations of each vitamin D metabolite (nmol/L) were plotted against the relative errors . To estimate the LOQ for our new assay, dilutions of the lowest stable-isotope-labelled calibrator standards for both vitamin D metabolites  based on leftover protein extract from neonatal DBS. We demonstrated that the method has acceptable precision and accuracy and is sensitive and robust. As the assay was optimized to use only 30 uL of the leftover protein extract from DBS , it is possible to combine this new assay with other analytical assays, such as genotyping and protein quantification. With only minor adjustments, this approach could also be used for other small molecules of interest in an efficient manner. 2 and 25OHD3 [When interpreting results from this new assay, there are several important limitations to take into account. Neonatal hematocrit is known to influence assays based on DBS , and infd 25OHD3 . Therefo2 and 25OHD3 from protein extracts facilitates the assessment of a panel of vitamin D-related measures . We believe that our new assay will facilitate this type of integrated research in neonatal archived DBS.Although several other sensitive LC-MS/MS vitamin D quantification assays for DBS already exist, none have reported quantification of vitamin D metabolites in protein extracts from DBS. The quantification of the 25OHD


The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC Coronaviruses (CoVs) known to infect humans have caused three major disease outbreaks within the past two decades. Severe acute respiratory syndrome (SARS) CoV caused the SARS epidemic from 2002 to 2004; the Middle East respiratory syndrome (MERS) CoV caused outbreaks in 2012, 2015, and 2018; and SARS-CoV-2 caused the global CoV disease 2019 pandemic (COVID-19) . The enz7-methyltransferase (N7-MTase) and exoribonuclease (ExoN) domains [7GpppA) specific 2′-O- methyltransferase (2′-O-MTase). Both enzymes use S-adenosylmethionine (SAM) as a methyl group donor  inhibitors ,16,17,18Here, we aim to identify novel drug-like compound classes that inhibit the SARS-CoV-2 MTases nsp14 and nsp16 and could serve as starting points for lead optimisation. New inhibitors were identified via high-throughput virtual screening (HTVS) of a wide chemotype space compound library. Molecular docking was enabled by the availability of several SARS-CoV-2 nsp16/nsp10 heterodimer crystal structures  in complDespite the poor binding site sequence similarity between nsp14 and nsp16 D, the shThe HTVS hit aromatic rings that bound in the amino acid binding sub-site was in most cases a heterocycle that mimicked the carboxylic group, whereas the aromatic groups that bound in the hydrophobic mRNA sub-pocket were structurally more diverse. Binding in the terminal guanosine sub-pocket was particularly pronounced for nsp14 hits, which formed interactions with residues Phe401, Tyr420, Phe426, and Phe506 F. It is It has been shown that inhibitory activity of the cap MTase inhibitors can be determined using radioactive MTase assay ,41, LC-M50 values of ≈50–100 μM , which is a common issue with nucleoside derivatives ,26,27,28Since identified SARS-CoV-2 MTase inhibitors are rather small molecules with acceptable LEs and show drug-like properties, they can serve as templates for further chemical elaboration into lead-like molecules. To explore the chemical space around these compounds, and gain additional insight on the potential compound development route, commercially available hit analogues were purchased. Additionally, for comparison with the current best-in-class, commercially available SAM analogues that are well known to inhibit MTases were also purchased.The MolPort in-stock compound database was screened for ZINC23398144 and ZINC38661771 analogues, and 34 and 20 compounds, respectively, were identified. Based on the docking score and chemical diversity, 11 ZINC23398144 analogues and three ZINC38661771 analogues were purchased, and their binding potency was determined see . The ana50 values in range from 0.27 up to 0.41 kcal/mol, which is comparable to SAH and SFG. The top-scoring SAM analogues show large structural diversity and do not provide clear SAR , docked into the target structures, and 11 chemically diverse top scoring SAM analogues were purchased from MolPort. The top scoring adenosyl group containing analogues showed activity in low μM level see , and lig SAR see ; besides SAR see .ZINC15 in-stock drug-like compound library  of 6.9 mThe prepared library was docked in the crystal structure of SARS-CoV-1 nsp14   analogues was created by filtering the MolPort in-stock database for compounds with a Tanimoto similarity metrics coefficient above 0.7. The library of the SAM analogues was created by filtering the MolPort in-stock compound library for compounds that contain an adenosyl group. Ligands were prepared as described before.Docked poses were visualised using PyMOL .The stability of the docked pose for the top-scoring HTVS hits was evaluated by running the MD simulations and calculating the ligand RMSD throughout the simulation against the docked pose. The MD simulation systems of selected inhibitor–enzyme complexes were prepared by placing the molecules in dodecahedral boxes with at least 1.5 nm distance to the box walls. The TIP3P water model was used to solvate the complex. Sodium and chloride ions were added to neutralise the systems and reach 150 mM salt concentration. Forcefields for the inhibitors were based on the general AMBER force field (GAFF) and were generated using Ambertools . Amber03The stability of the docked pose was evaluated by calculating the ligand on protein RMSD value for ligand heavy atoms using Gromacs. Compound was considered as stable if the RMSD was below 2 Å for more than 90% of simulation time in at least 7 parallel runs. Stable compounds were purchased commercially. Simulations were visualised using VMD .Protein expression and purification, as well as homogeneous time-resolved fluorescent energy transfer (HTRF) assay, SARS-CoV-2 nsp16/nsp10 methyltransferase substrate RNA production, cytotoxicity, and cell permeability testing was performed as described before .E. coli expression system in pET28a vector. NdeI and NotI restriction sites were used for this construct, and the construct contained an additional N-terminal His-6x tag with thrombin cleavage site. Nsp10 and nsp16 were ordered from the ATCC repository as NR-52425 and NR-52427 catalogue items, respectively. Nsp10 and nsp16 constructs were cloned in the pMCSG53 vector and contained additional N-terminal His-6x tag with tobacco etch virus protease cleavage sites.Nsp14 gene sequence were purchased as a gene synthesis from General Biosystems with codon optimisation for E. coli BL21-DE3 cells. Cells were maintained in LB medium supplemented with 30 μg/mL kanamycin, and the protein expression was performed in 2xTY medium supplemented with 30 μg/mL kanamycin. Cells were grown at 37 °C to OD590 0.7 and shaken at 200 rpm, cooled at 20 °C for 30 min, and induced with 1 mM IPTG. Induction was performed overnight for approximately 16 h at 20 °C with shaking at 200 rpm. Biomass was collected by centrifugation and stored at −20 °C.Protein expression was performed in 2, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, and 50 mM imidazole. Then, the lysate was centrifuged for 30 min at 11,000× g. The proteins were purified from lysate with IMAC using either batch or column Ni2+-NTA agarose. The Ni2+-NTA agarose were previously equilibrated in 50 mM Tris-HCl (pH 9.0), 0.5 M NaCl, 10 mM 2-mercaptoethanol, 2 mM MgCl2, 5% (v/v) glycerol, and 50 mM imidazole and then were washed with the same buffer. Proteins were eluted with a buffer containing 50 mM Tris-HCl (pH 9.0), 0.5 M NaCl, 10 mM 2-mercaptoethanol, 2 mM MgCl2, 5%(v/v) glycerol, and 1 M imidazole.Procedures for nsp10, nsp14, and nsp16 purification were identical. Note that reducing conditions during purification procedures are extremely important for the stability of nsp16 and nsp14. The purification protocol was adopted from previous study  with modNsp10 and nsp16 for nsp16/nsp10 assay were cleaved overnight with TEV protease by adding it to the protein solution at a ratio of 1 mg per 30 mg of cleavable protein.2 and 5%(v/v) glycerol. Then, the peak fractions were pooled. In the case of nsp16/nsp10, a separation of uncleaved protein and TEV protease was performed at this point by passing this material through Ni2+-NTA agarose column.Then, the proteins were further purified with size exclusion chromatography on a Superdex 200 column in 20 mM HEPES (pH 8.5), 0.5 M NaCl, 10 mM 2-mercaptoethanol, 2 mM MgClNsp16 and nsp10 were mixed in equimolar ratios and concentrated with ultrafiltration to a concentration of 2 mg/mL. SAM was added to a concentration of 2 mM and the mixture was incubated overnight at 4 °C to allow proper complex formation. The next day, the mixture was dialyzed against 20 mM HEPES (pH 7.5), 0.15 mol/L NaCl, 5% glycerol, and 1 mM TCEP for 2 h to remove excess SAM, and then concentrated to 5 mg/mL with ultrafiltration. Then, the solution was flash-frozen in liquid nitrogen and stored at −20 °C.2, 0.01% Tween20. Then, 4 µL of purified recombinant protein nsp14 at 0.4 µM or nsp16/nsp10 at 3 µM final concentration were added in the assay wells, containing previously dispensed inhibitors. The reaction was started by preparing a mix containing 4 µM GpppG  or ≈5 μM m7GpppA-RNA (for nsp16/nsp10) and SAM at 10 µM final concentrations in a volume of 4 µL, which was incubated at 37 °C for 20 min (2 h for nsp16/nsp10).MTase activity was determined with an EPIgeneous Methyltransferase Assay kit by assaying the conversion of SAM to SAH according to the manufacturer’s instructions. The enzymatic reaction was performed in white ProxiPlate-384 Plus microplates using a final reaction volume of 10 µL. The reaction buffer was composed of 40 mM Tris-HCl pH 8.3 (pH 8.0 for nsp16/nsp10) and 100 mM NaCl (or 10 mM KCl only for nsp16/nsp10), 1 mM DTT, 2 mM MgCl50 values were determined for compounds showing higher than 50% inhibitory effect at 100 µM concentration. For the detection of released SAH, 2 µL of EPIgeneous detection buffer one was added in order to stop the enzymatic reaction. After 10 min of incubation at room temperature, detection reagents were added: first, 4 µL of a 1/16 dilution of SAH-d2 conjugate and then 4 µL of Anti-SAH-Lumi4-Tb at a 1/100 dilution. HTRF signals were measured using a Hidex Sense (Finland) microplate reader using an excitation filter at 337 nm and fluorescence wavelength measurements at 620 and 665 nm, an integration delay of 150 ms, and an integration time of 400 ms. Results were analysed by calculating a two-wavelength signal ratio:All compounds were dissolved in 100% DMSO  and were tested at 100 µM concentration in duplicate. IC50 value was calculated using the program Graph Pad Prism 8.0.The mean HTRF Ratio for each sample was calculated as:2, 0.5 µM GNMT, and compounds 1 nM to 10 µM. The reaction was started with a mix containing glycine and SAM at 5 mM and 10 µM final concentrations respectively and incubated at 25 °C for 30 min. Released SAH was detected, and the compound IC50 values were determined as described above.The selectivity of the compounds was tested using human Glycine N-Methyltransferase  using the same EPIgeneous Methyltransferase Assay kit. Reaction mixtures (volume 10 µL) contained 20 mM Tris-HCl (pH 8.6), 2 mM MgClRNA oligonucleotide substrate for the nsp16/nsp10 assay was acquired through in vitro transcription with a Thermo Fisher TranscriptAid T7 High Yield Transcription Kit, using a DNA oligonucleotide Co25F 5′-TAC AAA GCT TCA GTA ATA CGA CTC ACT ATT ATA GAA CTT CGT CGA GTA CGC TCA A as a sense strand, and a DNA oligonucleotide Co25R 5′-TTG AGC GTA CTC GAC GAA GTT CTA TAA TAG TGA GTC GTA TTA CTG AAG CTT TGT A as a complimentary strand, to allow the transcription from the class II T7 promoter 5′- TAATACGACTCACTATTA 3′ and to obtain the RNA oligonucleotide m7G(5′)ppp(5′)Co25: m7G(5′)ppp(5′)AUAGAACUUCGUCGAGUACGCUCAA.DEPC-treated water6 µL5x TranscriptAid reaction buffer4 µLATP, Tris buffered, 30 mM1 µL CTP, Tris buffered, 100 mM1.5 µL GTP, Tris buffered, 100 mM1.5 µL UTP, Tris buffered, 100 mM1.5 µL Cap analog G(5′)ppp(5′)A, 100 mM1.2 µL Co25 template DNA, double stranded, preheated at 95 °C for 10 min, then slowly cooled down to 35 °C1 µg (0.5 µL)TranscriptAid enzyme mix2 µLThe in vitro transcription reaction was set up as follows:The reaction was incubated for 6 h at 37 °C, products were desalted via Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL in DEPC-treated water, and then stored at −20 °C. A co-transcriptionally capped m7G(5′)ppp(5′)-Co25 oligonucleotide was obtained using 1.5 mM final concentration of ATP and 6.0 mM final concentration of G(5′)ppp(5′)A RNA Cap Structure Analog obtained from New England Bio Labs inc. Oligonucleotide was purified from template DNA by digestion with DNAse I for 15 min at 37 °C and subsequent desalting via Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, and 0.5 mL in DEPC-treated water. RNA oligonucleotide concentration was measured using a Nanodrop 2000 spectrophotometer, yielding concentrations of 2.5 ± 0.5 µg/µL and the oligonucleotides were analysed by 20% polyacrylamide denaturing (7 M urea) gel electrophoresis, where they appeared as a diffuse band around 8.3–8.6 kDa .2 atmosphere at 37 °C.A549 human non-small cell lung cancer cell line, HepG2 hepatocarcinoma cells, and BALB/c 3T3 clone A31 murine fibroblast cell line for the study were obtained from ATCC . Cells were propagated in DMEM medium  supplemented with 1% penicillin (100 U/mL)–streptomycin (100 μg/mL) and 10% foetal bovine serum  in case of A549 and HepG2 or 10% calf serum  in case of BALB/c 3T3. All cultivations were performed in a humidified 5% CO3 cells per well, HepG2 at density 1.2 × 104 cells per well. After 24 h incubation, test compounds in a concentration range of 0.8 to 200 μM were added. Cultivation in the presence of test compounds was done for 48 h. Afterwards, the plates were washed with phosphate-buffered saline (PBS) , and 25 µg/mL NR solution  was added. In case of BALB/c 3T3 cells, NR was diluted in 5% foetal calf serum containing media, while for A549 and HepG2 cells, it was diluted in 5% foetal bovine serum containing media. After 3 h incubation, the plate was washed with PBS, and the NR taken up by viable cells was extracted using desorbing fixative (50% ethanol/1% acetic acid/49% water). Absorbance at 540 nm was measured using a Tecan M200 Infinite Pro microplate reader. Cytotoxicity was expressed as a concentration-dependent reduction of the uptake of NR, compared to the untreated controls, and the IC50 value for each compound was calculated.The cytotoxicity of the compounds was tested in A549, HepG2, and BALB/c3T3 cell lines by neutral red (NR) uptake assay. A549 and BALB/c3T3 cells were seeded in 96-well plates at a density of 8 × 104 and 4 × 104 cells per well at concentrations 1 × 104 and 2 × 104 cells/mL media for the testing of each compound, each in three replicates. After the incubation, the cell cultivation media and cell lysates were collected. After the removal of media, cells were washed with ice-cold PBS  and lysed for 30 min in 500 µL per well ice-cold RIPA buffer . Samples were stored at −80 °C until analysis.To test cell permeability, each compound was added to A549 cell culture at a concentration of 20 μM and incubated for 24 h. Cells were seeded in 6-well plates at a density of 2 × 10v/v) was add to 200 μL of each cell lysate and culture media sample. Then, the samples were centrifuged , and the supernatants (300 μL) were removed from each sample and placed in vials for LC/MS/MS analysis. Calibration curves for all analytes were produced by analysis of standard solutions over a concentration range of 0.2 to 20 μM.The quantitative determination of tested compounds in cell lysates and culture media was performed on a Waters MICROMASS QUATTRO microTM tandem mass spectrometer combined with Acquity UPLC system. Acquity UPLC BEH C8 (2.1 × 50 mm × 1.7 μm) column was used at a flow rate of 0.25 mL/min. The column oven was set at 30 °C, and the sample injection volume was 5 μL. The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (acetonitrile). The gradient elution program from 5% B to 98% B in 5 min was applied for analysis. Tandem mass spectrometer in positive electrospray mode was used for quantification. The TargetLynx software was applied to process LC/MS/MS data. Samples for LC/MS/MS analysis were prepared as follows: 200 μL of acetonitrile/methanol mixture (3:1 In conclusion, the adenosyl group containing analogues is a quick way to obtain potent SARS-CoV-2 MTase inhibitors; however, these compounds show poor selectivity and cell permeability. The identified drug-like nsp14 and nsp16 inhibitors also suffer from poor selectivity; however, the cell permeability of these compounds is slightly better. The compounds identified explore a wide range of chemotypes that interact with the adenine binding subsite and either the methionine or mRNA base binding sub-site of the MTases. Despite underwhelming selectivity, we believe that the identified inhibitors can serve as solid starting points for inhibitor development against the well-conserved nsp14 and nsp16 of SARS-CoVs.


Finally, no cross-reactivity with other viruses was detected and the test displayed a viable shelf-life of 5 d when stored at 4 °C.High-frequency testing is urgently needed to help prevent the spread of COVID-19. Here, we introduce Low-cost Electrochemical Advanced Diagnostic (LEAD), a diagnostic test that detects SARS-CoV-2 within 6.5 min, costs $1.50 per unit, and uses easily assembled materials such as human angiotensin-converting enzyme 2, modified graphite leads, and a plastic vial. LEAD presents sensitivity comparable to the gold-standard methods  and displays an excellent performance profile using clinical saliva  and nasopharyngeal/oropharyngeal  samples. No cross-reactivity was detected with other coronavirus or influenza strains. Importantly, LEAD also successfully diagnosed the highly contagious SARS-CoV-2 B.1.1.7 UK variant. The device presents high reproducibility under all conditions tested and preserves its original sensitivity for 5 d when stored at 4 °C in phosphate-buffered saline. Our low-cost and do-it-yourself technology opens new avenues to facilitate high-frequency testing and access to much-needed diagnostic tests in resource-limited settings and low-income communities.COVID-19 has led to over 3.47 million deaths worldwide and continues to devastate primarily middle- and low-income countries. High-frequency testing has been proposed as a potential solution to prevent outbreaks. However, current tests are not sufficiently low-cost, rapid, or scalable to enable broad COVID-19 testing. Here, we describe LEAD , a diagnostic test that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within 6.5 min and costs $1.50 per unit to produce using easily accessible and commercially available materials. LEAD is highly sensitive toward SARS-CoV-2 spike protein (limit of detection = 229 fg⋅mL Since its onset, the COVID-19 pandemic has led to 3.47 million deaths and costed our economy billions of dollars . DiffereMost rapid tests commercially available for diagnosing COVID-19 are antigen-based and exhibit low sensitivity, which limits their ability to accurately identify asymptomatic individuals –8, thus 6912Here, we report LEAD , a technology for rapid COVID-19 diagnosis that uses highly accessible and inexpensive materials. The do-it-yourself (DIY) test consists of a transducer made of graphite leads modified with human angiotensin-converting enzyme 2 (ACE2) receptor and a plastic vial A–C. ThisN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with ACE2, EDC-NHS-ACE2, to enable anchoring between the amine groups of AuNPs-cys and the EDC-NHS-ACE2, yielding ACE2-AuNPs-cys after 30 min at 37 °C. The electrodes were then incubated with BSA at 37 °C for 30 min in order to block the electrode’s remaining active sites after immobilization of ACE2 (6]−3/−4) enabled diagnosis of healthy samples versus those that were infected with SARS-CoV-2  and human ACE2, its receptor in the human body , 18 FigA. Thus,  of ACE2 B. BSA is of ACE2 . Next, wRS-CoV-2 C.4 solution with a maximum absorption band at 309 nm. After AuNP-cys formation, a wine color was obtained displaying a UV-vis absorption band at 532 nm. Successful formation of the spherical AuNP-cys solution was confirmed by scanning electron microscopy (SEM) images (Experiments were then performed to characterize the biosensor. ) images B, presen) images C. The Au) images D after tCT) of 57.8 ±2.5 Ω, indicating a small resistance toward redox conversion and a high electron transfer on the electrode surface. This result was in agreement with the high ip of 303.3 ±13.6 µA shown for the same electrode by CV plot (black line). Next, we modified the WE with GA (red line), leading to an increased RCT to 383.1 ± 8.3 Ω and decreased ip to 165.8 ± 6.1 µA. These data indicate that GA acts as an electrical insulator hindering the electron transfer at the interface of the WE by preventing the redox probe from reaching the GPE surface (−1 in our Fourier transform infrared (FTIR) spectroscopy analysis  (CT (47.6 ± 1.3 Ω) and increased ip (410.5 ± 13.7 µA) compared to the previous functionalization step 6]3−/4−, providing a preconcentration of the redox probe close to the electrode interface. This led to an improved electrochemical response, i.e., higher current peak . This stability allowed AuNP-cys synthesis to be carried out weekly, facilitating the large-scale modification process of GPE.The electrochemical behavior of each functionalization step A was ana surface , 25. AuN surface . The ACEFig. S1) , 27. Theion step A. The hiion step , 29, thu−1 H2SO4 after different periods of immersion in the AuNPs-cys suspension . Indeed, an anodic peak was observed at +880 mV and a cathodic peak at +522 mV, which correspond to the redox processes of Au (III) within the electrode surface . We next optimized the period of electrodic exposition to AuNP-cys suspension to be 80 min  based on the peak current of the anodic process derived from oxidation of the gold adsorbed on the GPE/GA surface. Subsequently, we immobilized the recognition element ACE2 onto the surface of the nanoparticles-functionalized GPE using EDC and NHS (magenta line). This led to increased RCT values (182.6 ± 2.2 Ω) and decreased ip values (247.2 ± 4.1 µA) compared to the previous functionalization step , confirming the proper anchoring of our recognizing element (ACE2) to the electrode surface, which led to the hindrance of the faradaic processes of [Fe(CN)6]3−/4− taking place at the modified GPE surface. As a final functionalization step, we immobilized BSA (green line) to block the remaining unmodified electrodic area to avoid nonspecific and undesired adsorption of other molecules . This step resulted in the highest RCT values (985.7 ± 12.7 Ω) and lowest ip (177.4 ± 3.2 µA), suggesting a continued decline in the charge transfer kinetics after anchoring the AuNPs-cys due to the insertion of nonconductive materials .The electrodic functionalization with AuNPs-cys was evaluated through the redox behavior of the adsorbed AuNPs by CVs recorded using 0.1 mol⋅L6]−3/−4. This means that the specific interaction between SP and ACE2 partially blocked the probe’s access to the surface of the WE .We used square wave voltammetry (SWV) for SARS-CoV-2 detection. This technique is highly sensitive, especially for detecting reversible redox species , such asf the WE . To achi−12 to 1 × 10−10 g⋅mL−1. The results were expressed as 6]3−/4−) after incubating the sample on the electrode surface and Next, we evaluated the optimal incubation time for detecting SARS-CoV-2 in clinical samples by evaluating the analytical sensitivity parameter obtained from dose–response curves at very low SP concentrations A. The exe sample A. Five m−1 phosphate-buffered saline (PBS) (pH = 7.4) under optimized experimental conditions  induced by the highly specific interaction between the SP and the ACE2-modified GPE  obtained at each concentration were plotted as a logarithmic function of the SP concentration  of 0.994.The SWV signals , using 35−1 and 0.91 pg⋅mL−1, respectively. Therefore, our device enabled the rapid detection of SP at very low concentrations (less than picograms per milliliter), providing high sensitivity , rapid testing time (6.5 min), and a very low production cost ($1.50 per test). The testing time was recorded to be 6.5 min considering the sample incubation period (5 min), the time required to record two SWVs , and the washing step with PBS after incubating the sample (30 s).The limit of detection (LOD) and limit of quantification (LOQ) of LEAD were calculated according to the four-parameter logistic (4PL) curve (sitivity C and a l2 to 106 plaque-forming units (PFU)⋅mL−1 ⋅mL−1 E and F. sitivity , 39.LC= R2 = 0.954) with Ct values ranging from 22.3 to 34.3 .Our electrochemical biosensor was applied for SARS-CoV-2 detection in clinical samples containing a wide range of viral loads. The threshold cycle (Ct) of the RT-PCR data for all clinical samples analyzed ranged from 21.5 to 34.3 Ct. It is important to highlight that our results (current suppression – ΔI) presented a high linear correlation (−1 [Fe(CN)6]−3/−4 after incubating the sensor with 1 × 10−12 g⋅mL−1 of SP prepared in 0.1 mol⋅L−1 PBS (pH = 7.4). A relative SD of 4.31% was obtained for the analytical signal (current suppression of the redox probe) using six sensors (n = 6) from different batches, indicating excellent reproducibility .We performed reproducibility assays of our device to ensure that different test batches performed similarly. To verify the reproducibility of the manufacturing and functionalization processes of LEAD we recorded SWVs in the presence of 5 mmol⋅L−1 PBS (pH = 7.4) at a concentration ranging from 1 × 10−12 g⋅mL−1 to 1 × 10−9 g⋅mL−1 of SP  over 7 d. Analytical curves were generated in 0.1 mol⋅L−1 of SP D. Sensor−1 of SP F. Intere−1 of SP F. These 1N1 , Influenza-B/Colorado, herpes simplex virus-2, and murine hepatitis virus (MHV). No cross-reactivity was detected against any of these viruses as indicated by the response to all strains, which presented a current suppression (ΔI) lower than the cutoff value of 10 µA obtained by SWV for the lowest SP concentration detected  obtained from inpatients from the Hospital of the University of Pennsylvania after heat inactivation. All samples were confirmed as COVID-19 positives and COVID-19 negatives by RT-PCR. To evaluate the diagnostic efficacy of LEAD, we tested 103 NP/OP  and 10 s−1 and 2.07 PFU⋅mL−1 for free SP in PBS medium and inactivated virus in virus transportation medium [VTM] medium, respectively) and did not cross-react with other relevant viruses.We present a simple, inexpensive, and portable electrochemical biosensor that enables diagnosis of COVID-19 within 6.5 min using 50 µL of sample and highly accessible and commercially available materials , yielding a test that costs $1.50. The WE can be functionalized in less than 3 h and remains stable for over 5 d when stored in a PBS solution at 4 °C. LEAD displayed excellent sensitivity for detecting SARS-CoV-2 SP  including the highly infectious SARS-CoV-2 UK variant 1.1.7.B, indicating that our method does not require further adaptations to accurately diagnose the SARS-CoV-2 UK variant B.1.1.7. However, additional variants would have to be tested to assess the broad applicability of LEAD, particularly when considering emerging mutants. Based on its low cost, selectivity, and accuracy features, LEAD constitutes a potential candidate for high-frequency testing at the point of care and may enable population surveillance and pandemic outbreak control since the modification of the electrodes can be performed in batch and the method can be automated to facilitate mass production. Collectively, our technology may help limit asymptomatic spread through high-frequent testing, i.e., multiple times per week , since i40.3H2O) (99.99%), sodium borohydride (NaBH4) with ≥98% purity, cysteamine hydrochloride (cys) with 98% purity, phosphate buffer saline solution, pH = 7.4, and glutaraldehyde  were purchased from Sigma-Aldrich. Graphite pencils 0.7-mm and 0.9-mm diameter under the trade name of Pentel were purchased in a local store in Philadelphia. Ag/AgCl conductive ink was acquired from Creative Materials.All reagents used in this work were of analytical grade. The deionized water (resistivity ≥18 MΩ cm at 25 °C) was obtained from a Milli-Q Advantage-0.10 purification system (Millipore). ACE2 Fc Chimera, Human was obtained from GenScript. Spike protein was kindly donated by Scott Hensley, University of Pennsylvania, Philadelphia. EDC and NHS with a degree of purity ≥98%, gold(III) chloride trihydrate , each containing three electrodes. Graphite pencils (0.9 mm) were used as reference and counterelectrodes, and graphite pencils (0.7 mm) were used as the WE. The Ag/AgCl ink was painted over one of the graphites to create the reference electrode.−1. EIS was conducted in the frequency range from 1 × 105 Hz to 0.1 Hz using an amplitude of 10 mV and under open circuit potential. Both electrochemical techniques were used to characterize the electrochemical behavior of electrodes in each modification step. In SWV studies, potentials were scanned from −0.4 to 1.0 V, corresponding to a frequency of 80 Hz, amplitude of 75 mV, and step low of 8 mV. All electrochemical characterizations were performed in 0.1 mol⋅L−1 KCl solution containing 5.0 mmol⋅L−1 of the mixture [Fe(CN)6]3−/4− solution. All electrochemical experiments were carried out at room temperature (25 ± 3 °C).Electrochemical techniques, such as CV, were carried out in a potential window from 0.7 to −0.3 V with a scan rate of 50 mV⋅s−1 to 4,500 cm−1 and 32 scans were performed for each measurement.Morphological characterizations of the synthesized AuNP-cys dispersion and GPE before and after superficial functionalization with AuNP-cys/ACE2/BSA were performed using SEM images acquired using a JEOL 7500F HRSEM from the Singh Center for Nanotechnology (University of Pennsylvania). SEM images were recorded with 15,000 to 150,000× magnifications, acceleration voltage of 5 kV, and using SEI mode. The spectrophotometry analysis was performed using a PerkinElmer Multimode Plate Reader spectrophotometer (model EnVision). The absorbance of AuNP-cys dispersion was measured at 532 nm (at room temperature) over 7 d to evaluate its stability. FTIR spectra were obtained for GPE/AuNP-cys and GPE/AuNP-cys/ACE2 samples in a PerkinElmer Spectrum 2 equipped with a Diamond UATR 2 detector ranging from 500 cm−1) was dropped into 1.5 mmol⋅L−1 HAuCl4 in a final volume of 10.0 mL, under vigorous stirring for 20 min at room temperature. Subsequently, 10.0 µL of NaBH4 (10.0 mmol⋅L−1) was added and kept under stirring for 20 min in a light-protected environment at room temperature. The resulting yellow color changed to wine red as a consequence of the formation of AuNPs. Finally, the solution was stored at 4 °C in a refrigerator for up to 7 d, conditions at which it presents high stability when stored in the absence of light   , 44.3+) and a mercaptan group (−SH) in its extremities, the latter of which favorably binds onto the AuNPs surface through an Au–S bond. Thus, cys serves to provide greater stability to the AuNPs due to the electrostatic repulsion process among the amine groups, which generates free positive charges .The WE was polished with 2,000-grit sandpaper, and a contact area of 1.0-cm length by 0.7-mm diameter was obtained. Next, the GPE was kept immersed in a 25.0% (vol/vol) glutaraldehyde solution for 1.0 h as a first modification step. This process allowed the graphite surface to be functionalized with aldehyde groups. Then, the modified graphite substrate was kept in a AuNPs-cys solution with protonated amine groups from cys at pH 7.4, which enabled covalent anchoring of the AuNPs to the substrate by the formation of an amide bond . The AuN−1 EDC, 25 mmol⋅L−1 NHS, and 10 µg⋅mL−1 of ACE2 diluted in 0.1 mol⋅L−1 PBS (pH = 7.4) was incubated on the substrate containing GPE/GA/AuNPs-cys. After 30 min, the ACE2 immobilized onto the substrate reached the anchoring stability and provided a highly sensitive SWV response. In the presence of EDC and NHS, the carboxyl group of ACE2 bound to the primary amine of cys through a covalent bond. The reaction between carboxyl groups and EDC–NHS resulted in the formation of a stable ester, which undergoes nucleophilic substitution with the amine groups present on the substrate surface (AuNPs-Cys), resulting in the formation of an amide bond between the modified GPE/GA/AuNPs-cys surface and ACE2. In the final step, nonspecific sites present within the electrode surface were blocked by incubation in a 1% (mass/vol) BSA solution for 30 min. Subsequently, the solution containing 50 mmol⋅L−1 PBS sample containing SP was applied to the WE using a plastic vial and incubated for 5 min. After the incubation period, the WE was gently washed with 0.1 mol⋅L−1 phosphate buffer solution (pH = 7.4) to remove unbound virus or sample. Next, the electrodes were stored in a 2.0-mL cryogenic vial for subsequent electrochemical detection. One milliliter redox probe solution (5.0 mmol⋅L−1 [Fe(CN)6]3−/4− in 0.1 mol⋅L−1 KCl) was used for the voltammetric measurements and to detect the current suppression due to binding of SARS-CoV-2 SP to the biosensor. Subsequently, the electrochemical response was monitored using the SWV technique. Notably, this procedure was applied to other studies such as cross-reactivity, reproducibility, incubation time, and SARS-CoV-2 detection in clinical samples. The total diagnostic time was calculated to be 6.5 min considering the sample incubation period (5 min), the time required to record two SWVs , and the washing step after sample incubation (30 s).For diagnosing SARS-CoV-2, a volume of 50 μL of VTM or a 0.1 mol⋅L−1 SP solution. The stability of LEAD was evaluated by the analytical sensitivity value extracted from analytical curves in a concentration range from 1 × 10−12 to 1 × 10−10 g⋅mL−1 of SP in different conditions 25 °C, −20 °C, stored dry at 4 °C, and stored at 4 °C in PBS medium (pH = 7.4) over 7 d. All modified electrodes were stored inside capped plastic vials during the stability study.The reproducibility study was performed by recording the analytical signal (current suppression of the redox probe) obtained for six different sensors (from different batches) after incubation with 1 pg⋅mL8 PFU⋅mL−1 (coronavirus); H1N1, A/California/2009; Influenza B, B/Colorado; and HSV2, herpes simplex virus-2 . The highly contagious SARS-CoV-2 B.1.1.7 UK variant was used to assess the capability of LEAD to detect SARS-CoV-2 variants.The specificity studies were carried out using the following different viruses: MHV at 10−14 g⋅mL−1) in the dose–response curve . The Ct values obtained by RT-PCR for the clinical samples ranged from 21.5 to 34.3.The clinical performance of LEAD was assessed using clinical samples acquired from the Hospital of the University of Pennsylvania and deidentified prior to use. We set a current suppression (ΔI) cutoff value higher than 10 µA for diagnostic purposes in accordance with the analytical response obtained for the lowest concentration of SP detected (10se curve C, i.e., Supplementary File


FLT1P1) was previously reported to be upregulated in PE. In this study, we verified the effect of FLT1P1 and its cognate gene FLT1 on trophoblast cell proliferation and angiogenesis by using Cell Counting Kit-8 (CCK-8) assay, tube formation assay, and western blot analysis. Their binding to RNA binding protein dyskeratosis congenita 1 (DKC1) was validated by conducting RNA immunoprecipitation (RIP) and RNA pulldown assays. In this study, knockdown of FLT1P1 or FLT1 was found to promote cell proliferation and angiogenesis in trophoblasts. In addition, FLT1P1 recruited DKC1 to stabilize FLT1. Importantly, silencing FLT1P1 or DKC1 decreased the stability of FLT1. Rescue assays revealed that FLT1 overexpression reversed the effect of silenced FLT1P1. Overall, FLT1P1 cooperates with DKC1 to restrain trophoblast cell proliferation and angiogenesis by targeting FLT1.In preeclampsia (PE), preexistent maternal endothelial dysfunction leads to impaired placentation and vascular maladaptation. Long noncoding RNAs (lncRNAs) have been shown to participate in the placentation process. LncRNA fms-related tyrosine kinase 1 pseudogene 1 ( Preeclampsia (PE), with a global prevalence of estimated 2% to 8% of all pregnancies, is one of the causes of maternal and fetal mortality . It is rMEG3) promotes the apoptosis and suppresses the migration of trophoblast cells [MALAT-1) is expressed at a low level in PE and regulates JEG-3 trophoblast cell migration and invasion [PTENP1) modulate the level of its matched protein-coding gene phosphatase and tensin homolog (PTEN) [URAHP), promotes proliferation and regulates the pathogenesis of PE [PGK1P2) is involved in PE by acting as a competing endogenous RNA of phosphoglycerate kinase 1 [DKC1) is a nucleolar, RBP, which is highly conserved in eukaryotes [DKC1 as a RBP is negatively regulated by MEG3 that has been frequently reported to promote trophoblast migration and decrease apoptosis in PE [FLT1P1 (fms-related tyrosine kinase 1 pseudogene 1), as the pseudogene of VEGFR1 , which is also known as FLT1 (fms-related tyrosine kinase 1) [FLT1P1 was reported to be overexpressed in preeclamptic placentas [Long non-coding RNAs (lncRNAs) are defined as transcribed RNA molecules ranging from 200 to 100,000 nucleotides that do not code for any protein . It has invasion . As a suinvasion . It was g (PTEN) . Since tg (PTEN) ,15. Theris of PE . Dysfunckinase 1 . The strkinase 1 ,19. Dyskkaryotes . Previouis in PE . We founinase 1) . In addilacentas , but itsFLT1P1 in trophoblast cell proliferation and angiogenesis and mechanistically analyzing how FLT1P1 exerts its function in PE. We hypothesized that FLT1P1 may exert its function in PE by interacting with the RBP DKC1 to stabilize its cognate gene FLT1. Our study may provide a novel regulatory mechanism for exploration of the pathogenesis of PE.In this study, we focused on identifying the role of the pseudogene Normal placentas (n = 10) were obtained from full-term births after the cesarean section. Age-matched placentas were obtained from women with severe PE (n = 10) after cesarean section. All placentas involved in this study were collected by procedures of planned cesarean section without the aid of artificial labor. Placental tissues were obtained by a certified doctor by making a vertical incision across a normal area at the center, involving fetal and maternal placental surfaces. Tissues having calcified deposits or clots were excluded. The experiments were approved by the Ethics Committee of the Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University. All volunteers participating in this study signed written informed consent.Term placentas were collected from uncomplicated pregnancies after cesarean delivery. Isolation and culture of PTBs was done according to conventional methods. Specifically, we used a protocol based on the classic trypsin digestion and Percoll gradient centrifugation method, as previously described . Briefly2.HTR-8/SVneo (HTR8) and BeWo cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences . Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640  supplemented with 100 U/ml penicillin (HyClone), 10% heat-inactivated FBS, and 100 μg/ml streptomycin  in a humidified incubator at 37°C with 5% COFLT1P1, FLT1 or DKC1  and the scrambled negative control (sh-NC) were designed and synthesized by GenePharma . The plasmid vector expressing full-length FLT1 was generated by GenePharma to overexpress FLT1 and termed as pcDNA3.1/FLT1 (FLT1), and the empty vector (vector) was used as a negative control. Cells were seeded in 24-well plates at 2 × 105 cells/well and transfected with 40 nM shRNA vector or 0.2 μg overexpression vector following the instructions of Lipofectamine 2000  as described previously [The short hairpin RNAs (shRNAs) against eviously , and cel−ΔΔCt method [FLT1P1, forward: 5ʹ-AAGAACGCCGATTATGTGAG-3ʹ, reverse: 5ʹ-CAAGAGCCACCCATTTCAG-3ʹ; FLT1, forward: 5ʹ-CAAGATTGACTTGAGAGTAACCAG-3ʹ, reverse: 5ʹCTGGAATGGCAGAAACTGG-3ʹ; DKC1, forward: 5ʹ-GGTATAGTAGCCAAGATCAAGAG-3ʹ, reverse: 5ʹ-TTCTGACTTGCCTTTGGAC-3ʹ; GAPDH, forward: 5ʹ-TCAAGATCATCAGCAATGCC-3ʹ, reverse: 5ʹ-CGATACCAAAGTTGTCAT GGA-3ʹ.TRIzol  was used to isolate total RNA from trophoblast cells. Then, the first-strand cDNA was generated by ImProm-II Reverse Transcription System . RT-qPCR was conducted by using SYBR Green qPCR assay  and gene-specific primers. The relative gene expression was calculated by the 2t method , and gly3 cell/well. At 24 h, 48 h, and 72 h, each well was added with 10 μl CCK-8 solution for an additional 1 h of incubation at 37°C. Finally, a microplate reader  was used to read the absorbance value at 450 nm [The cell Counting Kit-8  was used to measure the viability of stably transfected trophoblast cells. In brief, cells were seeded in 96-well plates at 4 × 10t 450 nm .5 trophoblast cells in a medium containing 10% FBS were plated on top of presolidified Matrigel. Once seeded on Matrigel, capillary tubes and networks start to form. After 6 h of incubation, plates were examined with a microscope , and images were taken. The number of branching points was quantified using ImageJ plug-in according to the protocol angiogenesis analyzer from Gilles Carpentier.For the tube formation assay, as described previously , 24-wellFLT1 , and GAPDH  overnight at 4°C. After washing, the membranes were further incubated with secondary antibodies  for 1 h at room temperature. The blots were detected by using a chemiluminescence substrate . Immunoblot signals were quantified using Image Quant software .Western blot was performed as previously published . Total pDKC1 or control IgG (Millipore)-conjugated magnetic beads at 4°C for 6 h, and anti-IgG was used as a negative control. The beads were then washed with a washing buffer and the complexes were incubated with Proteinase K for 30 min at 55°C to remove the protein. Finally, immunoprecipitated RNA was purified and analyzed by RT-qPCR.For the RIP assay, the Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit  was used following the manufacturer’s protocols . The troFLT1P1 and its antisense RNA were transcribed in vitro based on the corresponding PCR product using T7 RNA polymerase (Ambio Life) and Biotin RNA Labeling Mix . The obtained product was purified using RNeasy Plus Mini Kit (Qiagen). For the pulldown assay, 20 µl Dynabeads M-280 Streptavidin beads  were activated and blocked with 10 µg/ml RNase-free BSA and yeast tRNA (Sigma) for 30 min at 4°C. Cell lysates were incubated with the beads at room temperature for 2 h, followed by pulldown, RNA extraction, and subsequent RT-qPCR quantification.As described previously , biotin-FLT1 mRNA stability, trophoblast cells were treated with 10 µg/ml actinomycin D (Sigma) after transfecting with sh-FLT1P1 or sh-DKC1 [FLT1 mRNA level.For detecting  sh-DKC1 . At 3 h,t test or one-way or two-way analysis of variance (ANOVA). P value less than 0.05 was considered statistically significant.Statistical analysis was performed using Graphpad Prism 5 software , and the results are presented as the mean ± standard deviation (SD). Each assay was repeated in triplicate. A comparison between groups was performed using Student’s FLT1P1 in trophoblast cell proliferation and angiogenesis and mechanistically analyzing how FLT1P1 exerts its function in PE. We hypothesized that FLT1P1 may exert its function in PE by interacting with the RBP DKC1 to stabilize its cognate gene FLT1. Our results showed that FLT1P1 and FLT1 play a vital role in PE by regulating trophoblast cell proliferation and angiogenesis. Moreover, FLT1P1 increases FLT1 mRNA stability via recruiting DKC1.In this study, we focused on identifying the role of the pseudogene FLT1P1 in PE progression using two trophoblast cell line (HTR8 and BeWo). As shown in FLT1P1 in HTR8 and BeWo cells was effectively knocked down by transfection with the specific shRNA (sh-FLT1P1) compared to the sh-NC. Next, the functional role of FLT1P1 in trophoblast cell proliferation and angiogenesis was assessed by CCK-8 assay, tube formation assay, and western blot analysis. According to CCK-8 assay, the proliferative ability of trophoblast cells was increased after knocking down FLT1P1 . Next, we investigated the biological relevance of n FLT1P1 . The resWo cells . Further . The sh-FLT1 was transfected into HTR8 and BeWo cells to silence FLT1. The results of RT-qPCR showed that FLT1 expression was significantly downregulated in the sh-FLT1-transfetced HTR8 and BeWo cells , DKC1 was predicted as an RBP that has the potential to interact with FLT1P1 and FLT1 (FLT1P1 (or FLT1) and DKC1 was further predicted at the RPISeq website (http://pridb.gdcb.iastate.edu/). The score of RF Classifier is over 0.5 and the score of SVM Classifier is over 0.8, indicating that FLT1P1 (or FLT1) stands a good chance of binding to DKC1 (DKC1 in FLT1P1 and FLT1 was obtained (FLT1P1 and FLT1 were both enriched in the beads conjugated with anti-DKC1 compared to anti-IgG in trophoblast cells (FLT1P1 (or FLT1) and DKC1, a biotin-labeled FLT1P1 (or FLT1) probe was used to perform RNA pulldown assay in HTR8 and BeWo cells. The results revealed that the DKC1 was significantly enriched in the FLT1P1 (or FLT1) sense probe compared with the sense probe (FLT1P1 (or FLT1) could bind to DKC1.Next, we investigated whether there is a regulatory mechanism between pression ,19. Thusand FLT1 . Then, t to DKC1 . As predobtained . RIP assst cells . To furtse probe . These rFLT1P1/DKC1 exerts function on FLT1 mRNA stability, we tested FLT1 mRNA expression with the treatment of Actinomycin D. The results showed that FLT1P1 knockdown decreased FLT1 mRNA expression under Actinomycin D treatment  expression is correlated with abnormal decidualization and might lead to the occurrence of PE [FLT1P1 is expressed at a high level in preeclamptic placentas [FLT1P1 might play an important role in the development of PE. The biological function of FLT1P1 has not been investigated previously. In this study, we found that FLT1P1 was upregulated in PTBs isolated from PE patients compared with healthy controls. The functional assays suggested that the downregulation of FLT1P1 altered the proliferative capacity of HTR8 and BeWo cells. Defects in decidualization cause inadequate placentation and angiogenesis, which could give rise to PE [FLT1P1 knockdown promoted the angiogenesis in the trophoblast, demonstrating the key role of FLT1P1 in PE.Pseudogenes, which are fairly common (~0.7% of DNA sequence) in the human genome, as well as lncRNAs are traditionally claimed to not yield functional mRNAs and not translated into proteins consequently, thus regarded as garbage fragments or dark matter in the genome ; howevered genes . Hexokince of PE . Howeverlacentas , implyinse to PE . VEGF isse to PE ,41. HereFLT1P1 shares molecular ancestry with the cognate gene FLT1 (VEGFR1) in humans and high primates [FLT1 (VEGFR1) is a type V protein-tyrosine kinase receptor that is crucial for cell proliferation and differentiation and is expressed in vascular endothelial cells, placental trophoblast cells, and peripheral blood monocytes. A soluble form of FLT1 is markedly increased during the last 2 months of gestation in those with PE compared with normotensive pregnant controls, which is involved in the endothelial dysfunction characterizing the pregnancy disorder of PE [FLT1 was upregulated in PTBs isolated from PE patients. FLT1 knockdown promoted the proliferation and angiogenesis in the trophoblast. One of the most commonly described biological feature of processed pseudogenes is the ability to influence the expression of their parental coding genes. The high sequence similarity between these RNA pairs sets up a certain level of competition for posttranscriptional regulators, including, among others, RBPs [FLT1P1 and FLT1. Through bioinformatics analysis, DKC1 was predicted to be an RBP interacting with both FLT1P1 and FLT1. DKC1 is a key RBP encoding a protein responsible for telomerase holoenzyme complex stability [DKC1 could bind to either FLT1P1 or FLT1. Moreover, the knockdown of FLT1P1 or DKC1 effectively decreased the mRNA stability and protein level of FLT1. Therefore, we demonstrated that FLT1P1 increases FLT1 mRNA stability by recruiting DKC1 in this study.Pseudogenes have a high sequence similarity to their parental protein-coding genes, which generates the potential for sequence-specific regulation . The pseprimates . The VEGprimates . FLT1 (Ver of PE . Here, ors, RBPs . Thus, wtability . In our FLT1P1 and FLT1 play a vital role in PE by regulating trophoblast cell proliferation and angiogenesis. Moreover, FLT1P1 increases FLT1 mRNA stability via recruiting DKC1. These results show that dysregulated FLT1P1 and FLT1 may be related to the occurrence of PE, suggesting that FLT1P1 and FLT1 could act as useful biomarkers for the diagnosis of PE. The present study is not without limitations. First, clinical samples need to be collected from PE patients to further verify the clinical significance of our findings. Second, the related signaling pathways targeted by the FLT1P1/FLT1remain unclear and require further investigations.In conclusion, we demonstrated that Click here for additional data file.