Alzheimer’s disease (AD) is the most common type of dementia and, after age, the greatest risk factor for developing AD is the allelic variation of apolipoprotein E (ApoE), with homozygote carriers of the ApoE4 allele having an up to 12-fold greater risk of developing AD than noncarriers. Apolipoprotein E exists as three isoforms that differ in only two amino acid sites, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). These amino acid substitutions are assumed to alter ApoE structure and function, and be responsible for the detrimental effects of ApoE4 via a mechanism that remains unclear. The hypothesis that a structural difference between ApoE4 and ApoE3 (and ApoE2) is the cause of the ApoE4-associated increased risk for AD forms the basis of a therapeutic approach to modulate ApoE4 structure, and we were therefore interested in screening to identify new chemical probes for ApoE4. In this regard, a high-yield protocol was developed for the expression and purification of recombinant full-length ApoE, and three diverse biophysical screening assays were established and characterized; an optical label-free assay (Corning Epic) for hit identification and microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) as orthogonal assays for hit confirmation. The 707 compounds in the National Institute of Health clinical collection were screened for binding to ApoE4, from which six confirmed hits, as well as one analogue, were identified. Although the compounds did not differentiate between ApoE isoforms, these data nevertheless demonstrate the feasibility of using a biophysical approach to identifying compounds that bind to ApoE and that, with further optimization, might differentiate between isoforms to produce a molecule that selectively alters the function of ApoE4. Apolipoprotein E (ApoE) is a 299-amino acid glycoprotein that is involved in cholesterol and lipid delivery. Its primarily function is the recognition of specific receptors, such as the low-density lipoprotein receptor (LDLR) that mediates the internalization of lipoprotein particles ,2. ApoliAPOE) exists as three different polymorphic alleles  that correspond to variations in the combinations of amino acids at residues 112 and 158 of the protein. While ApoE3 has a cysteine at site 112 and an arginine at position 158, ApoE2 has two cysteines and ApoE4 has two arginines at both sites, respectively [APOE is associated with an increased risk for Alzheimer’s disease (AD) [The apolipoprotein E gene (ase (AD) ,7,8,9, aase (AD) . The risData suggest that ApoE4 may contribute to AD pathogenesis through both amyloid beta (Aβ)-dependent and Aβ-independent pathways , althougin silico for molecules using the X-ray structure of the ApoE4 amino terminal domain. These small molecules, such as GIND-25 (Azocarmine G), are believed to disrupt the domain interaction [APOE genotypes and suggest an ApoE4 gain of a toxic function [in vitro effects but devoid of in vivo toxicity were developed by Merck Research Laboratories and are currently in preclinical trials to establish target engagement and a structure–activity relationship [The modulation of ApoE4 structure to change the protein into an ApoE3-like structure therefore represents a novel therapeutic approach, and tool compounds that directly bind to ApoE4 have been identified . A firsteraction , as welleraction . A seconeraction . These meraction . Most refunction . Many offunction . Howeverfunction . Pyrazoltionship ,29,30. AOur aim was to express and purify the different ApoE isoforms and use biophysical assays to enable screening for novel chemical probes. The assays that were optimized were an optical label-free assay (Corning Epic), microscale thermophoresis (MST), and isothermal titration calorimetry (ITC). The 707-compound National Institute of Health (NIH) clinical collection was screened against human full-length recombinant ApoE4, with the label-free assay being used as the primary screen, and MST and ITC being used for confirmation . Taking All materials were purchased from Sigma-Aldrich  or Fisher Scientific  and were the highest purity available. The codon-optimized ApoE4 gene was synthesized and cloned into a pET17b vector at the NdeI/HindIII site by Thermo Fisher Scientific (pET17b_ApoE4). A 6-histidine tag, thioredoxin (TRX), and human rhino virus (HRV) 3C protease cleavage site were upstream of the ApoE4 gene. E. coli Rosetta2 (DE3) cells  in Luria-Bertani (LB) broth supplemented with 1% (v/v) glycerol and 100 µg/mL ampicillin. Protein expression was induced in bacteria grown to an optical density of 600 nm (OD600) of 0.7–0.9 with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 2 h at 37 °C. Bacteria were then pelleted and resuspended in an ice-cold lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 240 mM NaCl, 5 mM MgCl2, 10 mM imidazole, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol, 0.05% (v/v) Tween-20, 7 U/mL DNAse I, using one protease inhibitor tablet per 50 mL, pH 8.0). Cell lysis was achieved by sonication and cell debris separated by centrifugation. Proteins were affinity-purified with the Talon  affinity resin. After equilibration of the Talon beads with the soluble component of the cell lysate, the beads were washed with a talon-binding buffer  glycerol, 0.05% (v/v) Tween-20, pH 8.0) and protein eluted with talon elution buffer  glycerol, 0.05% (v/v) Tween-20, pH 8.0). The elution fractions were then applied on a HiTrap heparin column  and extensively washed with either a size-exclusion buffer for ApoE3 and ApoE4  glycerol, pH 8.0) or a heparin-binding buffer for ApoE2  glycerol, pH 8.0). After on-column digestion with HRV 3C protease overnight at 4 °C, the cleaved TRX tag was eluted with a size-exclusion or heparin-binding buffer, and ApoE proteins were then eluted by applying a linear salt gradient. Elution fractions were pooled, applied on a HiLoad Superdex 26/600 pg 200 column , and all proteins eluted in a size-exclusion buffer. ApoE-containing fractions were concentrated to ~15 mg/mL by centrifugation using VivaSpin 20 concentrators  and protein stored at −80 °C in a size-exclusion buffer  glycerol, pH 8.0). All steps and purity of samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The identity of the ApoE isoforms was confirmed by mass spectroscopy.The ApoE2 and ApoE3 full-length variants were created by site-directed mutagenesis  using the pET17b_ApoE4 plasmid as template, and primers 5′-ggtgcagatatggaagatgtttgtggtcgtctgg-3′ and 5′-gccgatgatctgcagaaatgtctggcagtttatcag-3′, respectively. Bacterial ApoE proteins were expressed in 1-191) was created by inserting a stop codon into the pET17b_ApoE4 plasmid using primer 5′-gattcgtctgcaggcagaagcagctcaagcccgtct-3′. ApoE41-191 was expressed as for the full-length isoforms. The bacterial pellet was resuspended in ice-cold lysis buffer, sonicated, and cell debris separated by centrifugation. The supernatant was then loaded on a HiFliQ nickel–NTA column  and extensively washed with a binding buffer  glycerol). After on-column digestion with HRV 3C protease overnight at 4 °C, ApoE41-191 was washed out with a binding buffer and the TRX tag eluted, and therefore separated from the amino terminal domain, by applying a linear imidazole gradient. Elution fractions were pooled, applied on a HiLoad Superdex 26/600 pg 75 column , and all proteins eluted in a size-exclusion buffer. ApoE1-191-containing fractions were concentrated by centrifugation using VivaSpin 20 concentrators , and protein stored at −80 °C.The ApoE4 amino terminal domain (ApoE4E. coli Rosetta2 (DE3) cells in Luria-Bertani (LB) broth supplemented with 100 µg/mL ampicillin and 35 µg/mL chloramphenicol. Bacteria were grown to an OD600 of 0.6, induced with 0.4 mM IPTG, and protease-expressed overnight at 20 °C. Cells were pelleted and resuspended in an ice-cold lysis buffer , and cell disruption was achieved by sonication. Insoluble components were separated by centrifugation and GST-tagged protease, then affinity-purified using a GSTrap FF column . After passing the soluble lysis fractions through the column, the column was extensively washed with a binding buffer  and protease then eluted with 10 mM reduced glutathione. Protease-containing fractions were pooled, concentrated as described above, rebuffered into a storage buffer  glycerol), and stored at −80 °C.The glutathione S-transferase (GST) HRV 3C fusion protein was expressed in v/v) Triton X-100 + 2% (v/v) dimethyl sulfoxide , and the remaining reactive groups on the biosensor were then blocked by incubation with 50 mM ethanolamine in PBSTD for 5 min at room temperature (RT). The plate was washed three times with PBSTD and, after the final wash, 15 µL PBSTD were added to each well, sealed, and placed into the Epic reader for 1.5 h for thermal equilibration. To measure immobilization levels and/or take the baseline before compound addition after thermal equilibration, 10–15 total points were recorded with 4 × 3 s scans averaged per point.Corning Epic 384-well plates  were activated with 200 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 50 mM sulfo-N-hydroxysulfosuccinimide (sulfo-NHS) at 26 °C for 30 min. Five microliters of ApoE4 diluted to 300 μg/mL in 20 mM sodium citrate pH 5.6 were dispended into wells and incubated at 26 °C for 1 h. The microplate was subsequently washed three times with phosphate buffered saline (PBS) + 0.1% , the measurement was paused, the plate removed, and suramin added into the wells  in triplicate at different concentrations, ranging from 0 to 12 mM. The plate was sealed and placed back into the Epic for 30 min. Binding shifts were then measured over 15–20 points in total, with 4 averages per point. Responses were measured as shifts in reflected wavelength and were expressed in picometers (pm).v/v) Triton X-100 (resulting in 2% (v/v) DMSO) and then dispensed into the plate  after having read the baseline. Plates were left for 30 min in the machine and binding shifts measured as above. Suramin at 5 mM and 150 μM was used as a positive control and buffer-only was the negative control. Prioritized hits were tested in the form of a concentration response, and were diluted 1:2 in neat DMSO, then transferred into PBS + 0.1% Triton X-100 as above, and added into the wells  in duplicate at different concentration, ranging from 0.2 to 100 μM.The NIH collection  contains 707 compounds and was screened against ApoE4 using the Corning Epic. Screening was carried out at a compound concentration of 100 μM in two identical runs. Compounds at 10 mM in neat DMSO were diluted to 200 μM in PBS + 0.1% . Shifts were corrected for background by subtracting the averaged response of the buffer control. Samples with low protein-immobilization levels (<1800 pm) were excluded from data analysis and only plates with a Z′-factor above 0.5 were accepted. The Z′-factor is defined as:g for 5 min at RT before use, and then diluted to 200 nM in 20 mM HEPES, 500 mM NaCl, 10% (v/v) glycerol, and 0.1% Triton X-100 (MST buffer). Binding of hits was first tested in eight-point concentration response assays. Compounds at 5 mM in neat DMSO were diluted 1:2 in DMSO and then transferred into MST buffer. Diluted compounds in MST buffer were then combined with equal volumes of 200 nM labelled protein, resulting in a 2% (v/v) DMSO working concentration, 100 nM labelled ApoE4, and compound concentrations ranging from 0 to 100 μM. The mixture was pulse-centrifuged and incubated at RT for 5 min. Samples were then centrifuged at 16000× g for 5 min and soaked into standard capillaries in duplicates. To achieve full binding curves of compounds in which an effect on thermophoresis or initial fluorescence was observed, 12-point concentration responses were performed from 250 or 500 μM top final concentration in triplicate. For this, compounds were diluted 1:2 in neat DMSO from 12.5 or 25 mM, respectively, transferred into the MST buffer, and combined with equal volumes of 200 nM ApoE4. Microscale thermophoresis measurements were performed on the Monolith NT.115 (Nanotemper). Capillaries were thermally equilibrated at 25 °C for 5 min, and MST traces then collected at 20%–40% light-emitting diode (LED) excitation power and 20%, as well as 40% MST power. Data were analyzed on integrated analysis software . A sample denaturation test was performed for the samples that altered the initial fluorescence signal. The compound–ApoE4 mixture was centrifuged for 5 min at 16,000× g at RT and then mixed with equal volumes of 2 × denaturing buffer . Samples were denatured at 95 °C for 5 min, soaked into capillaries, and the initial fluorescence was measured. Binding affinities, as presented in ApoE4 was labelled in a size-exclusion buffer using the Monolith NT Protein Labeling Kit Red-NHS  following the manufacturer’s instructions, and the labelling reaction was performed with 20 μM ApoE4 and 40 μM labelling dye. The labelled protein was centrifuged at 16,000× 1-191 were dialyzed against PBS + 0.01% (v/v) Triton X-100 overnight at 4 °C using 3.5 K Slide-A-LyzerTM dialysis cassettes . ApoE, as well as ApoE41-191 at 300 μM was injected into the cell containing compound at 25–50 μM in PBS + 0.01% (v/v) Triton X-100 + 2% (v/v) DMSO. Titration experiments were carried out on a Microcal PEAQ-ITC  at 25 °C and 750 rpm, and consisted of an initial (1 μL) injection followed by additional 18 (2 μL) injections. Injection of the protein into PBS + 0.01% (v/v) Triton X-100 + 2% (v/v) DMSO was used as control experiment and integrated control heats were subtracted from the reaction heats. Data were analyzed on Microcal PEAQ-ITC analysis software  and data-fitted for one set of sites. Binding affinities as presented in ApoE isoforms, as well as truncated ApoE4 .E. coli as a TRX fusion protein with a six-histidine tag at the amino terminal domain, and a human rhino virus (HRV) 3C cleavage site between TRX and ApoE. ApoE was purified by a combination of immobilized metal affinity, heparin affinity, and size-exclusion chromatography. All purification steps were under nondenaturing conditions, and the TRX tag was removed by digestion with glutathione S-transferase (GST)-tagged HRV 3C protease. The purity of all ApoE isoforms was confirmed to be above 95%  was screened at 100 µM in two separate runs. Suramin was used as a positive control and, at concentrations of 150 µM and 5 mM, produced a mean response measured as shifts in reflected wavelength of 53 ± 11 and 146 ± 17 pm, respectively A. All plOut of the 707 screened compounds, 59 met the criterion to be considered a hit . Using the MST, we identified pamoic acid to be binding to ApoE4 instead of hydroxyzine, which was the presumed active constituent of hydroxyzine pamoate. Similar observations were made by Zhao et al. , who scrl-thyroxine), three selective estrogen receptor modulators , and one NSAID (meclofenamic acid). The binding of l-thyroxine to ApoE has previously been reported using a radio-ligand binding assay [l-thyroxine in the radio-ligand assay was shown to be inhibited by meclofenamic acid [l-thyroxine or meclofenamic acid to the amino terminal domain but we were able to confirm the binding of the l-thyroxine analog tafamidis to ApoE. Notably, we observed the binding of SERMs to ApoE, which has not previously been described. Selective estrogen receptor modulators are agonists or antagonists of the estrogen receptor, and have been considered as a possible treatment for women with AD. However, it is still a matter of debate if hormone therapy and SERMs have beneficial effects on cognitive function, and clinical studies gave inconsistent results [Six out of the eight drugs could be confirmed by ITC. All hits besides pamoic acid are in clinical use and include a hormone (ng assay ,53 with mic acid ,53, we c results .The hits identified in the present study do not show selectivity for ApoE4 versus ApoE2 and ApoE3, and binding to all ApoE isoforms with similar affinities was observed by ITC. This indicates that ApoE isoforms share structural features, and that compounds bind to conserved binding sites. Affinities are in the micromolar range  and it wIn summary, our screening trial successfully identified compounds as novel ApoE chemical probes, and our validated biophysical assays can be used for larger screening initiatives. Although none of the identified hits differentiated between ApoE isoforms, these methods nevertheless provide a novel means of identifying compounds that may be useful in defining the physiological functions of ApoE, and are more generally applicable to investigate other physiological interactions such as ApoE–receptor interaction.


Although a large fraction of high-grade serous epithelial ovarian cancers (OCs) expresses Estrogen Receptor alpha (ERα), anti-estrogen-based therapies are still not widely used against these tumors due to a lack of sufficient evidence. The histone methyltransferase Disruptor of telomeric silencing-1-like (DOT1L), which is a modulator of ERα transcriptional activity in breast cancer, controls chromatin functions involved in tumor initiation and progression and has been proposed as a prognostic OC biomarker. As molecular and clinico-pathological data from TCGA suggest a correlation between ERα and DOT1L expression and OC prognosis, the presence and significance of ERα/DOT1L association was investigated in chemotherapy-sensitive and chemotherapy-resistant ER+ OC cells. RNA sequencing before and after inhibition of these factors showed that their activity is implicated in OC cell proliferation and that they functionally cooperate with each other to control the transcription of genes involved in key cancer cell features, such as the cell cycle, epithelial-mesenchymal transition (EMT), drug metabolism, and cell-to-cell signaling, as well as expression of the ERα gene itself. Together with evidence from loss-of-function genetic screens showing that ERα and DOT1L behave as core fitness factors in OC cells, these results suggest that combined inhibition of their activity might be effective against ERα-expressing, chemotherapy-resistant ovarian tumors. Ovarian cancer (OC) represents a heterogeneous tumor, traditionally classified according to histologic and differentiation grade features . Among OEpigenetic mechanisms, such as histone modifications and DNA methylation, have emerged as contributing factors to carcinogenesis by playing a pivotal role in regulating the malignant phenotype. Therefore, it represents valuable therapeutic targets . The DisA correlation between OC prognosis and the presence of ERα has been recurrently considered but is still debated. Analyzing TCGA OC expression profiling datasets , we obseTo investigate this possibility, we adopted as experimental model of two cell lines, PEO1 and PEO4, isolated from the same individual and representing two stages of the disease, which are the first recurrence stage and the chemo-resistance stage ,21. BehaAmong these, the NRF2-mediated oxidative stress response, together with other oxidative stress pathways, has been implicated in OC resistance to chemotherapeutics . NRF2 haBased on the evidence that DOT1L is a co-factor and upstream regulator of ERα in breast cancer , we veriThen, to assess the effects of ERα and DOT1L inhibition on OC cell proliferation, MTT assays were performed before and after treatment with anti-estrogens  and increasing concentrations of EPZ. The results obtained confirmed the responsiveness of both PEO cell lines to the mitogenic effects of estrogen, which was demonstrated by the ability of anti-estrogens to inhibit cell proliferation c,i. On tSince H3K79 methylation by DOT1L is directly coupled to gene transcription , we thenThe heatmap of As previously shown in breast cancer , DOT1L iTo investigate whether the effects of DOT1L inhibition on ERα gene expression are mediated by a direct role of this enzyme on ESR1 gene transcription, we analyzed a regulatory region of this gene promoter region for the presence of ERα, DOT1L, and the active transcription marker H3K79me2 by ChIP–qPCR. Results shown in −8 and 10−9 M) and increasing concentrations of EPZ. Results show an additive effect of the combined treatment on cell proliferation and ERα levels   showed tTaken together, these results indicate that DOT1L inhibition can be effective against a sizeable fraction of ovarian tumors.ERα is a well-known cancer-associated gene, target of anti-estrogen based therapies in breast cancer, and expressed in a large OC fraction, where its prognostic value is still debated. OCs represent a highly heterogeneous group of cancers, with different etiology, phenotypes, molecular biology, and clinico-pathological features, including in the prognosis. Epithelial ovarian cancers (EOCs) represent the vast majority of these tumors, and, among them, HGSOCs are the most aggressive and account for almost 75% of all EOCs . As a maDespite active ERα-mediated estrogen signaling having been observed in OC ,35, whicWe report in this paper how inhibition of estrogen signaling by targeting this upstream ERα co-factor and regulator may represent an effective therapeutic approach against ERα-expressing OCs. Recently, it has been demonstrated that silencing ERα expression and activity through the use of epigenetically-acting compounds is a good option in breast cancer to overcome resistance to endocrine therapy ,41. In tIn conclusion, the results described in this study provide evidence of a functional cooperation between ERα and DOT1L, acting at the transcriptional level to modulate the expression of genes involved in OC cell proliferation and other key cellular functions. Since specific inhibitors of the two proteins show additive effects on OC cell growth, it is possible to assume that dual inhibition of estrogen signaling by simultaneous blockade of ERα and DOT1L may represent a way, worth exploring further, to improve the response and survival of patients suffering with ERα-positive OC, where endocrine therapy usually shows only moderate responses. DOT1L expression correlates with a worse clinical behavior of OCs and genome-wide in vitro genetic screens by the CRIPSR-Cas9 gene ‘knock-down’ revealing that this gene is important for proliferation and survival of >60% of the OC cell lines tested . The reskmplot.com). To determine groups presenting low-expression and high-expression of ERα and DOT1L, quartile filtering was applied. In detail, cases with expression levels below the first quartile were included in the ‘low expressing’ group, while those above the third quartile were included in the ‘high expressing’ one.Data from the TCGA Ovarian Serous Cystadenocarcinoma  dataset https://depmap.org) and from the Project Score website (https://score.depmap.sanger.ac.uk).CRISPR-Cas9 gene knock-down data have been downloaded from DepMap portal .Human ovarian cell lines ERα-positive PEO1 (ECACC 10032308) and PEO4 (ECACC 10032309) and ERα-negative PEO14 (ECACC 10032311) were purchased from the American European Collection of Authenticated Cell Cultures (ECACC). All cell lines were isolated from peritoneal ascites of serous ovarian adenocarcinomas and carry TP53 mutations . PEO1 ceThe following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα , rabbit anti-estrogen Receptor Alpha , rabbit polyclonal anti-DOT1L , β-actin , Rabbit anti-KMT4/DOT1L (ab72454), anti-Histone H3, total, (ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K27me3 (ab24684) from Abcam, anti-Rabbit IgG Isotype Control , and the anti-Mouse IgG antibody .Cells were treated with the following compounds: DOT1L inhibitors EPZ004777 (S7353), Pinometostat (EPZ5676)(S7062), SGC0946 (S7079), all from Selleckchem, and with 4-hydroxytamoxifen (4-OHT) , Fulvestrant  , and β-estradiol  or vehicles (DMSO and/or EtOH), according to different experimental settings.5–107 cells was added directly to the culture dish to lyse the cells by pipetting several times to homogenize. Total RNA was extracted using the standard RNA extraction method with TRIzol , according to the manufacturer’s instructions. Furthermore, 0.3–1 mL of TRIzol™ Reagent per 1 × 10Before use, RNA concentration was determined by using Quant-IT RNA Assay Kit-High Sensitivity and a Qubit Fluorometer  and its quality and integrity assessed with the Agilent 4200 Tapestation System .−8 M ICI 182,780, and their vehicles for nine days. Indexed libraries were prepared, using 1 μg of total RNA as starting material from biological triplicates, with TruSeq Stranded Total RNA Sample Prep Kit . Libraries were sequenced  at a concentration of 1.8 pM/lane on the NextSeq 500 platform (Illumina Inc.). The raw sequence files generated (.fastq files) underwent quality control analysis using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and quality-checked reads were then aligned to the human genome (assembly hg19) using a star [p value (adj-p) ≤ 0.05. Raw RNA sequencing data are deposited in the EBI ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) with accession number E-MTAB-8198.For RNA sequencing, cell lines were treated with 6.4 µM EPZ004777, 10g a star  with theg a star . A gene One μg of total RNA for each sample was reverse transcribed to cDNA using Random Hexamer . RTqPCRs were performed in triplicate on a Stratagene Mx3005P instrument (Agilent Technologies) using SensiFAST SYBR Lo-ROX kit (Bioline), according to the manufacturer’s instructions. Values were normalized to values relative to RPLP0 mRNA, analyzed in parallel.The primers used for qPCR are shown below. for RPLPO: Forward primer: CCATCAGCACCACAGCTTC     Reverse primer: GGCGACCTGGAAGTCCAACT for ERα: Forward primer: ACCCTCCATGATCAGGTCCA     Reverse primer: CTGGTTCCTGTCCAAGAGCAfor DOT1L: Forward primer: GTTCTACCAGCTACCTCCGAGCGTGCAGCGCTGCACGCTCGGAGGTAGCTGGTACAAC    Reverse primer: Protein extractions were performed after culturing in a normal growing condition with either vehicle (at the longer time point) or DOT1L inhibitors for 3, 6, 9, or 12 days. Media were refreshed every 3 days. 2 1.5 mM, NaMo4 1mM, and NaF 0.5 M). After incubation on ice for 15 min, samples were centrifuged at 13.000× g for 30 min and the supernatant, containing total proteins, was transferred to a new tube and diluted by adding two volumes of low salt buffer with respect to the high salt buffer previously added. For total protein extraction, cells were harvested and lysed in an equal volume with respect to the cell pellet of high salt buffer  glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1× protease inhibitor cocktail, and 1 mM PMSF), incubated for 30 min at 4 °C and centrifuged for 30 min at 4 °C. Nuclear proteins were then diluted to restore the physiological saline concentration. Protein concentration was established by a Bradford Assay.Cells were harvested in ice cold PBS and pellets were suspended in Hypotonic Buffer  to isolate a cytosolic fraction. Then, the nuclear pellets were dissolved in a Nuclear Lysis Buffer  and Wash Buffer , and suspended in Laemmli buffer.For ERα and DOT1L immunoprecipitations, 35 μL of equilibrated Dynabeads M-280 Sheep Anti-Rabbit IgG (Thermo Fisher Scientific) were conjugated overnight at 4 °C, respectively, with 2 μg of anti-ERα, anti-DOT 1L, and a Rabbit IgG Isotype Control. Immunoprecipitation was performed by incubating conjugated beads/antibodies at 4 °C for 1 hour with 500 μg of nuclear protein extracts. After incubation, beads were washed with IPP150 buffer , 2 mm PMSF, 0.02% (w/v) NaN3). Cells were lysed on ice for 10 min and nuclear separation was obtained by centrifugation at 6500× g for 10 min at 4 °C. Nuclei were then suspended in 0.2 N HCl. After an overnight incubation at 4 °C on a thermomixer, samples were centrifuged and the histone concentration in the supernatant was determined by using the Bradford Assay.At the indicated time points, cells were harvested, washed in ice cold PBS, and suspended in Triton Extraction Buffer , and electro-transferred onto a nitrocellulose blotting membrane . Following blocking with 5% skimmed milk in TBST buffer , the membranes were immunoblotted overnight with different primary antibodies.Each Ab was used according to the manufacturer’s instructions. After extensively washing with TBST, the primary Abs were detected by the appropriate horseradish peroxidase-conjugated secondary Abs  and revealed by chemiluminescence and autoradiography.3 cells per well and the experiment was repeated three times . After incubation for 3, 6, 9, and 12 days with either drugs or vehicles, cell proliferation was evaluated using 3--2,5-diphenyltetrazolium bromide (MTT)  at a final concentration of 1 mg/mL, according to the manufacturer’s instructions. Absorbance was measured at 570 and 620 nm (background) wavelengths by the VICTOR Multilabel Plate Reader .For cell proliferation analyses, cells were seeded in octuplicate into 96-well plates at a density of 2.5 × 105 cells were incubated with increasing concentrations of a compound for 3, 6, 9, or 12 days with ICI 182,780, EPZ004777 or the vehicle, by refreshing every 3 days. All assays were carried out in triplicate.For cell cycle analysis, 10Cells were then fixed in cold 70% ethanol at 4 °C, treated with RNase A (10 μg/mL) for 10 min at room temperature, and stained with 20 μg/mL Propidium iodide (Sigma-Aldrich) for 30 min. Flow cytometry was performed using a BD FACSVerse™  and data processed using the software ModFit LT™ . Results shown were obtained from three independent experiments.3 cells were seeded in triplicate in 6-well plates and treated for 9 days with different compounds, according to the experimental settings. After incubation, cells were fixed with 4% paraformaldehyde and stained with 0.1% (w/v) crystal violet in PBS. For quantitation, colonies were dissolved in 10% acetic acid before absorbance measurement at 595 nm. For each cell line, 5 × 10ChIP assays were performed by following the protocols previously detailed . Furthermore, 100 µL of equilibrated suspension of Dynabeads M-280 Sheep Anti-Rabbit IgG or Anti-Mouse IgG were incubated overnight at 4 °C with 10 µg of the indicated antibodies for the immunoprecipitations. Purified Rabbit IgG and Mouse IgG antibodies were used as negative controls. Crosslinking was performed with 1% formaldehyde at room temperature for 10 min and the reaction was stopped by adding glycine to a final concentration of 0.125 M. Bead washing, elution, reverse crosslinking, and DNA extraction were then performed as described . DNA conFor ChIP–qPCR, 2 ng of DNA were used to amplify indicated promoter regions with the following specific primers.ESR1: Forward primer: TGTGCGCCCTAACCAAAGG  Reverse primer: TGCTCCCAAAGTAGATAGACCCTHOXB3: Forward primer: CCACCTGCATAAGGGCAAAAT  Reverse primer: CTACAGGGCTAGGAATGAGGGTGM1: Forward primer: TGCCCAACCATTCCTTAGCA  Reverse primer: CTGCTGTTGAGACTTGCCCATWIST2: Forward primer: CGACTGTCCTTACACTGGCG  Reverse primer: CACAGCTAGTCTGCACCGCWT1: Forward primer: CGTATACCCTTGCTTTGCACC  Reverse primer: ATCATGGCCACTCCCCTACCChIPs were performed in triplicate and qPCRs were carried out in duplicate in each case.−8 and 10−9 M) and increasing concentrations of EPZ00477 after 9 days of treatment.Drug combination analysis was carried out by using Combenefit software  and dosewww.ingenuity.com) and investigation of modulated canonical pathways was carried out. The Circos plot was generated using GOPlot [The lists of differentially expressed transcripts were submitted to Ingenuity Pathway Software . Error bars represent mean ± SD of independent replicates. Comparisons between two groups were conducted by performing student’s In the present study, we provide evidence of a key role of DOT1L in OC, including the functional cooperation between the nuclear receptor ERα and this epigenetic writer in conveying estrogen signaling to modulate the transcription of genes involved in cell proliferation and other key functions of OC cells. Furthermore, the results provide evidence suggesting how combined inhibition of both these factors might be therapeutically effective in blocking estrogen signaling in chemo-resistant ovarian tumors.


The increase in cytokinin maintained the growth of Mg-deficient plants, while JA and JA-IEA were induced in response to carbohydrates accumulation. Altogether, our study reveals the vital role of Si under Mg deficiency by regulating plant primary metabolite and hormonal changes.The importance of magnesium (Mg) for plant growth is well-documented. Silicon (Si)-mediated alleviation of mineral deficiencies has been also reported in a number of plant species; however, there is no report on the relevance of Si nutrition in plants grown in Mg-deficient condition. Therefore, in the present work, an attempt was undertaken to study the role of Si nutrition in maize plants exposed to Mg deficiency. Plants were grown either under low (0.02 mM) or normal (0.5 mM) levels of Mg, with or without Si supplement. We have shown that Mg-deficient plants treated with Si maintained their growth and increased significantly the levels of chlorophyll and soluble sugars compared to those plants which did not receive Si. In addition, the concentrations of hexose-P, and glycolytic intermediate metabolites—mainly organic acids (isocitric and glutamic acids)—were increased in response to Si nutrition, which was associated with an increase in the levels of stress amino acids such as gamma-aminobutyric-acid (GABA), serine and glycine, as well as polyamines putrescine, which overall contributed to Mg stress tolerance. In addition, Si enhanced the levels of phytohormones cytokinin  To ensure proper growth and development, plants require nutrients. Restricting nutrient supply will lead to poor establishment, which will subsequently arrest proper growth . Among eMg concentrations in soil solutions are usually between 125 and 8.5 mM; however, plants, which are typically immobile encounter a lack of Mg in their natural environment . Mg defiSilicon (Si) is the second most abundant element in the earth’s crust, and is considered a beneficial element for agriculture production . The supHence, the aim of this study was to determine whether, and to what extent, Si application mitigates Mg deficiency in maize plants. We hypothesized that Mg deficiency would induce a decline in plant chlorophyll levels and impair phloem loading of sugar to the roots, which would result in photosynthesis inhibition and accumulation of sugar in the leaves, thus reducing plant growth. The application of Si to Mg-deficient plants increases chlorophyll level and biomass, enhances sugar accumulation and modulates hormonal changes, thus alleviating Mg deficiency. To accomplish the objective of this study and to investigate the cross-talk between Si and Mg, different physiological, biochemical and molecular approaches were performed.In the present study, Si supply did not influence the shoot fresh weights at normal levels of Mg (0.5 mM), whereas both concentrations of Si significantly increased shoot fresh weights under Mg- deficient conditions compared to Mg-deficient plants which did not receive Si A. ConverIn our study, we examined whether Si affected Mg uptake in the roots and its translocation to the shoots. As we expected, the total Mg in Mg-deficient plants as significantly decreased compared to Mg-sufficient plants for both roots and shoots A,B. AppaZmSUT1) and bidirectional sugar transporters  were induced by Si in Mg-deficient plants  significantly decreased compared to Mg sufficient-plants A. The lot plants .In general, the levels of Glu, Fru, and Suc were significantly increased in the shoots of Mg-deficient plants compared to Mg-sufficient plants D–F. InteWe further analyzed the levels of amino acids, organic acids and polyamines which are thought to change under mineral nutrition deficiencies . The levUnder normal Mg supply, the level of shoot GABA decreased with a lower level of Si, but was restored to the levels of Mg-sufficient plants with the higher concentration of Si D. UnlikeWe further measured glycolysis intermediate metabolites including organic acids which are involved in tricarboxylic acid cycle (TCA) cycle and amino acids synthesis. In the roots, we did not observe any particular changes in the levels of hexose-P and isocitric acid A,C. CompAmong the polyamines, putrescine (Put) and spermidine (Spm) were detected using HPLC device. In roots of Mg-sufficient plants, the level of Put was increased with Si supply, and this increase was statistically significant when the concentration of Si increased in the solution A. The roiso-pentenyladenine (IP), iso-pentenyladenine riboside (IPR), jasmonic acid (JA), and its derivate L-isoleucine (JA-ILE). The roots of Mg-deficient plants showed significantly higher levels of IP and IPR compared to Mg-sufficient plants  and 12-oxophytodienoate 1 (ZmOPR1) were significantly enhanced in the roots and shoots of Mg-deficient plants when a higher level of Si was supplied into the solution (ZmOPR1 was significantly increased in the roots of Mg-deficient plants with a higher level of Si (ZmD9 (dwarf plant 9) and ZmSLN1-like (slender 1) were also induced with Si supply in Mg-deficient plants compared to those plant which were solely exposed to Mg deficiency  upregulated by Si supply in response to Mg deficiency in both roots and shoots. This indicates that Si transcriptionally regulated JA biosynthesis pathway for the synthesis of JA in response to Mg deficiency  were germinated on vermiculite for 3 days in the dark, followed by a further four days in light conditions. After 7 days, the seedlings were transplanted to a 5.9 L tank in a greenhouse that was set to a 14/10 h day/night cycle at a day/night temperature of 28/25 °C with 40 to 50% relative humidity. Plants were divided into two batches: (i) Mg-sufficient (0.5 mM) plants were grown with a complete solution: Ca(NO3)2 1.3 mM, NH4NO3 0.7 mM, K2SO4 1 mM, MgSO4 0.5 mM, K2H2PO4 0.2 mM, CaCl2 0.5 mM, H3BO3 5 µM, MnSO4 2 µM, ZnSO4 0.5 µM, CuSO4 0.02 µM, (NH4)6Mo7O24 0.01 µM and EDTA, 2NaFe, H2O 200 µM. (ii) Mg-deficient plants were grown in the same nutrition solution, but with only 0.02 mM Mg. The nutrient solution was buffered to pH 5.9, and renewed every two days and continuously aerated. In order to avoid osmotic shock, seedlings were grown in 25% strength nutrient solution for the first 2 days, which was raised stepwise to 100% strength within seven days. One week after normal growth, two concentrations of Si (1.5 and 3 mM) were introduced to the culture solution. Monosilicic acid [Si(OH)4] was freshly prepared by passing sodium silicate solution through a column filled with cation-exchange resins, according to the method described by Yin et al. -phaseic acid (D-PA) were purchased from . ABA, [2H6]-ABA, IAA, and [2H5]-IAA were purchased from OlchemIn . Phytohormones were analyzed by an UHPLC-MS/MS system. Then, 10 mg FW samples were extracted with 70% methanol, 29% H2O, 1% formic acid containing isotope-labeled internal standards, and centrifuged at 12,600 rpm to collect the supernatant. After evaporation , the extract was re-suspended in 2% formic acid solution and purified using an SPE CX express plate of 30 mg/mL . The cytokinins were eluted with 5% ammonium hydroxide methanolic solution and samples were evaporated and re-suspended in 100 μL of 0.1% formic acid solution before injection into the system. Separation and detection conditions were determined using the same parameters described above.Isopentenyladenine (IP), isopentenyladenosine (IPA), trans zeatin (TZ), trans zeatin riboside (TZR), cis zeatin (CZ), cis-zeatin riboside (CZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR) were purchased from OlchemIn . Cytokinins were analyzed by an UHPLC-MS/MS system. Then, 20 mg FW samples were extracted with 70% methanol, 29% Hv/v) ethanol and incubated at 80 °C for 60 min. Crude extracts were centrifuged at 14,000 rpm for 10 min at 4 °C, and the upper phase was concentrated in a SpeedVac concentrator  at 45 °C for 180 min. The pellet was re-suspended in 0.75 mL deionized water and incubated at 80 °C for 60 min. After centrifugation, the second supernatant was added to the first and concentrated. Hexokinase (HK), phosphoglucoisomerase (PGI) and b-fructosidase were added successively to measure Glc, Fru and Suc, as described in Kim et al., 2013 [Soluble sugar determination was undertaken according to the method described by Kim et al., 2013 . Next, 1l., 2013 .2O. After centrifugation , the supernatant was recovered, evaporated and dissolved in 100 µL of ddH2O. Derivatization was performed using an Ultra Derivatization Kit AccQ tag, following the manufacturer’s protocol . The amino acid profile was determined by using UPLC/PDA H-Class system with BEH C18 100 ×2.1 mm column.For amino acid determination, 10 mg lyophilized dry matter was extracted with a solution containing of 400 µL of MeOH and 0.625 nmol/µL Norvaline, which was used as the internal standard . Extraction was stirred for 15 min, and was then re-suspended with 200 µL of chloroform and 400 µL of ddHv/v (−20 °C) containing 0.1% of perchloric acid (v/v) solvent were added. Samples were shaken with vortex for 20 min. Then, they were centrifuged using an Eppendorf Centrifuge 5427 R  for 15 min 12,700 rpm at 4 °C. Supernatants were collected and introduced in a new 2 mL Eppendorf tubes. A second extraction was performed adding 500 µL of water + 0.1% perchloric acid (v/v) to leaves and roots, shaken for 5 min with vortex, and centrifuged for 15 min with 12,700 rpm on 4 °C. Supernatants were mixed and centrifuged for 10 min in order to eliminate suspended particles. Finally, supernatants were diluted 3 times with water + 0.1% Formic acid (v/v) and introduced in 2 mL LC-MS vials. Metabolite analysis was achieved using an ultra-high performance liquid chromatography (UPLC) Acquity H-Class system , and high-resolution detection was performed by using a Xevo G2-S QToF mass spectrometer  equipped with an electrospray ionization (ESI) source. A Phenomenex Luna® Omega PS C18  column  was used to profile metabolites such as organic acids. The mobile phase, comprising water containing 0.5% formic acid (A) and methanol: water (70:30 v/v) containing 0.5% formic acid (B), was applied with the optimized gradient elution as follows: 100% A at 0–1 min, 100–20% A at 1–4 min, 20–0% A at 4–6.5 min, 0% A at 6.5–7.5 min, 0–100% A at 7.5–7.9 min, 100% A at 7.9–10 min. The flow rate was kept at 0.3 mL/min, column temperature was maintained at 35 °C. The injection volume for both columns was 10 µL and samples were maintained at 10 °C. The ESI source was used in negative ionization, source voltage was set to 2.5 kV and cone voltage was 30 V, whilst source temperature was maintained at 130 °C with a cone gas flow of 20 L/h. The desolvation temperature was at 550 °C, with a desolvation gas flow of 900 L/h. Leucine-Enkephalin was used as lockmass reference, (ion at m/z 556.2771 in positive mode), which was introduced by a lockspray at 10 μL min−1 for real-time data calibration. The MSE data were acquired in centroid mode using a scan range 50–800 Da, scan time 0.1 s, resolution was set at 20000 full width half maximum (FWHM), and a collision energy ramp 40–80 V.Metabolite analysis was performed as described previously by Ali et al., 2018 [2O/29% MeOH/1.0% formic acid (v/v/v) at −20 °C. Next, the tubes were stirred at room temperature for 30 min, and then centrifuged at 4 °C , and the supernatant was transferred into new Eppendorf tubes. The supernatant was transferred to a LC/MS vial for analysis. Polyamines were analyzed by an UHPLC–MS/MS system. The separation and detection were achieved using a Nexera X2 UHPLC system  coupled to a QTrap 6500+ mass spectrometer  equipped with an IonDriveTM turbo V electrospray (ESI) source.Polyamine extraction was achieved using 20 mg of frozen grounded leaves that were weighed in a 2 mL eppendorf tube . Extraction was carried out by adding 1 mL of a solution of 70% H® 8 RNA kit following the manufacturer’s protocol . The quality and yield of all RNA samples were analyzed and checked in a 4200 Tapestation , followed by DNase treatment and cDNA synthesis from 1 μg RNA using iScriptTM gDNA clear cDNA synthesis kit . Quantitative RT-PCR (qPCR) analysis was performed in a total volume of 10 μL using Universal SYBR Green Supermix  in Real-Time PCR Detection System . The qPCR reactions were performed in technical triplicates using independent cDNA reactions for each biological replicate and 300 nM of gene-specific primer pairs. Specific primers for all candidate genes were designed using Primer3 software (version 0.4.0) and are listed in GAPDH, CYCLOPHILIN, EIF4a and β-tubulin. All qPCR expression data were acquired and analyzed using CFX Maestro Software Version 1.0 .The roots and shoots sample (100 mg) of maize plants were grounded to a fine powder in the presence of liquid nitrogen and total RNA was extracted using a NucleospinZea mays were conducted under control and osmotic stress conditions, with 4 independent biological replicates consisting of 3 plants each. Data are represented as mean ± standard error (SE) for n = 4. The LSD test (R software) was employed to analyze all the data and marked by different letters when significantly different (p < 0.05).For individual treatment, hydroponic experimentations on In this study, for the first time, we report the cross talk between Mg and Si nutrition. We showed that Si supply to maize plants exposed to Mg deficiency regulated plant primary metabolites and modulated the phytohormones levels to cope with Mg deficiency, thus maintaining plants growth and development. The first response of Mg-deficient plants to Si nutrition was the accumulation of soluble sugars as compatible solutes, as well as the increase in hexose-P. This increase provided carbon flow toward glycolytic pathway, and consequently, increased the level of organic acids, synthesis of stress amino acids GABA, Gly and Ser as well as polyamines Put which all contributed to Mg stress tolerance. The second response to Si nutrition under Mg deficiency was associated with phytohormonal changes—mainly an increase in the levels of cytokinin, JA and its derivate JA-ILE. Higher cytokinin maintained the plants’ growth and development under Mg deficiency, as indicated by higher shoot fresh weight. Moreover, the increase in the level of JA and JA-ILE switched on the plant defense system (against probable pathogens attack) in response to substantial sugar accumulation in the shoots, which was associated with higher expression of the DELLAs which induced JA related genes. Overall, our findings revealed that the application of Si increases the tolerance of maize plants subjected to Mg deficiency by regulating primary metabolite and the hormonal balance .


WBSCR17 (Cfa6.6 and Cfa6.7), GTF2I (Cfa6.66) and POM121 (Cfa6.83) were identified in domestic dogs and gray wolves. Variations in these MEIs were significantly associated with a heightened propensity to initiate prolonged social contact or hypersociability. Using our dataset of 837 dogs, 228 of which had paired survey-based behavioral data, we discovered that one of the insertions in WBSCR17 is the most important predictor of dog sociable behaviors related to human proximity, measured by the Canine Behavioral Assessment Research Questionnaire (C-BARQ©). We found a positive correlation between insertions at Cfa6.6 and dog separation distress in the form of restlessness when about to be left alone by the owner. Lastly, assistance dogs showed significant heterozygosity deficiency at locus Cfa6.6 and higher frequency of insertions at Cfa6.6 and Cfa6.7. We suggest that training programs could utilize this genetic survey to screen for MEIs at WBSCR17 to identify dogs with sociable traits compatible with successful assistance dog performance.Assistance dog training programs can see as many as 60% of their trainees dismissed. Many training programs utilize behavioral assays prior to admittance to identify likely successful candidates, yet such assays can be insconsistent. Recently, four canine retrotransposon mobile element insertions (MEIs) in or near genes  Genome-wide approaches have increased in their application for exploring the molecular basis of animal behavior and personality. In contrast to many dog morphological traits, which have been successfully mapped to genetic variants ,2,3, effAssistance dogs are tremendously valuable with respect to their owner’s physical and mental wellbeing ,16. ThesWBSCR17 (Cfa6.6 and Cfa6.7), GTF2I (Cfa6.66), and POM121 (Cfa6.83), which are known to be involved in Williams–Beuren syndrome in humans [WBSCR17 and POM121 were associated with increased hypersociability, while MEIs within the gene GTF2I were associated with decreased hypersociability [WBSCR17 and POM121 were associated with increased breed groups by their stereotyped attention-seeking behaviors [cis-regulatory pathways. MEIs are highly methylated and may regulate adjacent genes [Recently, we discovered four canine retrotransposon mobile element insertions (MEIs) in domestic dogs and gray wolves that were significantly associated with a heightened propensity to initiate prolonged social contact, commonly referred to as hypersociability . These Mn humans . VonHoldiability . Additioehaviors . These Mehaviors . These Mehaviors ,24, and ehaviors . ThroughHere, we assessed patterns of MEI insertions of dogs with varying sociable personalities and explored the frequency of MEIs across various dog groups . The C-BARQ© assesses dog attachment seeking, which is described by an individual’s propensity to seek physical proximity to owners and heightened anxiety when separated from owners . In thisAll dog owners gave their informed consent for inclusion and participation of their dogs in the study. The study was carried out under the IACUC protocol number 2098 A-17.n: purebred = 656, mixed-breed = 104, unknown = 78), from 196 assistance dogs  and 642 pet dogs. For all DNA isolated from buccal cells or saliva, we completed a second purification step using a 1:2 ratio of DNA to AMPure XP magnetic purification beads . We followed previously published amplification methods to genotype and survey the insertional dynamics of four MEIs implicated in canine hypersocial behavior [We isolated DNA from 837 adult domestic dogs >1 year of age , 159 of behavior . We obtaOf 837 dogs with genetic samples, 228 also had paired detailed demographic  and behavioral data derived from 42 questions of the C-BARQ© (short version) . The C-BPrevious studies have shown that dog behavior varies with age. We conducted Spearman’s rank correlational tests to elucidate associations between dog age and sociability measures on the C-BARQ©. We also created four datasets of pet dogs based on demographics from the C-BARQ© to ensure that we accounted for differences in behavior that may be due to the age of the dog or how many years the dog was owned at the time of reporting. Each of these datasets has paired MEI genotype and behavioral C-BARQ© data. The datasets comprising of pet dogs were as follows: (1) 69 young adult dogs of 1–5 years of age; (2) 95 adult dogs over >5 years of age; (3) 65 dogs that have been owned for between 1 and5 years by the reporting owner; and (4) 98 dogs that have been owned for >5 years by the reporting owner . AdditioTo elucidate the potential order of importance of these loci in affecting sociability-related phenotypes, we employed conditional random forests ensemble algorithms using 20,000 bootstrap samples from the R package “party”  and funcn = 117) and those owned from 1–5 years (n = 115), since all assistance dogs containing C-BARQ© scores belonged to these groups. To assess whether relevant questions on the C-BARQ© tag the same behaviors as tested in vonHoldt et al. [Previously, hypersocial canine behavior was quantified using solvable tasks and sociability measures, including dogs’ propensity to spend time looking at a human relative to a nonsocial stimulus  and their propensity to spend time in proximity to familiar or unfamiliar humans  . To detet et al. , we modet et al. . Ridge rt et al. . We analt et al.  and implt et al. , which iO) and allele frequencies with the Hardy–Weinberg equilibrium (HWE) exact test function “Hwe.exact” in the R “genetics” package [We assessed differences in C-BARQ© behavioral scores and MEI copy number comparing assistance and pet dogs. Differences in C-BARQ© behavior and MEIs were assessed using Mann–Whitney U tests with a Bonferroni correction implemented with the R functions wilcox.test and “p.adjust” . To asse package . Between package . Paired We hypothesize that the MEIs will display allele frequency differences for breeds with respect to their genetic relationships with other breeds and their own unique breed history. We therefore grouped dogs into breed groups based on previous studies that identified the genetic relationships between breeds using phylogenetic trees. Several studies have reported breed groups that are highly genetically divergent and distinct. The primary signals of divergence are from Asian Spitz-type breeds, Arctic Spits-type breeds, Sighthounds, and African breeds, which branched separately from European-derived breeds ,35,36. Mn = 52), we randomly sampled 100 individuals from the recent-radiation group to ensure sample size differences did not introduce any biases. Only pure-bred dogs were used for this analysis.We assessed MEI insertion frequency in dogs belonging to either divergent or recent-radiation breed groups. We hypothesized that selection for human-directed hypersociability pre-Victorian-era breed radiation likely resulted in differences in sociability and MEI frequencies before and after the Victorian breed radiation ,39. As tR = −0.14, p = 0.03), a decrease in separation-related problems , and an increase in stranger-directed aggression   corresponded to a decrease in attachment and attention-seeking behavior (≤ 0.001) .2 measures from the VARIMP scores. We found the highest positive variable importance for attachment/attention-seeking (VARIMP = 0.003) and separation-related problems (VARIMP = 0.04) at locus Cfa6.6  and those owned for >5 years  (p = 0.242) and those owned from 1–5 years , have higher yet nonsignificant associations for MEI copy number at Cfa6.6 and increased hypersociability (p = 0.008) (p = 0.040) (p = 0.006), in addition to Q3  (p = 0.024) (p = 0.025) and those owned from 1–5 years  (p = 0.017) and those owned from 1–5 years    . Contrariability . When we= 0.008) . Higher = 0.040) . This as= 0.029)  and the = 0.024) . Higher = 0.029) . This as= 0.037) , in addi= 0.038) .n = 49) had significantly lower median scores on C-BARQ© questions negatively associated with social interest in strangers than pet dogs of the same age group (n = 69): stranger-directed aggression , dog-directed fear , and energy, which measures a dogs’ propensity to act in a playful, boisterous, and active manner   (German Shepherd assistance dogs of 1–5 years of age (ge: 0–4) a,c. Assige: 0–4) . O = 0.08, p = 3.2 × 10−4) relative to pet dogs at loci Cfa6.6 , where the proportion of observed frequency of the insertion allele was 96% in assistance dogs, compared to 61% in pet dogs (Bonferroni-adjusted p = 2.3 × 10−10) (p = 0.02) (p = 9.7 × 10−7)  relative to pet dogs at locus Cfa6.6 . In comparison to pet Retriever-clade dogs , assistance Retriever-clade individuals had a proportionally higher frequency of MEIs at Cfa6.6  and Cfa6.66 , and lower frequency of MEIs at Cfa6.83 . For both sets of assistance dogs, the population were not in HWE and presented an excess of genotypes homozygous for the MEI at Cfa6.6 (Assistance dogs had lower observed heterozygosity (H× 10−10) d. Additi = 0.02) e. Furthe × 10−7) f. Retriet Cfa6.6 ; Table 2p = 3.6 × 10−7), Cfa6.7 (Bonferroni-adjusted p = 6.4 × 10−7), and Cfa6.66 (Bonferroni-adjusted p = 8.0 × 10−7) (p = 8.8 × 10−8) (O = 0.276) and Cfa6.83 (HO = 0.057), due to higher frequency of homozygous genotypes for the insertion at Cfa6.6 (p = 1.6 × 10−3) and homozygous genotypes for the no insertions at Cfa6.83 (p = 0.029).Divergent dog breeds had higher frequency of inserted alleles at Cfa6.6 (Bonferroni-adjusted  × 10−7) a,c; howe × 10−8) d. AdditiWBSCR17, GTF2I, and POM121) on canine chromosome 6 (Cfa6) correspond to hypersocial behaviors in dogs as quantified by the C-BARQ©. We further investigated if the frequency of these MEIs differed between assistance and pet dogs to understand whether MEI screening of dogs could predict social behavior in adult behavior as reported by the C-BARQ©. Highly predictive genetic markers for social behaviors relevant to assistance dog training and placement may provide important benefits, including increased success rates, lower costs, and early behavioral interventions, allowing for better welfare and greater assistant dog availability. We discovered that locus Cfa6.6, a locus associated with WBSCR17, had the highest predictive power for C-BARQ© behaviors related to the hypersocial phenotype, which include higher levels of attention-seeking and separation-related distress among adult dogs of age 1–5 years. Increased frequencies of insertions at Cfa6.6 was associated with higher scores on the hypersociability-related C-BARQ© question Q22, which is consistent with previous work [WBSCR17 transcripts are known to be predominantly expressed in the cerebellum, hippocampus, thalamus, and cerebral cortex of the rat brain [WBSCR17 and behavior, although definitive proof of the functional link between WBSCR17 and sociability is yet to be established. Furthermore, vonHoldt et al. and colleagues [WBSCR17 expression than what was previously known.Our objective was to elucidate whether increased MEIs with hypersociability genes , filed with Princeton University.


Picea glauca, genotype WS77111), a coniferous tree widespread in the boreal forests of North America. This sequence contributes to genomic and phylogenetic analyses of the Picea genus that are part of ongoing research to understand their adaptation to environmental stress.Here, we present the complete chloroplast genome sequence of white spruce , a coniferous tree widespread in the boreal forests of North America. This sequence contributes to genomic and phylogenetic analyses of the Picea genus that are part of ongoing research to understand their adaptation to environmental stress.Here, we present the complete chloroplast genome sequence of white spruce ( Picea glauca) have evolved to cope with adverse environmental conditions (P. glauca (genotype WS77111) was published in 2015  needle tissue sample was collected in southeastern Ontario . Genomic DNA was extracted from 60 g of tissue by Bio S&T using an organelle exclusion method yielding 300 μg of high-quality purified nuclear DNA, as previously described . Briefly, 1 μg of genomic DNA was sonicated (Covaris LE220) in 62.5 μl to 400 bp and purified with PCRClean DX magnetic beads (Aline Biosciences). Illumina sequencing adapters were ligated overnight at 16°C. Pooled libraries were sequenced with paired-end 250-bp reads on an Illumina HiSeq 2500 instrument in rapid mode. Using this protocol, four libraries were generated, sequencing approximately 400 million reads from each one.k = 128 and kc = 3). Due to the large number of chloroplasts per cell, the chloroplast genome would be sequenced at a very high coverage over a full lane of data. Therefore, by subsampling the full data set, the coverages of the nuclear and mitochondrial genomes were lowered to a level where these sequences do not assemble well, while the coverage of the chloroplast genome was still sufficient enough for a high-quality assembly. The 43×, 88×, and 172× subsets produced the best ABySS assemblies , as determined by a QUAST analysis (v5.0.0) (NC_028594) . The assembly with the fewest aligning contigs (n = 14) and fewest misassemblies  was chosen for further scaffolding with the PG29 chloroplast genome, using LINKS v1.8.5 , producing subsets with 21×, 43×, 88×, 172×, 345×, 711×, 1,219×, and 5,619× coverage of the chloroplast genome. Each subset was assembled with ABySS v2.1.0 (_028594) , as it iS v1.8.5  for readPicea sp. chloroplast genomes as references  required manual annotation. The genome map in The complete genotype WS77111 chloroplast genome is 123,421 bp long, with a G+C content of 38.74%. Using GeSeq v1.65  with sevferences , 10, we Picea spp.The assembly of this new chloroplast genome will enable further analysis of the phylogeny and genetics of Picea glauca, genotype WS77111, is available in GenBank under accession number MK174379, and the raw reads are in the SRA under accession numbers SRX525336 and SRR1259605. The annotations used as references were from Picea abies (GenBank accession number NC_021456), Picea asperata (GenBank accession number NC_032367), Picea glauca genotype PG29 (GenBank accession number NC_028594), Picea morrisonicola (GenBank accession number NC_016069), and Picea sitchensis (GenBank accession numbers NC_011152 and KU215903).The complete chloroplast genome sequence of


Pueraria candollei var. mirifica, and 17β-estradiol attenuated ovariectomy-induced depression-like behavior, as indicated by a decrease in immobility times in the tail suspension test (TST) and the forced swimming test (FST), an increase in the uterine weight and volume, a decrease in serum corticosterone levels, and dose-dependently normalized the downregulated transcription of the brain-derived neurotrophic factor (BDNF) and estrogen receptor (Erβ and Erα) mRNAs. Like 17β-estradiol, puerarin also inhibited ovariectomy-induced suppression of neurogenesis in the dentate gyrus of the hippocampus (increased the number of doublecortin (DCX)-immunosuppressive cells). These results suggest that puerarin exerts antidepressant-like effects in OVX animals, possibly by attenuating the OVX-induced hyperactivation of the HPA axis and/or normalizing the downregulated transcription of BDNF and ER mRNA in the brain.Daily treatment of ovariectomized (OVX) ICR mice with puerarin, a glycosyl isoflavone isolated from the root bark of  Physiological fluctuations of estrogen in women are an important factor that affects mood, behavior, and cognition during their lifespan. A sharp decline in estrogen levels, which occurs during the anestrus cycle and menopausal transition, post-menopause, and surgical menopause, causes the most significant alterations in mood and cognitive function ,2. Thus,Preclinical studies demonstrated that bilateral ovariectomy and aging make female rodents more susceptible to stressful stimuli than control animals, inducing emotional deficits such as anxiety and depression. These symptoms can be reversed by supplementation with estrogen. A body of evidence suggests that such estrogen replacement therapy (ERT) can exert anxiolytic and antidepressant effects in menopausal females by modulating the hypothalamic–pituitary–adrenal (HPA) axis functions ,4. HowevPueraria candollei var. mirifica is a medicinal plant used in Thai traditional medicine for its rejuvenating and estrogenic effects , MeOH–H2O , MeOH (8 × 2 L), and EtOAc (5 × 2 L). The fractions eluted MeOH–H2O (1:1) were combined and evaporated under reduced pressure, to give a crude aqueous methanol extract (55.3 g), which was applied over a column of silica gel (1.1 kg) and eluted with mixtures of CHCl3–MeOH and CHCl3–MeOH–H2O, wherein 250 mL fractions were collected as follow: Fractions (Frs.) 1–3 , 4–20 , 21–25 , 26–54 , 55–63 , 64–68 , and 69 (MeOH). Frs. 37–42 were combined (15.53 g) and purified by reversed-phase high-performance liquid chromatography (HPLC) of the JASCO model 887-PU pump and a 875 UV variable-wavelength detector , using TSKgel ODS-80TS column  with an isocratic elution using a mixture of MeCN–H2O  as a mobile phase, at a flow rate of 45.0 mL/min, to obtain 2.98 g of puerarin (1) . Puerarin (1) was identified by comparison of its MS, 1H, and 13C NMR data  were used in the experiments. Animals were housed in paper-bedding cages and given food and water ad libitum. Housing conditions were a 12 h light–dark cycle (lights on: 07:00 –19:00 h) under temperature control (24 ± 1 °C) and constant humidity (65 ± 2%). All behavioral experiments were performed between 07:30 and 17.00 h, and each animal was used once, according to the Guiding Principles for the Care and Use of Animals  and were also approved by the Institutional Animal Use and Care Committee of the University of Toyama, Japan.®: 60 mg/kg; Ceva Sante Animale, Libourne, France), as previously described [2], OVX + puerarin 10 mg/kg/day [OVX + 1 (10)], and OVX + puerarin 100 mg/kg/day [OVX + 1 (100)]. There were 8–12 animals in each group. The body weights of the mice were recorded weekly, during the experimental period. The experimental protocols are summarized in Ovariectomy was conducted under pentobarbital anesthesia  . The 4th and 5th groups were OVX groups, which received, respectively, 10 and 100 mg/kg (po) of puerarin (1). A small volume of absolute ethanol (25 uL) was added to 0.5 g of puerarin (1), and then corn oil (25 mL) was added in order to give a final concentration of 20 mg/mL, and it was used as a stock solution. The reference standard drug, 17β-estradiol (2), was suspended in corn oil. The stock solutions were kept in a dark bottle, at room temperature, until used.The vehicle  and drugs were administered daily , at 8.00Mice were placed in the testing room for 1 h prior to the behavioral test for habituation. The order of the behavioral tests was as follow: Y-maze test (day 45), FST , and TST (day 51–52). One day after completion of the behavioral tests, the mice were decapitated. All tissues were collected immediately and kept at −80 °C throughout the experiment.In order to exclude false positive results from drug-induced hyperlocomotion in hopeless behavioral tests, locomotor activity was performed, using the Y-maze task. The Y-maze consisted of three equal arms that were 40 cm long, 18 cm high, and 3 cm wide at the bottom, and 12 cm wide at the top. The arms were positioned at equal angles, in a Y-shape. One hour after drug administration, the mice were individually placed on one arm, and the total arm entries were recorded manually, over an 8 min period, for measuring the locomotion activity .The FST is the method used to examine “learned helplessness and hopeless” behaviors in rodent models. Mice were placed individually in a transparent glass cylinder (12 cm in diameter and 25 cm high), which was filled with water, to the height of 10 cm, at 25 ± 2 °C. Twenty-four hours before the test, a pre-swimming test session was performed for 15 min. In the test session, each mouse received a vehicle or a drug treatment 1 h before the test. Immobility time was recorded during the 5 min period. Mice were considered to be immobile when they discontinued struggling and remained floating motionless in the water, making only those movements necessary to keep their head above the water.2/s, monitored by the SMART® system version 2.5 . Immobility times were recorded for the last 4 min during a 6 min period.The TST was developed as a rodent screening test for potential (human) antidepressant drugs. This test was conducted as previously described . Briefly1) exerts in vivo estrogenic effects. The weight and volume of the uterus, isolated from each group, was directly measured on a scale and recorded.A uterine hypertrophy test was performed, to examine whether the 8-week treatment of OVX mice with puerarin , and the blood samples were collected. Samples were left overnight at room temperature and then centrifuged at 3000 rpm, at 4 °C, for 15 min. The supernatant was collected and frozen at –80 °C, until used. Assay Max Corticosterone ELISA kit  was employed to measure the serum corticosterone levels according to the previously described procedure . The ser® reagent , according to the manufacturer’s instructions. First-strand cDNA was synthesized with oligo (dT) primers and M-MLV reverse transcriptase  and used as a template for QPCR. QPCR was conducted by using SsoAdvanced™ Universal SYBR® Green Supermix . The following primers were synthesized by Macrogen : amplification was performed by using the following gene-specific PCR primer sets as follows: (1)-β-actin: 5′-AAC GGT CTC ACG TCA GTG TA-3′ (sense) and 5′-GTG ACA GCA TTG CTT CTG TG-3′ (antisense); (2)-BDNF: 5′-GAC AAG GCA ACT TGG CCT AC-3′ (sense) and 5′-CCT GTC ACA CAC GCT CAG CTC-3′ (antisense); (3)-CREB: 5′-TAC CCA GGG AGG AGC AAT AC-3′(sense) and 5′-GAG GCA GCT TGA ACA ACA AC-3′ (antisense); (4)-ER-β: 5′-CAG TAA CAA GGG CAT GGA AC-3′ (sense) and 5′-GTA CAT GTC CCA CTT CTG AC-3′ (antisense); and (5)-ER-α: 5′-GAC CAG ATG GTC AGT GCC TT-3′ (sense) and 5′-ACT CGA GAA GGT GGA CCT GA-3′ (antisense). A melting curve analysis of each gene was performed each time after amplification. β-Actin mRNA was used as a control to which results were normalized. Relative expression was used to represent data.Mouse BDNF, CREB, ERα, and ERβ expression levels were analyzed by QPCR. Total RNA from the hippocampus was extracted, using TRIzolDoublecortin (DCX)-immunopositive cells in the hippocampal dentate gyrus region were measured as a marker of neurogenesis and immature neurons in the present study. After completing behavioral studies, animals were intracardiacally perfused with 4% paraformaldehyde (PFA) in PBS, under pentobarbital anesthesia (whole brains were removed and fixed in 4% PFA in PBS for 24 h). PBS with 30% sucrose was used as sucrose fixation for cryo-protection at 4 °C for 24 h, and then the brains were fixed on dry ice and stored at –80 °C, until sectioned. Coronal brain sections (10 µm) were sliced on a cryostat , fixed on glass slides, and stored at –20 °C, until stained. Brain slides were washed with PBS, and then antigen activation was achieved by incubating in Target Retrieval Solution , at 98 °C, for 30 min, and 10% normal goat serum in PBS was used as a blocking solution at room temperature for 1 h. Slides were then incubated with an anti-DCX antibody  at 4 °C for 3–6 h. Slides were washed with PBS six times, and then incubated with a goat anti-rabbit IgG antibody  for 1 h. Brain slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing medium. Images of brain slices were captured by using an upright optical microscope, under a 20 x objective  equipped with a digital camera . DCX-immunopositive cells were counted in the dentate gyrus area at –2.06 mm, relative to the bregma , in a blind manner.p < 0.05 were considered significant. SigmaStat® version 3.5  was used for statistical analyses.Data were expressed as the mean ± SEM and analyzed by a one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons among different groups. Differences with 1), a glycosyl isoflavone isolated from the root bark of the Thai medicinal plant Pueraria candollei var. mirifica, relieves a depressive-like behavior in OVX mice, by suppressing HPA axis hyperactivity, decreasing corticosterone serum levels, and mimicking estrogenic-like activity through the ERα and ERβ subtypes. Furthermore, puerarin (1) promotes hippocampal neurogenesis, possibly by upregulating BDNF mRNA expression, and increases the number of DCX-immunopositive cells. Thus, puerarin (1) has the potential for treatment of depression in estrogen-deficient women.The present study provides corroborative evidence, for the first time, that puerarin (


Gold and silver nanoparticles (ca. 10 nm diameter) with up to 5% (mol/mol) FluPep ligand remained as stable as the control of mixed-matrix-passivated nanoparticles in a variety of tests, including ligand exchange with dithiothreitol. The free FluPep ligand peptide was found to inhibit viral plaque formation in canine MDCK cells (IC50 = 2.1 nM), but was less potent than FluPep itself (IC50 = 140 pM). Nanoparticles functionalised with FluPep ligand showed enhanced antiviral activity compared to the free peptides. The IC50 value of the FluPep-functionalised nanoparticles decreased as the grafting density of FluPep ligand increased from 0.03% to 5% (both mol/mol), with IC50 values down to about 10% of that of the corresponding free peptide. The data demonstrate that conjugation of FluPep to gold and silver nanoparticles enhances its antiviral potency; the antimicrobial activity of silver ions may enable the design of even more potent antimicrobial inhibitors, capable of targeting both influenza and bacterial co-infections.The influenza (“flu”) type-A virus is a major medical and veterinary health concern and causes global pandemics. The peptide “FluPep” is an established inhibitor of influenza virus infectivity in model systems. We have explored the potential for noble-metal nanoparticle conjugates of FluPep to enhance its antiviral activity and to determine their potential as a delivery platform for FluPep. FluPep ligand is FluPep extended at its N-terminus with the sequence CVVVTAAA, to allow for its incorporation into a mixed-matrix ligand shell of a peptidol and an alkanethiol ethylene glycol consisting of 70% CVVVTol and 30% HS(CH The influenza (“flu”) type-A virus is a major health concern for humans and livestock animals. The primary mode of transmission is by the respiratory route. Flu infection occurs seasonally and can cause global pandemics, e.g., the 2009 H1N1 subtype swine influenza, which resulted in more than 18000 deaths worldwide . HoweverCurrently there are two common types of anti-influenza drugs, based on their mechanism of action. The first class are neuraminidase inhibitors such as Oseltamivir (Tamiflu). The second class are virus ion-channel blockers, such as Amantadine (Symetrel). The effectiveness of Tamiflu has been questioned , and in The infectivity of influenza A viruses, including the H1N1 subtype, is strongly inhibited by a peptide called FluPep . FluPep Noble-metal nanoparticles possess a strong plasmon absorbance, which allows for the detection at very low levels, using a range of approaches, from absorbance –18 to phThe mixed-matrix ligand shell of 70:30 (mol/mol) peptidol and alkanethiol ethylene glycol assembled on gold nanoparticles has a well-characterised stability with respect to ligand exchange and non-specific binding –27, but Gold nanoparticles with a ligand shell incorporating 5% (mol/mol) FluPep ligand had a very similar resistance to ligand exchange with DTT as the control mixed-matrix-protected gold nanoparticles. Their aggregation parameter was unchanged up to 5 mM DTT, even after 48 h incubation ,C. At 10When the peptide FluPep ligand was included in the ligand mix to functionalise the nanoparticles, its molar fraction in percent in relation to the matrix ligand should reflect its grafting density on the gold nanoparticles ,26,30–32Mixed-matrix gold nanoparticles did not to bind to either CM-Sepharose or DEAE-Sepharose . Thus, t50 values. In these experiments the IC50 value of the original FluPep sequence was found to be of the order of 140 pM  and vehicle (DMSO)-treated cells, the cell monolayer in the well was evenly stained . Countinf 140 pM , inset. measured . The soue extent .The addition of mixed-matrix-passivated nanoparticles as control had no detrimental effect on viral infectivity, since their addition to the cells did not change the number of plaques. However, when purified mixed-matrix-capped gold nanoparticles functionalised with 5% (mol/mol) FluPep ligand were added, there was a marked decrease in the number of plaques, i.e., a reduced virus infectivity . Thus, a50 value, which shows a 4.5-fold greater potency of nanoparticles functionalised with 5% (mol/mol) FluPep ligand, when compared to nanoparticles functionalised with just 0.03% (mol/mol) FluPep ligand  FluPep ligand, the number of plaques started to decrease at 20 pM gold nanoparticles and reached a minimum of approximately two to eight plaques at 200 pM . As the p ligand . The nanp ligand , so in ap ligand .Silver has well-established antimicrobial properties . It was Purification of the FluPep-functionalised silver nanoparticles was achieved by cation-exchange chromatography on CM-Sepharose. As the percentage of FluPep incorporated into the ligand shell increased, so did the percentage of nanoparticles bound to CM-Sepharose . This su+) ions that exert antimicrobial activity  were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) foetal calf serum (FCS) , 1% (v/v) 200 mM L-glutamine, 1% (v/v) 100 U/mL penicillin and 1% (v/v) 100 µg/mL streptomycin  and incubated in a humidified environment at 37 °C under 5% (v/v) CO6 cells/flask. Then, the cell monolayer was washed with 2 × 5 mL PBS, and virus (A/WSN/33 H1N1 subtype) was added at a multiplicity of infection (MOI) of 0.001 in 2 mL DMEM. Cells were incubated with virus for 1 h at 37 °C on a rocking platform. Virus-containing medium was removed and the cell monolayer washed with 2 × 5 mL DMEM, then 5 mL N-acetyl trypsin , 2.5 µg/mL in DMEM, was added and incubated for 24–48 hours at 37 °C until a significant cytopathic effect had developed, to a point where the cells were lifting from the flask substrate. Medium was removed and centrifuged for 5 min at 2500 rpm to remove cell debris and the supernatant, which represented the viral stock, was stored at −80 °C.MDCK cells were grown to 90% confluence in T25 tissue culture flasks , which corresponds to 7 × 106 cells/well) for two days. At confluence, monolayers of MDCK cells were then infected with a serial dilution of influenza virus inoculum (sufficient to obtain approximately 100 plaques per well) for 1 h at 37 °C on a rocking platform. An agarose overlay was prepared by mixing equal volumes of 2% (w/v) of pre-warmed (55 °C) low-melting agarose  and the overlay solution  bovine serum albumin , 1.4 mL L-glutamine, 2.6 mL 7.5% (w/v) NaHCO2, 1.4 mL 1 M HEPES, 1.4 mL (1% (v/v) 100 U/mL penicillin and 1% (v/v) 100 µg/mL streptomycin), 44.8 mL H2O and 5 µL N-acetyl trypsin) to give a final 1% (w/v) agarose mixture. After 1 h of incubation of the cells with the virus, the supernatant was removed from the plates and overlaid with 2–3 mL of the 1% (w/v) agarose overlay solution. The plates were left at room temperature for 15 min for the overlay to solidify and then inverted and placed in an incubator at 37 °C, 5% (v/v) CO2 for 3 days for plaques to develop. Cells were then fixed with 4 mL 10% (v/v) neutral buffered formalin  for 1 h, after which the formalin and overlay were removed and cells were stained with 0.1% (w/v in water) toluidine blue, rinsed in water, and left to dry before counting plaques.MDCK cells were grown in 6-well plates , 10Virus titre was determined for each preparation of virus. The virus titre, as plaque-forming units (PFU) per mL, was determined by serially diluting the virus stock and counting the number of plaques in duplicate wells of MDCK cells. Only wells containing between 10 and 100 plaques were counted to ensure the assumption that each plaque formed was due to one infective virus particle was met.File 1Additional experimental data.


Leishmania (NTD) endemic in 98 countries. Although some drugs are available, current treatments deal with issues such as toxicity, low efficacy, and emergence of resistance. Therefore, there is an urgent need to identify new targets for the development of new antileishmanial drugs. Protein kinases (PKs), which play an essential role in many biological processes, have become potential drug targets for many parasitic diseases. A refined bioinformatics pipeline was applied in order to define and compare the kinomes of L. infantum and L. braziliensis, species that cause cutaneous and visceral manifestations of leishmaniasis in the Americas, the latter being potentially fatal if untreated. Respectively, 224 and 221 PKs were identified in L. infantum and L. braziliensis overall. Almost all unclassified eukaryotic PKs were assigned to six of nine major kinase groups and, consequently, most have been classified into family and subfamily. Furthermore, revealing the kinomes for both Leishmania species allowed for the prioritization of potential drug targets that could be explored for discovering new drugs against leishmaniasis. Finally, we used a drug repurposing approach and prioritized seven approved drugs and investigational compounds to be experimentally tested against Leishmania. Trametinib and NMS-1286937 inhibited the growth of L. infantum and L. braziliensis promastigotes and amastigotes and therefore might be good candidates for the drug repurposing pipeline.Leishmaniasis is a neglected tropical disease caused by parasites of the genus   The parasites are transmitted to humans through the bite of infected phlebotomine sandflies from the Lutzomyia and Phlebotomus genera.  OR “drug name” [All Fields]) AND (“Leishmania” [MeSH Terms] OR “Leishmania” [All Fields]). After applying the literature search filter, the antileishmanial activity of these compounds were predicted using an in-house QSAR phenotypic model for L. infantum. The compounds predicted to be active by the QSAR model were purchased for in vitro experimental evaluation.The FASTA sequences of each prioritized target were used to interrogate two different publicly available databases that provide detailed information on drugs and their targets: DrugBank  and kina2.7L. (L.) amazonensis (MHOM/BR/PH8), L. (L.) infantum (MHOM/BR/1972/LD) and L. (V.) braziliensis (MHOM/BR/94/H3227) promastigotes as described elsewhere [L. (L.) infantum and L. (V.) braziliensis intracellular amastigotes.Fresh aliquots from 10 mM DMSO (Sigma-Aldrich)-diluted stock solutions of seven kinase inhibitors  were prepared and tested against 6 logarithmic-phase promastigotes were incubated with increasing concentrations of each kinase inhibitor  in triplicates. Viability was assessed by the MTT method and 50% of effective concentrations (EC50%) were determined by sigmoid regression analysis using Prism 5.0a (GraphPad Software. Inc.).Parasites were maintained at 26 °C in medium 199 (Sigma-Aldrich) as previously described . To perf5 BALB/c bone marrow derived macrophages (BMDM) were obtained [L. (L.) infantum and L. (V.) braziliensis stationary-phase promastigotes  for 24 h in 24 well plates and kept at 37 °C and 34 °C, respectively. The protocol was approved by the Animal Experimentation Ethics Committee (CEUA-UNICAMP #4535-1/2017). Established infections were incubated with 10, 15 and 30 μM of NMS-1286937 and Trametinib. Parasite burden was assessed by counting intracellular amastigotes in at least 200 BMDM per coverslip. Each assay was performed in triplicates and the reduction in amastigote number was compared to the untreated infection group (100%).To perform the amastigote assays, 4 × 10obtained  and infe33.13.1.1L. infantum proteome. From these original 197, 40 kinases were assigned to subfamilies, 109 to families, 18 to groups, and 30 remained unclassified. After manual curation and improvement of this draft kinome (see Methods Section), we obtained a total of 224 kinases , of which 157 were assigned to subfamilies, 64 to families, three to groups, and only one remained unclassified ; nuclear DBF2-related kinases ; phosphoinositide dependent protein kinase 1 ; protein kinase A ; and ribosomal S6 kinase . Two AGC kinases were not assigned to any family.A total of 195 ePKs were identified in L. e groups , among wn = 4); and the calcium-dependent protein kinase . The group CK1 had 7 members, of which 6 were identified as cell kinase 1 family (CK1) and one was from the tau tubulin kinase family (TTBK).The CMGC group is one of the most represented in L. infantum kinome, with 50 protein kinases classified in the following families: cyclin-dependent protein kinase ; cell kinase 2 ; CDC-like kinase  dual-specificity tyrosine-regulated kinase ; glycogen synthase kinase 3 ; mitogen-activated protein kinase ; resistance to complement killing ; and SR protein kinase . Forty-one PKs were assigned to the following families of STE group: MAP kinase kinase kinase genes, homologous to yeast Ste. 11 ; MAP kinase kinase kinase kinase genes, homologous to yeast Ste20 ; MAP kinase kinase genes, homologous to yeast Ste. 7 ; and STE-Unique (n = 1). The group “Other” contains several kinase families that do not fit within any of the main kinase groups. There were 62 kinases classified into the families of this group: aurora kinase family ; Bud 32 (n = 1); calcium/calmodulin-regulated kinase kinase ; IRA 1 kinase suppressor ; numb-associated kinase ; mitotic kinase ; PEK (n = 3); polo-like kinase ; SCY1 (n = 2); tousled-like kinase ; Unc51-like kinase ; VPS15 (n = 1); WEE (n = 2); and Other-unique (n = 14). Specific information on each classified kinase can be found in Supplementary Table S1 online. Moreover, a phylogenetic tree of the L. infantum ePK kinome groups ; calcium/calmodulin protein kinase-like ; CAMK-Unique (e groups  was consn = 5); phosphatidyl-inositol3 kinase related kinases ; right open reading frame kinase ; ABC1 (n = 5); alpha (n = 4); and CAK (n = 2). We identified four atypical protein kinases, of which one was classified into the A6 family and three were classified into the pyruvate dehydrogenase kinase family (PDHK).Besides ePKs, 24 protein kinase like (PKL) proteins were identified, and were distributed into phosphatidyl-inositol kinase . There are 33 highly expressed kinases in L. infantum which rank at the upper quartile of expression. At the lower quartile of expression there are 84 kinases. The other 139 kinases are constitutionally expressed. Most of the highly expressed kinases are members of the CMGC group, probably due to the diverse and important functions of MAPK cascades and CDKs [We also assessed the transcription profile of L. riTrypDB . A histoand CDKs .3.1.2L. braziliensis contained 195 kinases, 31 of which could be classified on the level of subfamilies; 114 on the level of families; 22 on the level of groups; and 28 protein kinases remained unclassified. After manual curation (see Methods), 221 proteins (193 ePKs and 28 PKL/ aPKs) were defined as protein kinases and classified into groups (n = 3); families (n = 61); subfamilies (n = 155); and 2 protein kinases remained unclassified ; NDR (n = 1); PDK1 (n = 3); PKA (n = 2); and RSK (n = 1). Two AGC kinases were not assigned to any family. The CAMK group had 23 kinases classified in the following families: CAMK1 (n = 5); CAMKL (n = 11); CDPK (n = 2); and CAMK-Unique (n = 5). The CK1 group had 7 kinases classified into CK1 (n = 6) and TTBK (n = 1) families. There were 49 proteins assigned to the following families of CMGC group: CDK (n = 12); CK2 (n = 2); CDKL (n = 2); CLK (n = 4); DYRK (n = 9); GSK (n = 2); MAPK (n = 13); RCK (n = 3); and SRPK (n = 2). Among the STE group, there were 41 kinases classified in the following families: STE11 (n = 31); STE20 (n = 2); STE7 (n = 7); and STE-Unique (n = 1). The “Other” group contained 60 kinases within the following families: AUR (n = 3); Bud32 (n = 1); CAMKK (n = 4); IKS (n = 1); NAK (n = 2); NEK (n = 23); PEK (n = 3); PLK (n = 1); SCY1 (n = 2); TLK (n = 1); ULK (n = 3); VPS15 (n = 1); WEE (n = 1); and Other-Unique (n = 14). One “Other” kinase was not assigned to any family.A total of 193 ePKs were identified in the L. e groups . The AGCbraziliensis proteome. They were classified in the following families: ABC1 (n = 5); alpha (n = 5); PIK (n = 4); PIKK (n = 6); RIO (n = 2); and CAK (n = 2). There were also four atypical protein kinases: three PDHKs and one A6. Since the kinomes L. infantum and L. braziliensis are very similar, their phylogenetic trees are very similar as well and therefore we decided not to report the tree for L. braziliensis. Specific information of each classified kinase can be found at Supplementary Table S2.Along with the ePKs, there were 24 PKLs identified in L. 3.2Leishmania species and the human kinome in order to identify candidate drug targets among the parasites kinome . Likewise, we performed a pairwise comparison of Leishmania kinases and H. sapiens homologs [L. infantum v H. sapiens), and from 21 to 69% (L. braziliensis v H. sapiens). This demonstrates that Leishmania kinases can be targeted by compounds that are unlikely to bind human kinases . Combining these complementary approaches led to the prediction of 30 potential new drug targets (Supplementary Table S4).Two bioinformatics approaches were used for target prediction: (i)protein essentiality and (ii) centrality measures of the kinome protein-protein interaction graph. For the first approach (i), we used RNAi validations of targets with lethal phenotype in closely related E-value threshold of ≤10−30 was adopted to provide high confidence for the data. This analysis predicted 11 targets associated with 42 drugs (see Supplementary Table S5). These drugs can possibly also target Leishmania since the sequence conservation between the human and Leishmania targets is high. Then, we conducted a literature search of the 42 drugs, in order to check which of them have not been tested in Leishmania yet. We found that 15 of those drugs have already been tested in Leishmania, some of them had IC50 lower than 10 μM [Leishmania were submitted to an in-house developed QSAR model for prediction of phenotypic activity against L. infantum. Seven of them were predicted to be active against L. infantum amastigotes  in a drug repurposing pipeline. In this step, the an 10 μM , demonststigotes . These c3.4infantum, L. braziliensis and L. amazonensis promastigotes. Selumetinib, refametinib, MEK162, MLN-8054 inhibited ~ 70% promastigote growth at concentrations higher than 75 μM, showing poor leishmanicidal activity. On the other hand, R1530, NMS-1286937 and Trametinib presented good to moderate antileishmanial activity against Leishmania spp.  infantum parasite burden whilst Trametinib showed a more pronounced effect at the same dose (~90% reduction) (L. (V.) braziliensis, both compounds reduced ~50% of intracellular amastigotes at 15 and 30 μM (Cytotoxicity assays showed that RG-1530 was toxic to bone marrow derived macrophages (BMDM) from BALB/c mice while NMS-1286937 and Trametinib led to ~15% viability reduction at 50 μM . For this reason, the antileishmanial activity of NMS-1286937 and Trametinib were evaluated against intracellular amastigotes. NMS-1286937 at 30 μM reduced approximately 50% of duction) . For L. nd 30 μM .Fig. 6Pe4Leishmania species important to human health. The draft kinome revealed 197 and 195 protein kinases in L. infantum and L. braziliensis, respectively. These numbers represent 2.35% and 2.08% of these species' genomes. The curation procedure further improved that number to 224 and 221, representing 2.67% and 2.35% of their genomes. The number of protein kinases is compatible with related trypanosomatid kinomes, which comprise approximately 2% of their genomes [Plasmodium falciparum (1.5%) [Schistosoma mansoni (1.9%) [www.kinase.com), which does not contain trypanosomatid kinomes, have a similar number of kinases compared with their genomes ranging from 1.5 to 2% [Our bioinformatics pipeline, expanding on established methods for kinase identification, enabled us to complete the first complete definition and classification of the kinomes of two  genomes  and is am (1.5%)  and Schii (1.9%) . Organis.5 to 2% .The large set of PKs in the genus suggests that phosphorylation is a very important process in parasite biology. There are representatives of six of the nine ePK groups are described by Manning et al. . TyrosinL. major kinome, the kinomes of L. infantum and L. braziliensis constituted a larger number of proteins  [major kinome study [In comparison with the previously elucidated  kinome) . This dime study  could onWe aimed to select the proteins with no orthologues in humans, however, in the target prioritization analysis, half of the selected proteins were clustered with human orthologues. Looking at the sequence identity of these proteins, we observed that all of them shared <60% of their identities. It has been shown that kinases with sequence similarities higher than 60% have very similar structures and the compounds that bind to their sites usually show similar structure-activity-relationships . Thus, iT. brucei, e.g., Aurora kinase 1(AIRK) [Leishmania species. L. major and L. donovani AIRK have been cloned and characterized [L. donovani cell cycle [T. brucei validation study [. CK1 has been identified as an hexokinase [L. major CK1 has been targeted by human casein kinase inhibitors [L. donovani [. The cyclin-dependent kinases 2 – CRK1 and CRK3 were experimentally validated in L. mexicana as essential proteins [in vitro activity [major and L. infantum. The crystal structure of LmajGSK3 was elucidated and eleven protein kinase inhibitors were tested against LinfGSK3, TbruGSK3, and HsapGSK3 enabling a structure-activity relationship comparison between the binding sites, which provides ways to predict inhibitors with binding modes that might be effective and selective [T. brucei MKK1 and MKK5 knockout mutants were not essential for the parasite's survival or virulence [L. mexicana, MKK1 mutants have a shortened flagellum [Our target prioritization approaches enabled us to find promising targets. Eight out of the 33 highly expressed kinases were prioritized as targets, meaning that our approaches were effective in selecting important targets. Many of those targets have been genetically validated in  1(AIRK) ; Casein  1(AIRK) ; Cyclin- 1(AIRK) ; Glycoge 1(AIRK) ; WEE-1 l 1(AIRK) ; and Pol 1(AIRK) . Many ofcterized ,52, theion study ,54. CK1 xokinase  capable xokinase . L. majohibitors  and the donovani . The cycproteins  with funproteins . Also, mactivity . Recombielective . Targetielective ; a studyelective . T. brucirulence . In L. mlagellum  and MKK5lagellum .Leishmania inhibitors. Exploration into the biological targets of parasites with orthologues in mammals has been avoided for decades, and it aims to eliminate possible problems of selectivity and adverse effects during the drug development process. However, the situation is radically different when this parasite protein is orthologous to a therapeutic drug target. In this case, orthologues can provide evidence of “druggability” [infantum, L. braziliensis and L. amazonensis promastigotes. Trametinib was the most potent compound against L. infantum and L. braziliensis amastigotes. This drug reduced ~50% of intracellular amastigotes at 30 μM for L. infantum and at 15 μM for L. braziliensis. Trametinib is a drug used for the treatment of anaplastic thyroid cancer [(MEK1 or MAPKK1), MEK2 activation, and MEK1 and MEK2 kinase activity. MEK proteins are upstream regulators of the extracellular signal-related kinase (ERK) pathway, which promotes cellular proliferation [Leishmania are also MAPKK1 and MAPKK2. Both share 34% similarity with human MAPKK1. This reinforces the possibility of repurposing trametinib to a different application with similar mechanism of action. NMS-1286937 is an inhibitor of Polo-like kinase 1 (PLK-1), a key component of the cell cycle control machinery with important roles in the mitotic entry, centrosome duplication, bipolar mitotic spindle formation, transition from metaphase to anaphase, cytokinesis, and maintenance of genomic stability [Leishmania, with 48% of identity. This compound has good oral bioavailability in rodent and non-rodent species and has proven antitumor activity in different preclinical models against solid tumors and hematologic malignancies [50 of these compounds were not considered optimal against the Leishmania species tested (EC50 < 10 μM) [in vivo efficacy of kinase inhibitors in experimental leishmaniasis model.Our drug repurposing approach led to the selection of 7 compounds with potential as ability” . Three od cancer . Its tarferation . The pretability . PLK-1 ignancies . R1530 tgnancies . R1530 ignancies . Althoug< 10 μM) , here weinfantum and L. braziliensis kinomes – most of the kinases were assigned at subfamily level. The functions of these kinases were catalogued and compared to human and other Leishmania orthologues. Finally, we predicted 30 protein kinases that have the potential to be good drug targets. This information will be useful for the discovery of new leishmanicidal compounds. The drug repurposing pipeline allowed us to find a kinase inhibitor currently indicated for cancer treatments that has the potential to be repositioned for the treatment of leishmaniasis. The drug trametinib and the compounds NMS-1286937 and RG-1530 presented good to moderate inhibition of L. infantum and L. braziliensis amastigotes and might be used as scaffolds for future hit-to‑lead optimization.In conclusion, the bioinformatics pipeline used in this work allowed a very thorough classification of L. Supplementary Table S1: Leishmania infantum kinases classification following bioinformatics pipelineSupplementary Table 1Supplementary Table S2: Leishmania braziliensis kinases classification following bioinformatics pipelineSupplementary Table 2Supplementary Table S3: Ortholologous kinases between L. infantum, L. braziliensis, L. major; and blast comparisons with human kinases.Supplementary 3Supplementary materialImage 1The following are the supplementary data related to this article.Each author has contributed significantly to this work. JVBB, PIPR, ENM, NF, and CHA conceived and designed the experiments. JVBB, ACS, and PIPR performed the bioinformatics analyses and computational experiments. NG and DCM conducted the experimental assays. JVBB, ACS, PIPR, NG, DCM, ENM and CHA analyzed the data. JVBB, PIPR, ENM, and CHA wrote the paper. All authors read, edited, and approved the final manuscript.None.