The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation. Vibrio outer protein S) and IbpA (immunoglobulin-binding protein A)—from Vibrio parahaemolyticus and Histophilus somni, respectively—revealed that the AMPylation of small Rho GTPases promotes bacterial pathogenesis .Recombinant Hisescribed . Specifi6-tagged WT α-syn was expressed and purified similarly as described above with the following modifications. WT α-syn was cloned into a pT7-7 plasmid and expressed in E. coli BL21-DE3-RILP (Stratagene) in LB medium containing 100 μg/mL of ampicillin to an optical density of A600 = 0.6. Protein expression was induced with 0.4 mM IPTG for 3–4 h at 30 °C. Lysis was performed on frozen pellets dissolved in lysis buffer. Lysed cells were centrifuged at 15,000× g for 50 min. Supernatants were poured over cobalt resin. Resin was washed with wash buffer. Tagged proteins were eluted with elution buffer. Eluted protein was further purified by size-exclusion chromatography in a buffer containing 100 mM Tris and 100 mM NaCl, pH 7.5. Fractions containing WT α-syn were verified for purity by SDS-PAGE and pooled together. Protein concentrations were measured spectrophotometrically at A280. Proteins were flash-frozen and stored at −80 °C in storage buffer.HisGST-tagged WT IbpA-Fic2 and Q61L Cdc42 were bacterially expressed and purified as previously described .2, pH 7.5) at 37 °C in the dark for the specified times. Reactions were stopped with 4X SDS loading dye, boiled for 5 min at 95 °C, and run on 12% SDS-PAGE gels. Gels were imaged on a Typhoon 9500 FLA imager  for fluorescence at 473 nm. Imaged gels were then stained with Coomassie Brilliant Blue, destained, and imaged for protein concentration. The Fl-ATP concentration experiment was performed as previously described, but with specified concentrations of Fl-ATP, and at a 10 min timepoint.For the E234G HYPE time-course experiment, 0.2 μM of Δ102 HYPE enzyme was incubated with 25 nM Fl-ATP in minimal AMPylation buffer  AMPylation buffer in the dark at room temperature for 10 min in 384-well black-bottom, black-walled microplates (20 μL reaction volume). Where specified, buffers were supplemented with 1% DMSO. Microplates were snap-centrifuged at 1000× g for 10 s. Microplates were loaded onto a BioTek BioStack NEO2  plate-reader and assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Where specified, enzymes were incubated with a 10× molar excess of substrate.Buffer comparison experiments were performed by incubating 0.2 μM of Δ102 HYPE enzyme with 25 nM Fl-ATP  in minimal or complete (50 mM HEPES (pH 7.4), 150 mM KCl, 1 mM CaClg for 10 s, then transferred to a BioTek BioStack NEO2 plate-reader. Reactions were assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Z’ and S/B  values were determined by fitting the data to Equations (1) and (2), respectively,pµ and nµ are the means of the positive (E234G HYPE) and negative (WT HYPE) controls, respectively; and pσ and nσ are the standard deviations of the positive and negative controls, respectively.A Multidrop 384 reagent dispenser  was used to add 0.2 or 0.4 μM Δ102 HYPE, then 25 nM Fl-ATP  to 384-well black/black microplates (20 μL reaction volume). Both enzyme and nucleotide were dissolved in minimal AMPylation buffer. AMPylation reactions were incubated for 10 min at room temperature in the dark. Microplates were snap-centrifuged at 1000× 1280 (Sigma Aldrich), The Spectrum Collection (Microsource Discovery Systems), MEGx , and NATx . The compounds went into columns 3–22 of each plate, while columns 1, 2, 23, and 24 were reserved for an equivalent volume of 100% DMSO controls.An Echo Liquid Handler  was used to transfer 200 nL of DMSO-dissolved compounds via acoustic-coupled ejection from 10 mM source plates into 384-well black, flat-bottom, black-walled microplates, for a final compound concentration of 10 μM. Compounds were sourced from the following libraries: LOPACA Multidrop 384 reagent dispenser was used to pipette 0.4 μM of Δ102 WT HYPE  dissolved in minimal AMPylation buffer into columns 1–22 of the activator plates, and columns 1 and 2 of the inhibitor plates as positive controls. Then, 0.4 μM of Δ102 E234G HYPE was similarly added to columns 3–24 of the inhibitor plates, and columns 23 and 24 of the inhibitor plates as positive controls. Enzymes were then incubated with either compounds or DMSO for 10 min at room temperature.g for 10 s. Microplates were loaded onto a BioTek BioStack stacker and read in succession for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Fluorescence polarization values were converted to fractional activation (FA) or fractional inhibition (FI) values according to Equations (3) or (4), respectively:pµ and nµ are the means of the positive (E234G HYPE) and negative (WT HYPE) controls, respectively; and x is the measured value of fluorescence polarization.A Multidrop 384 reagent dispenser was used to pipette 25 nM of Fl-ATP  into all wells of each plate, giving a final reaction volume of 20 μL. Plates were then incubated for 10 min at room temperature in the dark, followed by a snap-centrifugation at 1000× Microplate “cherry-picking” validations were performed as described above.g for 10 s, then transferred to the BioTek BioStack NEO2 plate-reader. Reactions were assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. EC50 and IC50 values were determined by fitting polarization values to Equations (5) and (6), respectively,maxY is the maximum polarization signal; I is the μM concentration of activator or inhibitor; n is the Hill slope; EC50 or IC50 are the inflection point concentrations; and minY is the baseline polarization response.A Multidrop 384 reagent dispenser  was used to pipette 0.4 μM of Δ102 WT or E234G HYPE  dissolved in minimal AMPylation buffer with 0.1% Triton X-100  into designated microplate wells. In addition, 10 μM of DMSO-dissolved compounds  or an equivalent volume of 100% DMSO was manually pipetted into their specified wells. Enzymes were incubated with compounds or DMSO for 10 min at room temperature. The reagent dispenser was then used to pipette 25 nM of Fl-ATP  into each well, followed by a 10 min incubation at room temperature in the dark. Plates were snap-centrifuged at 1000× 2, pH 7.5) with or without 0.1% Triton X-100  as specified. All reactions were quenched with 4X SDS loading dye and boiled for 5 min at 95 °C, and the samples were run on 12% SDS-PAGE gels. Gels were imaged on a Typhoon 9500 FLA imager  for fluorescence at 473 nm. Imaged gels were then stained with Coomassie Brilliant Blue, destained, and imaged for protein concentration.Here, 1 μM of Δ102 or Δ45 WT HYPE  were preincubated with 10 μM of DMSO-dissolved compound  or an equivalent volume of 100% DMSO at room temperature for 10 min. Reactions were run for 1 h at 37 °C in the dark, and began with the addition of 1 μM Fl-ATP . Alternatively, 0.4 μM of Δ45 or Δ102 E234G HYPE were preincubated with 10 μM of DMSO-dissolved compound  or an equivalent volume of 100% DMSO at room temperature for 10 min. Reactions were run for 1 h at 37 °C in the dark, and began with the addition of 25 nM Fl-ATP . All reactions were performed in minimal AMPylation buffer  web server. Following program requirements, a protein structure and ZINC  database accession number were provided [Molecular docking to predict protein-compound co-structure and ∆G was done with the SwissDock (provided . Apo WT provided .A1) and aurintricarboxylic acid (I2). Though their activities against multiple known human targets restrict usage for therapeutic intervention, these compounds nevertheless remain promising candidates for hit-to-lead optimizations. Moreover, the use of these hits as research tools will aid in the mechanistic elucidation of HYPE’s cellular role in the UPR and other disease-associated pathways. Ongoing follow-up studies on remaining HTS hits not initially counterscreened are sure to provide additional activators and inhibitors of HYPE-mediated AMPylation. The high-quality assay described herein is also fully scalable for HTS processing and lends itself to the screening of larger, more diverse chemical libraries with more drug-like potential.Despite residing at the regulatory intersection of several disease pathways, HYPE has yet to be targeted for an HTS campaign. Here, we present a novel, robust, dual screening assay capable of monitoring changes in HYPE autoAMPylation in vitro. Further, this assay has been optimized for high-throughput scale and demonstrates reproducibility in identifying target hits. In conjunction with our post-HTS counterscreens, we have established a reliable pipeline for hit validation. Indeed, we have discovered at least two promising modulators of HYPE’s adenylyltransferase activity—calmidazolium (


Carthamus tinctorius L. in the Apis mellifera colonies of northwestern China. Scant information is available regarding its chemical composition and biological activities. Here, for the first time, we investigated this honey’s chemical composition and evaluated its in vitro antioxidant and anti-inflammatory activities. Basic physicochemical parameters of the safflower honey samples in comparison to established quality standards suggested that safflower honeys presented a good level of quality. The in vitro antioxidant tests showed that extract from Carthamus tinctorius L. honey (ECH) effectively scavenged DPPH and ABTS+ free radicals. In lipopolysaccharides (LPS) activated murine macrophages inflammatory model, ECH treatment to the cells inhibited the release of nitric oxide and down-regulated the expressions of inflammatory-relating genes . The expressions of the antioxidant genes TXNRD, HO-1, and NQO-1, were significantly boosted in a concentration-dependent manner. ECH decreased the phosphorylation of IκBα and inhibited the nuclear entry of the NF-κB-p65 protein, in LPS-stimulated Raw 264.7 cells, accompany with the increased expressions of Nrf-2 and HO-1, suggesting that ECH achieved the anti-inflammatory effects by inhibiting NF-κB signal transduction and boosting the antioxidant system via activating Nrf-2/HO-1 signaling. These results, taken together, indicated that safflower honey has great potential into developing as a high-quality agriproduct.Safflower honey is a unique type of monofloral honey collected from the nectar of  Carthamus tinctorius L.) honey is brewed by honeybees collecting safflower nectar, which is a local speciality honey type of Xinjiang Providence, China. This honey is mainly produced in the Balruk Mountains of Xinjiang (82°12′–83°30′ longitude in the East and 45°24′–46°3′ latitude in the north), and contains a variety of natural sweeteners with bioactive components [Honey used widely as a food and medicine . It is omponents ,12,13, wmponents . NeverthRecent decades, several studies reported on the anti-inflammatory and antioxidant activities of honey, which are mainly attribute to its abundant phenolic and flavonoid contents ,16. HoneCarthamus tinctorius L. safflower honey were collected to carry out a screening of physical and chemical indicators and the extract from Carthamus tinctorius L. safflower honey (ECH) were obtained for preliminary characterizations on major main phenolic acids and flavonoid components based on the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS). Furthermore, we tested the in vitro free radical scavenging activities by ECH and its anti-inflammatory and antioxidant potentials were evaluated in in LPS-activated Raw 264.7 murine macrophages.In the current study, β-actin were purchased from Cambridge Bio . The NanoDrop 2000 Ultra Micro Spectrophotometer was purchased from Thermo Fisher Scientific . Gallic acid, sodium nitrite, the Prime Script TM RT Master Mix kit and TB Green® Premix Ex Taq TM were purchased from Biotech Co. Ltd. . Protein loading buffer, 20% SDS, electroporation solution and Tris-HCl buffer were purchased from Solarbio . BCIP/NBT alkaline phosphatase developer and 40% Acr-Bis were purchased from Beyotime Biotechnology . The CCK-8 kit was purchased from Dojindo Laboratories . Amberlite XAD-2 resin was purchased from Sigma-Aldrich Trading Co. Ltd. . Other chemicals, including the LPS (Escherichia coli 0127: B8) and alkaline phosphatase-conjugated secondary antibody (anti-rabbit IgG) and the standards applied in the chemical analysis were purchased from Sigma .Primary antibodies including IκB-α, p-IκBα, and NF-κB-p65 were purchased from Cell Signaling Technology . Nrf-2, HO-1, and Apis mellifera L. colonies during the safflower (C. tinctorius L.) flower season in 2018. Each honey from the single hive was considered as one sample, 500 g each. The apiaries were located in the Yumin County, Tacheng City, China, which were belongs to bases of Jiangsu Rigao Bee Products Co., Ltd. . We collected the capped comb honeys in the day, and transfer them to the refrigerator at 4 °C, afterwards the honey samples were taken to the lab and storage at −20 °C until usage. The moisture, free acid, amylase, hydroxymethylfurfural, fructose, glucose, sucrose, and ash contents of the Xinjiang safflower honey samples were determined by the method specified in CODEX STAN 12-1981. Melissopalynology was applied to analyze the botanical origin of the pollens in the safflower honeys, following pervious published method [Safflower honey samples were obtained from three d method . The typC. tinctorius safflower honey (ECH) refers to the method of Mu et al. [w/v), and used an ultrasonic instrument for 30 min until it dissolved. The activated XAD-2 resin was weighed to 800 g, treated with ultrasonic to make it free of air, then mixed with Xinjiang safflower honey aqueous solution, mixed evenly for 1 h, and left standing overnight. We discarded the supernatant, added XAD-2 resin into the glass column, washed with 2 volumes of hydrochloric acid water (pH = 2) and 3 volumes of ultra-pure water, and then eluted with 8 volumes of ethanol to collect the eluate, evaporated the ethanolic extract to a solid residue with a rotary evaporator with vacuum, and then dissolved the residue in 15 mL of pure water. This aqueous solution was extracted with 20 mL of ethyl acetate. We collected the ethyl acetate layer, repeated this four times, for each extraction for 20 min. The collected ethyl acetate layer was blown dry with nitrogen to obtain the resulting ECH, and placed in a −20 °C refrigerator for storage. We dissolved the ECH with an appropriate amount of ethanol for the further usages.The preparation of the extract from u et al.  and was The HPLC-QTOF-MS analysis method of ECH was established by our laboratory . ChromatMass spectrometry conditions were conducted with an electrospray (ESI) ion source in negative ion mode The typical parameters of the mass spectrometer were as follows: drying gas temperature, 350 °C; drying gas flow rate, 6 L/min; sprayer pressure, 35 psi; capillary voltage 3500 V; atomizing gas temperature, 350 °C; and atomizing gas flow rate, 9 L/min. Qualitative and quantitative analysis were carried out by accuracy mass and extracted ion chromatography (EIC). Polyphenolic compounds ion chromatograms were extracted by Mass Hunter Qualitative Analysis software (Agilent Technologies) for ECH.For the determination of the DPPH· clearance rate, we referred to WU et al.’s method , and mad50.The removal ability of the sample is expressed by IC+ clearance rate, we referred to YANG et al. [For the determination of the ABTSG et al.  and made50.The removal ability of the sample is expressed by IC2 incubator [Raw 264.7 cells were a gift from Professor Hu Fuliang, college of Animal Science, Zhejiang University. Raw 264.7 cells were cultured with DMEM high glucose medium containing 10% heat-inactivated fetal bovine serum and double antibodies (streptomycin (100 µg/mL) and penicillin (100 IU/mL)) at 37 °C Incubation was in a 5% CO5 cells/mL, 100 µL per well, incubated overnight in a 5% CO2 incubator at 37 °C, until the cell adherence reached 70–80%. The cells were incubated with 2.5 µg/mL, 5 µg/mL, 10 µg/mL, 15 µg/mL, or 20 µg/mL ECH for 24 h, and the culture medium was aspirated. The cells were washed with new cell culture medium for two to three times, and a blank control group was set. We added 10 µL CCK-8 reagent and cell culture medium into each well, incubated for 3 h, and then the absorbance at 450 nm was determined [Raw 264.7 cells in the logarithmic growth phase were inoculated into 96-well plates with 1 × 10termined ,39.5 cells/mL, 500 µL per well, incubated in a 37 °C, 5% CO2 incubator, until the cell adherence reached 70–80%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we successively added 2.5 µg/mL and 5 µg/mL ECH for 1 h, followed by LPS (1 µg/mL) for 24 h. The cell culture fluid was collected, centrifuged at 5000 r/min for 10 min, and the supernatant was collected. We mixed 100 µL of each sample with an equal volume of Griess reagent and added these samples to a 96-well plate, and incubated at room temperature for 10 min. We measured the absorbance at 540 nm.The NO concentration was measured with a Griess reagent. We referred to the method of Lee et al. and adjusted . Raw 2645 cells/mL, 500 µL per well, incubated in a 37 °C, 5% CO2 incubator, until the cell adherence reached 70–80%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we successively added 2.5 µg/mL and 5 µg/mL ECH and incubated for 1 h, and then induced with LPS (1 µg/mL) for 6 h. The cells were collected, and then the total cell RNA was extracted using a CarryHelix RNA extraction kit, and its concentration and purity were determined with a NanoDrop 2000 ultramicro spectrophotometer. Using 1 µg of extracted total RNA as a template, the PrimeScript® TM RT Master Mix reverse transcription kit was used to perform reverse transcription of the cDNA, and the product was placed in a −20 °C refrigerator for use. Real-time quantitative PCR was performed with a TB Green® Premix Ex TaqTM kit. The total volume of the reaction system was 10 µL: TB Green® Premix Ex TaqTM 5.0 µL, cDNA template 0.2 µL, RNase Free dH2O 4.4 µL, upstream and downstream primers 0.2 µL each, related to use. The primer sequences are shown in Raw 264.7 cells in the logarithmic growth phase were inoculated into 24-well plates with 1 × 105 cells/mL, 1 mL per well, and incubated in a 37 °C, 5% CO2 incubator, so that the cell adherence reached 90%, and we set the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group we successively added 2.5 µg/mL and 5 µg/mL ECH and incubated for 1 h, and then induced with LPS (1 µg/mL) for 0.5 h. The collected cells were washed twice with PBS, and inhibited with NP-40 protein lysate Cellular protein was extracted from the reagent, and the concentration of the extracted protein was measured by BCA. The protein samples of each group of cells were detected by immunoblotting. The total sample loading was 20 µg of total protein, with β-actin as a reference, through 12% SDS-PAGE gel electrophoresis, transfer, hybridization, alkaline phosphatase color development and other steps to obtain the hybridization bands of IκBα, P-IκBα, Nrf-2 and HO-1 [Raw 264.7 cells in the logarithmic growth phase were inoculated in 6-well plates with 1 × 10and HO-1 .5 cells/mL, incubated in a 37 °C, 5% CO2 incubator overnight, and we set up the DXMS positive control group, blank control group, LPS treatment control group, and experimental group. To the experimental group, we added 5 µg/mL of ECH and incubated for 1 h. Then, we induced with LPS (1 µg/mL) for 0.5 h. A methanol-acetone mixture  was used as the fixing solution for 30 min. Permeabilization was performed with 0.5% Triton X-100 for 30 min, blocked with 10% serum blocking solution for 30 min at room temperature. We added primary antibody (NF-κB-p65) (1:50 dilution) and secondary antibody goat anti-rabbit (IgG) (1:500 dilution) and incubated for 1 h, with DAPI  (1:2000 dilution) stained nuclear processing coverslips, observed with a laser confocal scanning microscope, and analyzed the results [Raw 264.7 cells in the logarithmic growth phase were inoculated into the slide confocal small dish with 1 × 10 results .t-test, and a p value of <0.05 was considered as statistically significant.All experimental data were obtained from at least three repeated experiments. Data are presented as the Mean ± SD, and the statistical significance between two groups was determined using Student’s All data regarding the physical and chemical indicators of Xinjiang safflower honey see , using tHPLC-QTOF-MS detection of ECH was carried out with 24 phenolic flavonoid standards. As shown in + free radical scavenging experiments are commonly used to evaluate natural antioxidants [+ free radical was 81.88 ± 0.54 µg/mL.DPPH and ABTSoxidants . It can By treating Raw 264.7 cells with ECH in concentrations from 2.5 to 20 µg/mL and using the CCK-8 reagent to detect cell viability, we found the most suitable ECH concentration to ensure that adding ECH concentration to Raw 264.7 cells produced no obvious toxic effects see . CompareA typical Griess method was used to determine the amount of NO released by different treatment cells see . DexametNext, RT-qPCR was applied to detect the expression levels of inflammation and oxidation-related genes in LPS-induced Raw 264.7 cells see . After LThe expression of inflammatory and anti-oxidation-related proteins in LPS-induced Raw 264.7 cells was detected by western blot . The expA laser confocal scanning microscope was used explore the effect of 5 µg/mL ECH on the nuclear localization of NF-κB-p65 in LPS-induced inflammatory cells see . CompareUsing the methods recommended in the European Community Directives, we found that all the physical and chemical indexes results of Xinjiang safflower honey samples were in line with the requirements. Hydroxymethylfurfural is a substance produced by honey during storage or heating. It is an important criterion for evaluating the quality of honey ,44. As tAs polyphenols endowed honey with distinct color, taste, flavor and biological activities. Polyphenol analysis is therefore considered as an important tool for determining the quality of honey. A total number of 14 polyphenolic compounds were preliminary characterized using the HPLC-Q-TOF/MS analysis, and the sum of each component content was 14.83 mg/kg honey. Solid phase extraction (SPE) using the Amberlite XAD-2 resin is one of the most widely used extraction methods for isolating individual phenolic and flavonoid compounds in honey . In the + free radical scavenging capacity experiments used the IC50  as the criterion for evaluating the antioxidant activity. The literature showed that due to the rich polyphenol content, honey has a very significant antioxidant effect. Five types of honey in Croatia showed high DPPH· free radical scavenging activity , In eig52 mg/mL  and seveIn this experiment, ECH can significantly reduce the mRNA levels of the related inflammatory factors iNOS, IL-1β, TNF-α and MCP-1, while up-regulating the expression of the antioxidant genes HO-1, TXNRD and NQO. Due to the expression of iNOS when suppressed, the amount of NO released is reduced accordingly. Through the detection of IκBα phosphorylation and the NF-κB signaling pathway, we found that ECH was likely to inhibit the phosphorylation of IκBα to produce P-IκBα, thereby inhibiting the activation of the NF-κB signaling pathway and reducing inflammation. The Nrf-2/HO-1 signaling pathway demonstrated a protective effect against oxidative stress. Nrf-2 can regulate the gene expression of HO-1 at the transcription level, while HO-1 can inhibit the production of inflammatory factors in LPS-stimulated Raw 264.7 cells and protect the cells under oxidative stress . We meas3-H15 bond of gallic acid is easy to break to scavenge free radicals, providing antioxidant effects [ECH is rich in phenolic acids and flavonoids and also has good anti-oxidation and anti-inflammatory effects. Thus, we speculated that the existence of these compounds provides a material basis for the antioxidant and anti-inflammatory activities of safflower honey extract. Vanillic acid can play an anti-inflammatory and analgesic role through anti-oxidation and inhibiting the production of pro-inflammatory cytokines associated with NF-κB . Protoca effects . Gallic  effects . Caffeic effects . Ferulic effects . We foun effects . Myricet effects . Rutin [ effects  and nari effects  alleviat effects . Kaempfe effects . Based oIn the present study, we must explain the limitations of our experiment. First, as the identified polyphenols might had multiple isoforms and exist in the honey in a very complex form, their quantifications in our honey samples might have some bias. Second, phenolic compounds recovery from XAD-2 resin could to be estimated which can partly counteract the strong matrix effects in the honey. Third, in this study we only preliminary detected several polyphenolic compounds in our safflower honey samples, we still did not propose a “marker” compound in our honey samples, which is with great importance for the standardization and quality control on this special honey type. Nevertheless, our research group are keeping working on the isolation and identification on the marker compound of the safflower honey, which is helpful to discriminate from other commercial honey types. In such work accurate recovery rate will be examined on the single maker compound.Carthamus tinctorius L. honey (ECH). ECH showed a strong ability to scavenge DPPH·and ABTS+·free radicals in vitro. ECH also inhibited the phosphorylation of IκBα to activate the NF-κB signaling pathway, promoted the expression of antioxidant genes, thereby inhibiting inflammatory process induced by LPS. Our research provides basic information into development on this special honey in the future.In summary, we found that the main physical and chemical indicators of safflower monofloral honey samples, a local specialty honey type of Xinjiang Providence, China were in-line with the requirements of EU or Codex standards. We also preliminary characterized 14 polyphenolic compounds in the extracts from


Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured  Female reproductive senescence results from the depletion of a finite ovarian follicle pool that is assembled during fetal life. These follicles are produced from primordial germ cells which transition to oocytes as they undergo loss of pluripotency, proliferate with incomplete cytokinesis forming germ cell nests, and enter into meiosis. The initiation of meiosis is one of the defining features of germ cell differentiation and occurs in fetal life in female mammals. Although meiosis is comprised of two rounds of cell division, only prophase of meiosis I occurs during fetal oogenesis, with arrest occurring before completion of the first division.Stra8 (stimulated by retinoic acid gene 8), which is required for pre-meiotic DNA replication  family of compounds and can inhibit the enzymatic activity of at least one of the aldehyde dehydrogenases, thus preventing the conversion of retinol to retinoic acid . Howeverad libitum according to the UK Home Office and local University of Edinburgh ethical standards. To obtain fetuses for ovary culture experiments, mouse breeding harems were set up and females checked for the presences of a copulation plug; this was designated as embryonic day 0.5 (e0.5).Experiments involving mice were approved by the University of Edinburgh Animal Research Ethics Committee and performed according to the UK Animal (Scientific Procedures) Act 1986. WT CD-1 mice were maintained and bred in an environmentally controlled room on a 12 h light:12 h darkness photoperiod from 0700 h each day and fed 2. For the first 3 days of culture (days 0–3), culture media contained Dulbecco’s Minimal Essential medium  supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 μM β-mercaptoethanol and 1% sodium pyruvate. For subsequent days of culture, media were replaced with a simple culture medium consisting of α-MEM (Fisher Scientific) supplemented solely with 3 mg/mL BSA. Medium was replaced with fresh medium every 72 h. To inhibit retinoic acid production, fetal ovaries were cultured with doses of WIN 18,446  ranging from 0.01 µM to 5 µM or equivalent concentration of DMSO solvent for the first 3 days of culture; medium was replaced on day 3 of culture thus removing the drug. For rescue experiments, 0.7 µM of retinoic acid (Merck) was added to culture media in combination with WIN 18,446.Pregnant timed-mated females were obtained at e13.5 and culled by cervical dislocation. Fetal ovaries with attached mesonephros were dissected from female embryos; the day of dissection was designated as day 0 of culture. Ovaries with attached mesonephros were cultured for either 2, 3 or 12 days on a 2% agar block in a 35 mm petri dish, incubated at 37°C, 5% CO2. To investigate an independent effect on follicle activation, ovaries were cultured with 1 µM of WIN 18,446 for 3 days. Subsequently, ovaries were transferred to drug-free medium, which was replaced every 48 h. Ovaries were cultured for a total of 6 days.Ovaries were dissected from postnatal day 0 (P0) mice and cultured on floating polycarbonate membranes  in 24-well plates containing α-MEM (Fisher Scientific) supplemented with 3 mg/mL BSA, incubated at 37°C, 5% CO Stra8 F GTTTCCTGCGTGTTCCACAAG, Stra8 R CACCCGAGGCTCAAGCTTC, Rarβ F TGTTTACCTGTTCACAAGCCA, Rarβ R GGAGGAGACCGGAACAAGTT, Dazl F tggaccgaagcatacagaca, Dazl R actgcccgacttcttctgaa, Sycp3 F cagagccagagaatgaaagca, Sycp3 R atttgccatctcttgctgct, and see Mapk1 (Mitogen-activated protein kinase 1) and Canx  sequences. Each cDNA sample was analysed in triplicate. Target genes were normalised to the geometric mean expression of Canx and Mapk1 , homogenised using a motorised pellet pestle, and RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturer’s instructions. RNA was reverse transcribed to cDNA using concentrated random primers and Superscript III reverse transcriptase (Fisher Scientific) according to manufacturer’s instructions, and the cDNA synthesis reaction was diluted appropriately before proceeding. Primers for quantitative RT-PCR (RT-qPCR) were designed to amplify all transcript variants and are exon-spanning. Primer pair efficiencies were calculated with the LinReg PCR applet (Single immunostaining for Tra98 (germ cell specific antigen) was used to identify germ cells for counting after 3 days of culture with WIN 18,446. Double immunostaining for Msy2 and Tra98 was used to assess oocyte maturation on day 12 of culture . Cultured fetal ovaries were fixed for 2 h in Bouins solution. After processing, tissue was sectioned at 5 µm, dewaxed, rehydrated and antigen retrieval carried out in 0.01 M citrate buffer pH 6. Endogenous peroxidase activity was blocked with Dako REAL Peroxidase-Blocking solution (Agilent). Tissues were blocked in PBS containing 5% BSA and 20% normal goat serum and then incubated overnight at 4°C with primary antibodies diluted in blocking serum , Msy2: 1 in 400 ). Single immunostaining for Tra98 was carried out using ImmPRESS reagent  according to manufacturer’s instructions, and slides were counterstained with hematoxylin before mounting. For double immunofluorescence, tissues were incubated with a peroxidase-conjugated antibody for 30 min at room temperature, before visualisation using Tyramide Signal Amplification (TSA) (PerkinElmer) according to manufacturer’s instruction. The first primary antibody (Msy2) was then removed by a second antigen retrieval performed by microwaving slides in boiling 0.01 M citrate buffer for 2.5 min. After cooling, slides were blocked as before and incubated overnight at 4°C with anti-rat Tra98 (1 in 400). Secondary antibody and visualisation steps were carried out as mentioned previously, before counterstaining with DAPI and mounting. Images were captured using an Axioscan slide scanner (Carl Zeiss) and a 710 LSM confocal microscope (Carl Zeiss) with Zen 2009 software.n, five ovaries were pooled to ensure enough oocytes for quantification and oocyte chromosome spreads were prepared as described previously (Ovaries were taken at day 2 of culture (that is 48 h post culture setup), and for each Oocytes were staged, blinded to treatment, as outlined in Fetal (e13.5) ovaries were fixed for 2 h in Bouins solution on days 3 and 12 of culture, and newborn (p0) ovaries were fixed for 2 h in Bouins solution on day 6 of culture, paraffin wax-embedded, sectioned and stained for Tra98 (germ cell marker) or with hematoxylin and eosin. Assessment of germ cell number was carried out on 5-µM thick tissue sections and every sixth section, spanning the entire volume of the ovary , with the Abercrombie correction factor being applied to raw counts to estimate total germ cell number per ovary . Assessms.e.m. and were analysed using GraphPad Prism 7 software (GraphPad Software Inc.). Mann–Whitney U-test and Kruskal-Wallis test statistics were carried out as appropriate. A P value of <0.05 was considered statistically significant.All data are shown as mean ±Stra8 and Rarb on day 2 of culture (Stra8 (P = 0.008), and a clear dose response was observed with the 5 µM dose, causing approximately 97% reduction in Stra8 expression . Furthermore, the addition of retinoic acid increased Stra8 expression 0.5 fold above what was observed in vehicle-cultured ovaries (P = 0.014). Rarb expression was also reduced by WIN 18,446 with a similar dose response , to a similar level of expression in vehicle-cultured ovaries  (P <0.0001)   . Given t(per se)  and C. Q<0.0001) . Culture46 only)  and E. A46 only) . Taken tAs the Tra98/Msy2 quantification data was collected at the end of the culture period, we investigated whether this accelerated progression could be observed at an earlier point in culture. Oocyte chromosome spreads were therefore prepared from ovaries on day 2 of culture to capture oocytes in early meiotic prophase I. For sub-staging, oocytes were stained with antibodies against components of the synaptonemal complex: axial element protein Sycp3, and transverse filament protein Sycp1, as well as the recombination protein Rpa. A total of 258 oocytes from vehicle cultured-ovaries and 250 from ovaries cultured with 1 µM WIN 18,446 were analysed, blinded to the treatment group, using this dose only to maximise likely effects. Oocytes at three different stages were identified: leptotene, with short fragments of axial element but no synapsis; zygotene, with nuclei containing some regions of axial element undergoing synapsis, but this was not complete along the axial length; and pachytene, which had at least one pair of fully synapsed chromosomes. No diplotene oocytes were observed. Rpa recombination foci were present at all stages identified, but were less prevalent in pachytene oocytes .Figure P = 0.029) and a significant increase in the proportion of pachytene oocytes (P = 0.029). In WIN 18,446-cultured ovaries, on average 25.0% (±4.7) of oocytes were in leptotene, 21.9% (±2.6) in zygotene, and 53.2% (±3.5) in pachytene  of oocytes were in leptotene, 38.7% (±1.7) in zygotene, and 43.7% (±1.5) in pachytene . While tachytene . These dP = 0.032 and P = 0.040, respectively)    and D. O = 0.06) . As the  = 0.06) . Thus, tStra8 transcripts with treatment with WIN 18,446 , it wou et al. also observed increased folliculogenesis in Sycp1-deficient ovaries, with primordial follicles assembled earlier and in greater numbers than in control ovaries and more primary follicles indicating increased initiation of follicle growth  and the Biotechnology and Biological Sciences Research Council (BB/R015635/1 to R A A and R R).R R and R A A designed the experiments. R R, H L S and E W carried out experiments. R R wrote the manuscript. All authors contributed to data interpretation, editing the manuscript, and its final approval.


To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM  domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However,  In viewividuals –5. Next viduals –. Two cenviduals ––10. Targiduals ––. The seciduals ––, 12, 13.Two distinct protein domains are present in IDOL: an N-terminal FERM (band 4.1/ezrin/radixin/moesin) and a C-terminal RING domain , 14. In 18–IDOL/MYLIP locus and circulating LDL levels in humans   . To studFig. S1). We then analyzed by differential scanning fluorimetry (DSF), also known as ThermoFluor, whether the IDOL purification profile was influenced by a combination of different buffer and salt conditions. In most conditions, this resulted in a profile characterized by a double transition, suggesting heterogeneity , whereas only at low pH (pH 4–6) and intermediate NaCl concentration (500–750 mm), IDOL melting curves are characterized by a single transition .We noticed that size-exclusion chromatography (SEC) profiles of human IDOL were characterized by multiple peaks, suggesting different oligomerization/aggregation states . This revealed that IDOL runs as a dimer or larger oligomers, with peak widths that suggest polydispersity. Among the different conditions tested, its monodispersity is optimal in a buffer containing 20 mm MES, pH 6, and 500 mm NaCl. Because monodispersity can promote crystallization  to further analyze the effects of pH and NaCl concentration on IDOL homogeneity  (C).Using these optimized conditions for purification, we tested the ability of IDOL to promote polyubiquitin chain formation in alone , 17, ful(UBE2D3) C.D). In contrast to efficient IDOL-stimulated ubiquitination of the LDLR in mammalian cells (D). Additionally, despite extensive attempts, no significant binding interaction could be measured with a variety of LDLR-derived peptides in fluorescence polarization or surface plasmon resonance experiments (data not shown), further supporting the notion that additional interactions, conformational changes, or post-translational modifications may be required for efficient ubiquitination.We next evaluated the activity of full-length IDOL protein against its substrate, the LDLR, using a fluorescently labeled LDLR peptide containing the IDOL recognition and ubiquitination sites (LDLR residues 811–835). We observed IDOL-dependent ubiquitin modification of the LDLR peptide, and this activity was not present on a random lysine-containing peptide or with an alternative E3 ligase, RNF168 D. In conan cells , 14, 17,an cells D. AdditiA). The constructs were eluted as symmetric peaks from SEC columns in purification and showed well-defined melting transitions, indicating well-folded proteins . We obtained diffracting crystals for two different constructs: IDOL FERM domain, which spans residues 1–344 (FERM344), and the third lobe of the FERM domain, F3ab (residues 183–283).To investigate the mechanism underlying the IDOL-LDLR interaction, we therefore proceeded with structural studies. Unfortunately, neither the original purified full-length IDOL nor the protein purified under the optimized buffer conditions crystallized to allow structural determination of the full-length IDOL protein. Therefore, we purified a series of IDOL FERM domain truncations for crystallization A. The co12121, with cell dimensions a = 56.37 Å, b = 69.54 Å, c = 74.32 Å, α = β = γ = 90°, with three subunits in the asymmetric unit and a solvent content of 43%. We collected a single crystal data set, which diffracted to 2.35 Å resolution and solved F3ab crystal structure by molecular replacement using the equivalent region from DAL-1 (PDB ID: 2HE7) as a search model. Alternate model building in Coot  as search model. After several cycles of alternate model building in Coot and structure refinement in BUSTER and Refmac . Previous sequence analysis  divided into three subdomains to accentuate the extension away from the F3ab lobe: F3c′ (residues 279–300), F3c″ (residues 301–332), and F3c‴ (residues 333–344), with the latter not traceable in the electron density map (A).The F1, F2, and F3 regions of FERM domains A. Previoanalysis  had suggsity map A.344 subdomain F1 spans residues 1–80 and adopts a ubiquitin-like fold, consisting of a five-stranded β-sheet, a short 310 helix, and an α helix. Strands β1 and β5 are the first and the last β strand in the F1 subdomain, respectively, but form consecutive strands in the F1 β-sheet, resulting in an overall compact F1 domain. Subdomain F2 spans residues 81–185 and is all helical. It is composed of a four-helix bundle characteristic of the acyl-CoA–binding protein fold. The last subdomain in the traditional FERM cloverleaf is subdomain F3ab, encompassing residues 186–278. This domain resembles a phosphotyrosine-binding domain, characterized by the pleckstrin homology superfold, with seven antiparallel β-strands forming two orthogonal β-sheets capped by a C-terminal α helix . After the α7 helix, the FERM344 structure has a partially unstructured region from residue 301 until residue 317, followed by helix α8 (residues 318–332) (F3c″), which is the last residue traceable in the electron density map.The FERM344 and the three copies in the asymmetric unit of the F3ab crystal structure. There is a conformational difference in two β-turns (β6-β7 and β8-β9) in the FERM344 structure due to crystal contacts, which results in a loss of β-sheet character for strand “β9” in this structure . Moreover, there are clear differences in the F3c′ region, where the FERM344 crystal structure has an α helix (α7), following the F3ab, which spans residues 279–300 and folds back onto the F3ab. This region has lost helical conformation in all three copies of the F3ab structure, and in one case (F3ab monomer B) even the previous helix α6 is partially unraveled .The F3ab conformation is well-conserved between the FERM344 domain packs as a tetramer in the crystal created by crystallographic symmetry operations . Its extended F3c′-F3c″ region (residues 279–332) interacts extensively with F1 and F3 of a symmetry-related molecule, burying a substantial surface area of 2537 Å2 (B). This dimer has smaller crystallographic interfaces to another identical dimer , generating a tetramer, with a central four-helical bundle formed by helix α7 (F3c′ subdomain) .The FERM 2537 Å2 B. This dC, all the FERM variants presented in this study behave as monomers, indicating that the oligomerization is only relevant under crystallization conditions. The monomeric state of FERM344 was also confirmed by small-angle X-ray scattering (SAXS) analysis (see below) (D and E)).Such packing and especially the large dimer interface could indicate that oligomerization is important for IDOL function. Therefore, we investigated the solution behavior of different IDOL constructs, using SEC-MALS. As shown in Fig. 2Having obtained the IDOL FERM domain structure, we proceeded to compare it with other FERM domains in the PDB.A). Some FERM domains, such as radixin, have an inositol trisphosphate (IP3)-binding site between the F1 and F3ab subdomains. Analysis of the surface properties of the IDOL FERM domain shows that the general positive character of this surface is somewhat conserved (B), but not all coordinating residues are. There are three lysines  involved in the IP3 binding in radixin (PDB ID: 1GC6) (59 and Arg61) and one serine (Ser262), respectively, with a very different arrangement.First, we analyzed the surface charge properties of the IDOL FERM domain A. Some Fonserved B, but noD: 1GC6) , whereasA). Superposition of 27 structures shows that the characteristic cloverleaf structure is well-conserved in FERM344 and that a helix similar to the F3c′ region is also observed in human moesin (PDB ID: 1E5W) (1ISN) (6CDS) (1J19 and 2EMS) , and we could not identify an equivalent of the F3c″ subdomain in other FERM-containing proteins.Using SSM , we perfFig. 4D: 1E5W) , mouse m) (1ISN) , human m) (6CDS) , and mound 2EMS) , 42. SmaA) to assess the orientation of F3c′ and the extended conformation of F3c″ separately.We wondered whether this unusual conformation of F3c relative to the FERM domain is relevant in solution. We used SAXS to characterize a series of FERM domain variants see A to assegR) (D). However, only the FERM278 variant has its peak centered closed to √3, indicative of a particularly compact structure, whereas all the other constructs have their maxima shifted to higher values of q × gR (around 1.85 for FERM304 and FERM332 and around 2.25 for FERM344), suggesting properly folded but more elongated conformations  . Next, wRg)  to assesRg) D. Howevermations .P(r), a weighted histogram of all possible pair distances between pairs of atoms within a particle (E). By extrapolating the r values at which P(r) goes to zero, this analysis estimates the maximum dimension (Dmax) of the biomolecule in solution. FERM304, FERM332, and FERM344P(r) functions have a similar profile, with a noticeable tail, short for FERM304 and FERM332 and somewhat longer for FERM344. This suggests that the F3c′-F3c″-F3c‴ region extends away from the body of the FERM F1-F2-F3ab and is characterized by a certain degree of flexibility. Dmax values derived from the P(r) function for FERM278 (68 Å) and FERM304 (84 Å) are in very good agreement with their maximum dimensions calculated from the FERM344 crystal structure, suggesting that the F3c′ helix closely interacts with the F1-F2-F3ab core also in solution. The Dmax for FERM332 (84 Å) is not larger than that of FERM304, indicating that in solution, the F3c″ domain folds back onto the F1-F2-F3ab globular core instead of adopting the extended conformation seen in FERM344 crystal structure. The extended Dmax for FERM344 (110 Å) shows that the last 12 C-terminal residues are highly flexible. We compared the SAXS data with calculated scattering profiles, computed from our crystal structure of the FERM domain, including only the relevant residues of FERM278, FERM304, and FERM332. The calculated data are in good agreement with the corresponding experimental scattering data, with FERM304 having the worst fit   , 44–47, nd B) 44–, 28.344 structure, the F3c′ is helical and positioned over the F3ab domain, obscuring the access to the peptide binding site. If this particular position is retained in full-length IDOL, it could prevent the interaction with the LDLR intracellular tail, suggesting a possible form of autoinhibition. Because our SAXS data suggest that this conformation is maintained in solution, it is possible that this is indeed the case. This could potentially explain the low affinity and activity observed for full-length IDOL against the LDL receptor peptide in our in vitro binding and ubiquitination assays. Because the expression construct used to crystallize F3ab has an incomplete F3c′, helix α7 is unfolded and helix α6 is shorter in all of the three F3ab monomer structures. In fact, in two of the three copies in the asymmetric unit, this unraveled region contacts another F3ab molecule , where it takes on the role of the interacting peptide. The unraveled peptide binds in the F3ab peptide-binding site, making an interaction that closely resembles the β-β association observed in many FERM-substrate peptide complexes (C and D). This confirms that the F3ab subunit is intrinsically capable of forming the peptide interaction observed in other FERM receptor interactions. However, in the FERM344 structure, the α7 helix obscures access.In the FERMomplexes , C and D193, Lys199, and Arg259) were shown to be necessary, in vitro, for the recognition and interaction of IDOL with negatively charged phospholipid vesicles  is critical. Alternatively, post-translational modifications of IDOL may be required for optimal recognition of the LDLR, or some form of membrane association could trigger the necessary conformational change to unmask the peptide-binding site. Ultimately, it will be interesting to see whether IDOL autoinhibition resembles the autoinhibition observed in other FERM domain proteins or if it expands the mechanisms employed for this purpose.It will be interesting to see whether the orieniation of the F3c′ helix is relevant in full-length IDOL. If so, this could possibly be a novel form of FERM domain autoinhibition, explaining the low ubiquitination activity of IDOL on an LDLR peptide. It is possible that the conformation of the LDLR tail in a more physiological setting  was cloned into the pET-NKI-hisSUMO2 expression vector of the NKI LIC (ligation-independent cloning) suite (278), residue 304 (FERM304), residue 332 (FERM332), and residue 344 (FERM344). The E. coli codon–optimized gene of the human F3ab subdomain (residues 183–283) with an N-terminal TEV-cleavable His6 tag was purchased from GenScript and cloned into a pET28(a) vector using NcoI and XhoI restriction enzyme sites. The insect cell (full-length IDOL) and bacterial (FERM variants and F3ab subdomain) expression constructs used in this work are summarized in Fig. 1A.Full-length human IDOL (residues 1–445) was cloned with a TEV-cleavable N-terminal Hisg) suite . From thSpodoptera frugiperda Sf21 cells cultured in Sf900II SFM medium at 27 °C were infected with baculovirus (multiplicity of infection = 2) at a cell density of 2.5–3.0 × 106 cells ml−1 and harvested after 72 h. Cell pellets were resuspended in 50 mm Tris, pH 8.0, 500 mm NaCl, 1 mm TCEP, 15 mm imidazole, Benzonase, and 1 tablet of cOmplete™ protease inhibitor mixture (Sigma–Aldrich) per 50 ml of lysis buffer and lysed by sonication for 2.5 min on ice. The lysate was than centrifuged  and loaded on a 5-ml HisTrap HP column . The resin was washed with lysis buffer supplemented with buffered 25 mm imidazole, and the protein was eluted with lysis buffer supplemented with 500 mm imidazole at pH 8. The elution fractions were pooled; diluted 1:3 with 20 mm Tris, pH 8.0, 1 mm TCEP; immediately loaded on an anion-exchange column ; and eluted with a linear NaCl gradient. IDOL fractions were concentrated and loaded on a size-exclusion chromatography column  previously equilibrated with 20 mm Tris, pH 8, 200 mm NaCl, 1 mm TCEP. Peak fractions were concentrated to about 6 mg ml−1, aliquoted, and snap-frozen in liquid nitrogen for storage.For full-length IDOL, RE. coli cells. Cells were grown in TB medium, in the presence of kanamycin and chloramphenicol, at 37 °C while shaking until the A600 was 1.2–1.5. Cultures were then induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and allowed to grow overnight at 18 °C. Cells were harvested by centrifugation (5000 × g for 20 min at 4 °C) and resuspended in lysis buffer containing 50 mm Tris, pH 8, 500 mm NaCl, 1 mm TCEP, 25 mm imidazole, DNase, and one tablet of cOmplete™ protease inhibitor mixture (Sigma–Aldrich) per 50 ml of lysis buffer. The purification protocol for all the FERM variants was derived from Ref. m Tris, pH 8.0, 500 mm NaCl, 1 mm TCEP, 15 mm imidazole. The resin was then washed with lysis buffer supplemented with 25 mm imidazole (pH 8), and the protein was eluted with lysis buffer supplemented with 500 mm imidazole (pH 8). SUMO tag was removed by pooling the elution fractions and incubating with SENP2 protease for 45–60 min at room temperature while dialyzing against 20 mm Tris, pH 8.0, 150 mm NaCl, 1 mm TCEP, followed by a reverse affinity chromatography step. Once cleaved, FERM variant proteins were individually buffer-exchanged to 20 mm MES, pH 6.8, 150 mm NaCl, 1 mm TCEP and immediately loaded on a cation-exchange column  and eluted with a linear NaCl gradient. FERM fractions were then concentrated and loaded on a size-exclusion chromatography column  previously equilibrated with 20 mm MES, pH 6.8, 300 mm NaCl, 1 mm TCEP. Peak fractions were concentrated to about 6–10 mg ml−1, snap-frozen in liquid nitrogen, and aliquoted for storage.All FERM variant plasmids were transformed into Rosetta2 (DE3) T1E. coli cells. Cells were grown in lysogeny broth medium, in presence of kanamycin, at 37 °C while shaking until the A600 was 0.6–0.9. Cultures were induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside and allowed to grow overnight at 18 °C while shaking. Cells were harvested by centrifugation and resuspended in lysis buffer containing 50 mm Tris, pH 8, 300 mm NaCl, 10% glycerol, 1 mm TCEP, 25 mm imidazole supplemented with Benzonase and one tablet of cOmplete™ protease inhibitor mixture and lysed by cell disruption. The lysate was than centrifuged and loaded on a 5-ml HisTrap HP column . The column was washed with lysis buffer, and the protein was eluted with lysis buffer supplemented with 350 mm imidazole at pH 8. The eluted protein was then incubated with TEV protease overnight at 4 °C while dialyzing against 50 mm Tris, pH 8, 300 mm NaCl, 10% glycerol, 1 mm TCEP, 25 mm imidazole, followed by a reverse affinity chromatography step. Cleaved F3ab protein was concentrated and loaded on a size-exclusion chromatography column  previously equilibrated with 50 mm MES, pH 6.5, 500 mm NaCl, 1 mm TCEP, 10% (v/v) glycerol. Peak fractions were concentrated to about 6 mg ml−1, snap-frozen in liquid nitrogen, and aliquoted for storage. In all cases, after the size-exclusion chromatographic step, the purity and homogeneity of each protein was assessed by SDS-PAGE.FERM F3ab pET28(a) plasmid was transformed in BL21(DE3) Thermal unfolding profiles of full-length IDOL (25 μl at 1 mg/ml) were recorded, in the presence of different buffer and salt compositions using DSF in a MyiQ RT-PCR system (Bio-Rad) by monitoring the fluorescent intensity of SYPRO orange  with a temperature gradient of 0.5 °C/min between 15 and 95 °C and a dwell time of 10 s/step.m MES, pH 6.8, 300 mm NaCl, 1 mm TCEP to the desired concentrations. Measurements were taken over a temperature gradient of 1 °C/min between 20 and 95 °C, using an excitation power of 10%. The measured 350 nm/330 nm intensity ratios were plotted versus temperature. The mT values were determined from the maximum of the first derivatives of these spectra using Prometheus NT.48 internal software.Temperature-dependent conformational changes were measured using nano-DSF in a Prometheus NT.48 (NanoTemper Technologies). Each FERM variant was diluted in 20 mm protein solution was injected into a Superdex 200 10/300 column , previously equilibrated with 20 mm MES, pH 6.8, 300 mm NaCl, and 1 mm TCEP. Molecular weights of main peaks were determined using the manufacturer's software (ASTRA) and assuming a specific refractive index increment (dn/dc) of 0.185 ml g−1. Chromatographic profiles and molecular weights were plotted using Prism 7 (GraphPad).SEC-MALS measurements were performed on an ÄKTApurifier 100  connected to a tri-angle detector MiniDAWN Tristar (Wyatt Technologies). For each experiment, a volume of 100 μl of 100 μSDS gels used were all precast 4–12, 10, or 12% BisTris gels (Thermo Fisher Scientific) run in MES or MOPS buffer (Thermo Fisher Scientific) and blotted on polyvinylidene difluoride membrane  with standard blotting buffer. Blocking of the Western blots was done in 5% skim milk (Merck Millipore) in PBS-Tween 20. Antibodies used for Western blotting analysis were as follows: for ubiquitin, P4D1 , MYLIP/IDOL . The secondary antibody used was swine anti-goat horseradish peroxidase–conjugated from BioSource International, catalog no. ACI3404, 1:1000–2000 dilution.In vitro ubiquitination reactions were carried out at 37 °C for the time indicated for each experiment, in the presence of 25 mm Tris, pH 8, 150 mm NaCl, 3 mm MgCl2, 1 mm TCEP. Concentrations used were 5 mm ATP, 200 nm E1, 250 nm E2, 15 μm ubiquitin, 50 nm E3 ligase (IDOL or RNF168), and, if present, 15 μmTAMRALDLR_peptide (residues 811–835) or a 15 μm concentration of a random TAMRActrl_peptide (GPLATSTPKNNG). Reactions were stopped by the addition of SDS-PAGE loading buffer and analyzed by immunoblotting.Recombinant human UBA1, UBCH5C, RNF168, and ubiquitin were purified as reported previously . UBC13/U344 was concentrated using an Amicon Ultra centrifugal filter (Millipore) with a 10-kDa nominal molecular mass cutoff. Eventual dilutions were carried out using the SEC buffer . All the crystallization experiments were carried out using Mosquito (TTP Labtech) and 96-well two-drop MRC crystallization plates (Molecular Dimensions). Initial crystallization screenings identified few promising hits from the ComPAS suite (Qiagen) screen set up at 20 °C. After a few rounds of condition optimization, we were able to generate the crystal used in this study. Explicitly, the FERM344 crystal used to solve the structure was obtained with the sitting-drop vapor diffusion method at 4 °C, by mixing 100 nl of concentrated FERM344 protein at 5.8 mg ml−1 with 200 nl of reservoir solution containing 100 mm Tris, pH 8.5, 20 mm MgSO4, 4% (w/v) ethylene glycol, and 12% (w/v) 2-methyl-2,4-pentanediol. Except for this condition, FERM344 protein also crystallized in several other similar conditions, which all contained the following components: 100 mm Tris buffer, pH 8–8.5, 10-20 mm MgSO4 plus one or two alcohols at low percentage. Pyramidal-like crystals started appearing after 4–5 days and stopped growing after 2–3 weeks. Crystals were then cryoprotected via transfer into a drop of reservoir solution added with 20–25% (v/v) ethylene glycol. After incubation in the cryoprotectant solution, the crystals were looped and flash-cooled in liquid nitrogen before data collection.Purified FERM−1 in a buffer containing 500 mm NaCl, 10% glycerol, 1 mm TCEP, and 50 mm MES, pH 6.5. Initial crystallization screening was done using the commercial Index (Hampton Research) and JCSG+ (Molecular Dimensions) screens with 150 + 150 nl vapor diffusion drops at 20 °C using a Mosquito LCP robot (TTP Labtech). Crystals from the most promising condition, containing 100 mm Hepes, pH 7.0, and 10% (w/v) PEG 6000, were flash-frozen in liquid nitrogen using 25% (v/v) glycerol as cryoprotectant and prescreened using an FR-E+ SuperBright rotating anode (Rigaku Corp.).The purified F3ab domain was concentrated to 5.3 mg ml344 were collected at a wavelength of 0.966 Å at 100 K at the beamline MASSIF-1 ID30A-1    in solution.X-ray diffraction data for FERM France) , 52, equ package . For det package , an auto package , using 3ge   was usedlProbity   . A final model composed of Gly186–Ala283 (chain A), Tyr185–Tyr275 (chain B), and Tyr185–Val280 (chain C) was refined to an R/Rfree of 25.4/28.4%. Full data collection and refinement statistics can be found in gR and Dmax for FERM278, FERM304, and FERM332 were calculated with MOLEMAN . SSM (RRID:SCR_004727) and the RAPIDO web server (http://webapps.embl-hamburg.de/rapido/) , equipped with a PILATUS 6M detector (DECTRIS). Data corresponding to the F3ab crystal structure presented in this work were collected up to 2.5 Å resolution. The data were indexed and integrated with MOSFLM  and scaln PHASER , using tD: 2HE7)  as a sea MOLEMAN . FERM278rk). SSM  at the Erapido/)  were use278 at 5.87 mg ml−1 (187 μm), FERM304 at 7.73 mg ml−1 (226 μm), FERM332 at 5.71 mg ml−1 (157 μm), and FERM344 at 9.86 mg ml−1 (255 μm). 45 μl of each centrifuged sample were loaded onto a Superdex 200 Increase 3.2/300 column , previously equilibrated in 20 mm MES, pH 6.8, 300 mm NaCl, 1 mm TCEP. The scattering profiles from all the proteins were collected in 620 3-s frames.SAXS diffraction data were collected at 12.1 keV at beamline B21 (Diamond Light Source), equipped with a PILATUS 2M detector . Sample-to-detector distance was 4 m. Individual samples were injected at the following concentrations: FERMgR in reciprocal space was calculated with Guinier approximation . P(r) functions and estimation of the Dmax were measured with GNOM  database with accession codes 6QLY and 6QLZ, respectively. Other data will be stored in the NKI repository, available upon request from Titia Sixma (t.sixma@nki.nl).Model coordinates and relative structure factors for IDOL FERM


Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN129, and Rhodococcus sp. IN306 and mixed culture M1 developed based on those strains at 1:1:1 ratio was analyzed. The effectiveness of individual strains and of the mixed culture was assessed based on carried out respirometric tests and chromatographic analyses. The Rhodococcus sp. IN306 turned out most effective in terms of 18 PCB congeners biodegradation (54.4%). The biodegradation index was decreasing with an increasing number of chlorine atoms in a molecule. Instead, the Mycolicobacterium frederiksbergense IN53 was the best TPH degrader (37.2%). In a sterile soil, contaminated with PCBs and TPH, the highest biodegradation effectiveness was obtained using inoculation with mixed culture M1, which allowed to reduce both the PCBs (51.8%) and TPH (34.6%) content. The PCBs and TPH biodegradation capacity of the defined mixed culture M1 was verified ex-situ with prism method in a non-sterile soil polluted with aged petroleum hydrocarbons (TPH) and spent transformer oil (PCBs). After inoculation with mixed culture M1, the PCBs were reduced during 6 months by 84.5% and TPH by 70.8% as well as soil toxicity was decreased.Biodegradation is one of the most effective and profitable methods for the elimination of toxic polychlorinated biphenyls (PCBs) and total petroleum hydrocarbons (TPH) from the environment. In this study, aerobic degradation of the mentioned pollutants by bacterial strains  The meation (1) . After t2O5, 19.1% of K2O in the form of K2SO4, 4.4% of MgO in the form of soluble MgSO4, as well as microelements  to obtain optimum proportions of nutrients C:N:P = 100:10:1. Nutrients , NO3-N , and PO4-P  were determined by Perkin Elmer Lambda 40 spectrophotometer. Before the inoculation, the total number of heterotrophic aerobic bacteria in non-sterile soil D was 5.9 ± 5.1·105 CFU·g−1 dry mass. The soil prepared in this way was inoculated with mixed culture M1 with a density of 109 CFU mL−1 (2 L mixed culture M1/50 kg soil D). After 6 months of the experiment, the number of bacteria in D6 soil was 4.1 ± 4.6 × 107 CFU·g−1. The PCBs and TPH biodegradation was monitored by means of chromatographic analyses (every 30 days during 6 months) and ecotoxicological tests. Control was non-sterile Soil D non-inoculated with mixed culture M1.Studies on PCBs and TPH biodegradation in non-sterile soil (Soil D) were carried out by means of the ex-situ prism method. The soil at an amount of 50 kg was placed on a specially designed test stand, ensuring constant temperature (20–25 °C) and humidity (20–25%) during the experiment . A propeTen grams of dried soil were placed in an Erlenmeyer flask and extracted using a mixture (80 mL) of n-hexane and dichloromethane  at 1:1 ratio . The PCB−1), 280 °C , 280–320 °C (temperature increase 28 °C min−1), and 320 °C . The carrier gas was helium (He) at a constant flow of 3.5 mL·min−1. The identification and quantitative analyses of 18 PCB congeners , PCB52 , PCB77 , PCB81 , PCB101 , PCB105 , PCB114 , PCB118 , PCB123 , PCB126 , PCB138 , PCB 153 , PCB156 , PCB157 , PCB167 , PCB169 , PCB180 , and PCB189  were carried out using a certified reference material namely: PCB congeners in the SQC068-50g soil . A certified PCB-209 standard  was used as a biomarker.PCB congeners were analyzed by means of gas chromatography (GC), using a Perkin Elmer Clarus 500 chromatograph with an electron capture detector (ECD). The conditions of GC chromatograph operation were as follows: a capillary column of fused silica (Rtx-XLB 30 m × 0.32 mm ID)  with a low-polarity proprietary phase (0.50 µm thick), with the application of the following temperature parameters: injector temperature 280 °C; ECD detector temperature 320 °C; oven temperature programme: 120 °C , 120–280 °C  in three series . The extraction of petroleum hydrocarbons was carried out by means of the sonification method in an ultrasonic bath Sonoswiss SW 6H, at an ultrasonic frequency of 30 kHz . Polar s−1), 105–285 °C (temperature increase 5 °C min−1), and 285 °C . The carrier gas was He at a constant flow of 20 mL·min−1. For the quantitative determination of total petroleum pollutants content (TPH), a set of Tusnovic Instruments calibration standards was used (certified standard: BAM K010). In turn, certified sets of Supelco and Restek standards  were used to quantitatively determine individual n-alkanes being components of petroleum pollutants. A certified standard C30 17α(H), 21β(H)-hopane No 08189  was used as a biomarker.TPH were analysed by means of gas chromatography (GC) using a Perkin Elmer Clarus 500 GC chromatograph with a flame ionisation detector (FID). The conditions of chromatograph operation were as follows: a capillary column of fused silica (RTX-1: 30 m × 0.53 mm) , applying the following temperature parameters: injector temperature = 290 °C, FID detector temperature = 320 °C, oven temperature programme: 30 °C , 30–105 °C ,21β(H)-hopane  was used. The biomarker application of biomarkers in biodegradation studies allows to eliminate analytical errors in chromatographic determinations of individual analyte groups. Normalized values were used to develop a mathematical first-order model describing biodegradation course, according to Equation (2) , (C/Cx)0—normalized analyte concentration at the starting point, and t—process duration [d].A simplified mathematical model of polychlorinated biphenyls and petroleum hydrocarbons biodegradation during the performance of tests by the ex-situ method, was developed. PCB-209 biomarker  was applied to normalize concentrations of the following analytes: PCBs, TriCB, TetraCBs, PentaCBs, HexaCBs, and HeptaCBs. To normalize concentrations of: TPH, ΣnCtion (2) ,64.C/Cx2) defining the measuring points fit to theoretical curves of the biodegradation process: PCBs, TriCB, TetraCBs, PentaCBs, HexaCBs, HeptaCBs, TPH, ΣnC11–nC22, and ΣnC23–nC40.The analysis of non-linear regression using Equation (2) enabled determination of the first-order biodegradation constant (k) and the correlation coefficient  inoculated with mixed culture M1 (the ex-situ prism method) was performed. A set of new generation tests, which bioindicators belong to various trophic levels, was used. The specification of toxicological tests used in this studies is presented in p < 0.05.Statistical analysis was performed with Statistica 13.3 . Standard deviation (SD), relative standard deviation (RSD) (%) and Pearson correlation coefficient were calculated. Statistically significant differences were evaluated with a one-way ANOVA followed by a post-hoc pairwise Tukey test (when the ANOVA produced significant results). Significance was set at Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN129, Rhodococcus sp. IN306, and mixed culture M1 developed based on these strains. The results of both respirometric tests and chromatographic analyses revealed that chosen bacterial strains with potential PCB decomposition capacity are actually capable of removing this type of pollution from the soil, as well as they are capable of decomposing petroleum hydrocarbons . Rhodococcus sp. IN306 appeared as the most effective PCB-degrader among the studied strains. Moreover, IN306 is very closely related to Rhodococcus jostii RHA-1 . It was shown that the degree of PCB congeners biodegradation was decreasing with increasing number of chlorine atoms and depended on their spatial structure. Petroleum pollutants, in particular n-alkanes, were degraded by Mycolicibacterium frederiksbergense IN53 in the most efficient way. Its effect on low-, medium- (nC10–nC22) and long-chain compounds (nC23–nC40) was the greatest among all tested bioaugmentation variants. In turn, mixed culture M1 developed on the basis of tested bacterial strains, featured satisfactory biodegradation capacity both for individual PCB congeners and TPH. The action of mixed culture M1 was verified in a semi-technical scale experiment under ex-situ conditions on a soil polluted with aged petroleum hydrocarbons (TPH) as well as spent transformer oil (PCBs). The effectiveness of this treatment was confirmed by the results of both chromatographic and toxicological tests.The study was aimed at the assessment of bacterial strains application possibilities:


Fecal immunochemical test (FIT), DNA mutation, DNA methylation, and microbial dysbiosis all showed promising in colorectal cancer (CRC) non-invasive detection. We assessed CRC detection with an assay combining all these strategies and investigated the effect of clinical features on the performance of this comprehensive test.Fusobacterium nucleatum and Parvimonas micra using qPCR method. We used a linear support vector classification model to analyze the data.We performed a multidimensional analysis study using stool samples collected from 108 patients with CRC, 18 patients with colorectal adenoma, and 36 individuals with no evidence of colorectal disease. The multidimensional analysis of stool samples including FIT, stool DNA (sDNA) tests for three methylated genes  and three mutated genes  using next generation sequencing as well as detection of stool bacteria level of The sensitivity of FIT alone was 69.4% for CRC and 11.1% for adenoma. Separately, the sensitivity of the detection of intestinal bacteria, DNA mutation, and DNA methylation for CRC was 58.3, 50.0, and 51.9%, respectively. The combination of FIT and sDNA tests had a sensitivity of 81.5% for CRC  and 27.8% for adenoma with 94.4% specificity. Sensitivity of the multidimensional test to detect CRC with stage II (84.6%) and III (91.9%) CRC was relatively higher (88.2%) than that of patients with stage I (60.0%) and stage IV (75.0%) (P = 0.024). The rate of CRC detection increased with tumor size (P = 0.008) and age (P = 0.04). Interestingly, the rate of CRC detection was higher in smoking persons than non-smokers with marginal significance (P = 0.08).The multidimensional assay of stool samples combining FIT and stool DNA tests further improved the diagnostic sensitivity for CRC. This could provide new approach for improvement of CRC screening and further demonstrations are warranted. Colorectal cancer (CRC) is the third most common cancer with over 1.2 million new patients per year and the fourth leading cause of cancer-related death worldwide . The NurDespite the supporting evidence, recommendations, and availability of several screening tests, a large proportion of the U.S. population is not up to date with screening. For instance, screening compliance in the Nottingham trial was only around 60%, which signified that those tests still necessitate improving . TherefoMore and more study revealed colorectal cancer arises from accumulated genetic and epigenetic alterations , 7. The The Ethical Committee and Institutional Review Board of the Fudan University Shanghai Cancer Center reviewed and approved this study protocol. All patients signed written informed consent.Fusobacterium nucleatum and Parvimonas micra). To assess the performance of multidimensional stool analysis, stool samples were distributed in balanced to training and validation datasets. A linear support vector classification model was built based on the training set, and then the validation set was evaluated by the model with a pre-selected cut-off.In this study, we established a multidimensional analysis using stool samples for the detection of CRC or colorectal adenoma. Stool samples were collected before tumor removal of CRC or adenoma patients. The control stools were collected from control individuals with no evidence of colorectal disease. FIT was tested once the samples were received. Multiple stool DNA (sDNA) test was performed including three methylation markers , three mutation genes , and two bacteria relative levels  8th edition . All CRCAll stool samples were collected 7 days after diagnostic colonoscopy but before the removal of CRC or adenomas if there is any . Also, aBefore whole stool samples were buffed with STE, some stools were collected to q-FOB sample collection tube and tested with Fecal Occult Blood Gold Gel Stripe (W.H.P.M.INC) according to manufacturer’s instruction.Stool DNA was extracted following the manufacturer’s instruction of E.Z.N.A. Stool DNA Kit (Omega). Humanized DNA was quantified using standard curve method. Stool DNA was diluted with 1,000 fold; 10, 1, 0.1, 0.01, 0.001 ng/µl NA12878 DNA was used to establish the standard curve. Primers targeted to hLINE-1 was used to quantification, the sequence of primers used is listed in Ten ng humanized DNA was input for mutation assays, primers for target region amplify was in 4 μM final concentration. The 75 μl PCR mix was composed of 37.5 μl Phusion Blood Direct PCR Master Mix, 3 μl primer pool, and 34.5 μl sDNA. The thermal cycling comprised of 98°C 3 min, following 25 cycles of 98°C 15 s, 62°C 30s, and 72°C 30s, final extension at 72°C 3 min. PCR product was purified by 75 μl AMPure XP Beads and elute with 30 μl Low TE. Second PCR was performed with 12.5 μl Phusion Blood Direct PCR Master Mix, 6.25 μl nuclease free water, 0.75 μl DMSO, 2.5 μl index primers (5 μM), and 3 μl first PCR elute. The thermal cycling comprised of 98°C 3 min, following eight cycles of 98°C 15 s, 55°C 30s, and 72°C 30s, final extension at 72°C 5 min. Then 25 μl nuclease free water was added to each well after PCR, then purified by 40 μl AMPure XP Beads and elute with 20 μl Low TE. The libraries were quantified and loaded to Illumina Miseq or Nextseq sequencer for sequencing. Target sequence Table S2Ten ng humanized DNA was input for HpaII digestion, another 10 ng humanized DNA without HpaII digestion used as control. Digestion was treated in 37°C 3 h, then 80°C 20 min for enzyme inactivation. Digestion product was beads purified and elute with 20 μl low TE. Primers for target region amplify was in 4 μM final concentration, primers for KRAS region were used as reference. The 75 μl PCR mix was composed of 37.5 μl Phusion Blood Direct PCR Master Mix, 3 μl primer pool, 20 μl eluted DNA, 13.75 μl nuclease free water, and 0.75 μl DMSO. The thermal cycling comprised of 98°C 5 min, following 25 cycles of 98°C 15 s, 66°C 30s, and 72°C 30s, final extension at 72°C 3 min. PCR product was purified by 75 μl AMPure XP Beads and elute with 11 μl Low TE. Second PCR was performed with 12.5 μl Phusion Blood Direct PCR Master Mix, 0.75 μl DMSO, 2.5 μl index primers (5 μM), and 9.25 μl first PCR elute. The thermal cycling comprised of 98°C 3 min, following eight cycles of 98°C 15 s, 55°C 30s, and 72°C 30s, final extension at 72°C 5 min. Then 25 μl nuclease free water was added to each well after PCR, then purified by 40 μl AMPure XP Beads and elute with 20 μl Low TE. The libraries were quantified and loaded to Illumina Miseq or Nextseq sequencer for sequencing. The sequence of primers for library preparation-methylation detection is listed in Fusobacterium nucleatum, Parvimonas micra, and universal 16S were performed in parallel. The 20 μl PCR mix was composed of 10 μl KAPA SYBR FAST qPCR master mix, 5 μl diluted sDNA, 1 μl primers , and 4 μl nuclease free water. The thermal cycling comprised of 95°C 5 min, following 40 cycles of 95°C 15 s, 58°C 25s, and 72°C 30s with florescence take. Fusobacterium nucleatum, Parvimonas micra relative level was calculated with delta Ct method with universal 16S as reference. The sequence of primers for Fusobacterium nucleatum, Parvimonas micra, and universal 16S detection is listed in Diluted sDNA with nuclease free water to final 0.2 ng/μl, qPCR assays for Paired end reads were merged to single end reads by pear (0.9.6) with parameter “-j 4 -v 20 -t 30 -n 30” to recover high quality original DNA fragments. Adapter and primer sequences at the end of reads were trimmed by trim_galore (0.4.0). Reads from each sample were mapped to the reference sequence hg19 using Burrows-Wheeler Aligner  . Only the samples with greater than 50,000 total sequencing reads and 10,000 on-target reads were chosen for further analyses. GATK3.4.0 was applied to detect mutations . Integrative Genomics Viewer  was used for visual inspection of the aligned reads. Normalized methylation value of each target region was calculated as follows:Fn relative level, Pm relative level, Tumor load (KRAS),Tumor load (BRAF),Tumor load (PIK3CA), Tumor load (known markers), chr7:140453136:A:T, chr12:25398285:C:A, chr12:25398285:C:G, chr12:25398285:C:T, chr12:25398284:C:G, chr3:178936095:A:C, chr3:178952085:A:T, chr12:25398282:C:A, chr3:178952085:A:G, chr12:25398281:C:A, chr12:25398255:G:T, chr3:178936082:G:A, chr3:178936091:G:A, chr3:178936092:A:G, chr3:178936095:A:T, chr12:25398284:C:A, chr12:25398284:C:T, chr12:25398281:C:T, chr3:178936092:A:C, chr3:178952003:G:A, chr12:25398275:C:T, Septin9 methylation, NDRG4 methylation and BMP3 methylation.”Samples were divided into training dataset and validation datasets. Features involved were: “Q-FIT, Age, The values of each feature were scaled between 0 to 1 with MinMaxScaler. After scaling, linear support vector classification model was built with the training dataset (sklearn 0.22.1). Putting specificity prior to sensitivity, we set the threshold as the maximum value of prediction value of normal samples minus a margin of 0.005 in training dataset.The study comprised 162 samples from 108 patients with CRCs, 18 patients with colorectal adenoma, and 36 healthy control with no evidence of colorectal disease (NED). The median age was 58 years (IQR: 26–86) and 58% (n = 94) were male. Respectively, 80 samples were randomly selected and used for training and the other 82 samples were used for validation. Detailed clinicopathological characteristics in the training and validation sets were listed in Fusobacterium nucleatum and Parvimonas micra increased from NED, adenoma to CRC groups , mutations in four regions of three genes , and bacteria relative levels of  adenoma Table 3.vs 73%, P = 0.04). Also, the CRC detection rate increased with the tumor size (P = 0.008)  Table 4.In our study, combination of FIT and sDNA was addressed for potential role in detection of CRC and adenoma, with 94.4% combined specificity. Our result indicated that this multidimensional stool model consisting of FIT, three methylation markers, three mutation genes, and two bacteria relative levels reached 94.4% specificity and 81.5% sensitivity of CRC. So far, our study is the first study on combined multidimensional sDNA assay including fecal DNA mutation, DNA methylation, and bacterial in Chinese population, with relative sensitivity of 81.5% for CRC and specificity of 94.4%.Fusobacterium species, Porphyromonas asaccharolytica, and Peptostreptococcus stomatis explored the suitability of intestinal microbiota in CRC detection. Nevertheless, the ROC of these metagenomic classifiers were between 0.73 and 0.84  . Given tAge was demonstrated as clinical characteristic related to the sensitivity of multidimensional set in our study (P < 0.05). Defined as presence of methyl groups at CpG dinucleotides, DNA methylation was increasing with age. Further study illustrated that a small number of these certain CpG sites were highly associated with age, which even could be used for predicting age . Also, aNotably, the rate of CRC detection was significantly higher in smoking persons than non-smokers in our study, which evidenced the advantage of this multidimensional test for colorectal tumor screening in smoking population. This may due to lifestyle changes  and immuIn line with knowledge that the low incidence of CRC in cancer screening, specificity is another important indicator for evaluating screening tools and reduces burden of screening follow-up colonoscopy for participants. Up to now, the specificity of sDNA in Chinese population was varied from 87 to 98% , 26. In Also, there are several limitations in our study. First, as a retrospective study on cancer screening, our study only included colorectal cancer patients and healthy individuals, which did not include other colorectal non-neoplastic diseases such as ulcerative colitis and other gastrointestinal cancers such as gastric cancer. Moreover, there have been commercial stool DNA detection methods that have been used in clinical practice. However, head-to-head comparison with such commercial multitarget DNA test in feces (Cologuard™) was lack for screening for CRC . FIT tesThe multidimensional assay of stool samples combining FIT and stool DNA tests further improved the diagnostic sensitivity for CRC. This preliminary study could provide a new approach for improvement of CRC screening. Further demonstrations on a large-scale study especially including more healthy population are warranted.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethical Committee and Institutional Review Board of the Fudan University Shanghai Cancer Center. The patients/participants provided their written informed consent to participate in this study.SC, HaoW, SM, HuiW, RL, and GC had the idea for this study. SM, WX, WD, and LH collected specimens. PZ, FP, and HuiW performed the experiments. SC and GC supervised the acquisition of the data. ZS and CM performed NGS data analysis. SM and HuiW undertook the statistical analysis. SC and GC provided statistical advice. All authors contributed to interpretation of the results. SM, LH and HuiW wrote the article. GC, SC, HaoW, and RL revised the article and other authors contributed to the content. All authors contributed to the article and approved the submitted version.This study was supported by the Grant of Science and Technology Commission of Shanghai Municipality (No. 17411951100) and the National Key Research and Development Program of China (grant number:2019YFC1315800).HuiW, PZ, FP, ZS, CM and RL were employed by company Singlera Genomics (Shanghai). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intrathecal injection of voltage-sensitive calcium channel blocker peptide toxins exerts analgesic effect in several animal models of pain. Upon intrathecal administration, recombinant Phα1β exerts the same analgesic effects as the those of the native toxin. However, from a clinical perspective, the intrathecal administration limits the use of anesthetic drugs in patients. Therefore, this study aimed to investigate the possible antinociceptive effect of intravenous recombinant Phα1β in rat models of neuropathic pain, as well as its side effects on motor, cardiac (heart rate and blood pressure), and biochemical parameters.Escherichia coli expression. In rats, neuropathic pain was induced by chronic constriction of the sciatic nerve and paclitaxel-induced acute and chronic pain. Mechanical sensitivity was evaluated using von Frey filaments. A radiotelemeter transmitter  was placed on the left carotid of mice for investigation of cardiovascular side effects. Locomotor activity data were evaluated using the open-field paradigm, and serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were examined.Male Wistar rats and male Balb-C mice were used in this study. Giotto Biotech® synthesized the recombinant version of Phα1β using 50 of 0.02 (0.01-0.03) mg/kg, and reached the maximal effect (Emax = 100% antinociception) at a dose of 0.2 mg/kg. No significant changes were observed in any of the evaluated motor, cardiac or biochemical parameters. Intravenous administration of recombinant Phα1β toxin induced analgesia for up to 4 h, with EDOur data suggest that intravenous administration of recombinant Phα1β may be feasible for drug-induced analgesia, without causing any severe side effects. Pain, one of the most common symptoms in human diseases, remains one of the most poorly understood sensations, with insufficient clinical management despite the large number of pain-relieving drugs available on the market. To overcome this problem, numerous studies have been conducted to elucidate neuronal pathways of pain and develop new treatments . A consiPhoneutria nigriventer, such as PnTx3-6. This toxin was patented under the name Phα1β. It causes blockage of the mammalian VSCCs expressed in HEK cells, leaving LVA (T-type Ca2+ channel) unaffected [Our group has extensively described different toxins isolated from purified venom of the spider affected . Phα1β  divided into 12 groups (n = 6) and 30 adult male Balb-C mice  divided into five groups (n = 6). All animals were acclimatized in the laboratory for at least 2 h before testing. They were used only once throughout the experiments. While in the home cage environment, the animals were allowed free access to water and food. The room temperature was maintained at 22 ± 1°C, and the room illumination was on a 12/12-hour light/dark cycle. The experiments were carried out following the current guidelines for the care of laboratory animals, and adhered to the ethical guidelines for the investigations of experiments on conscious animals [Escherichia coli expression [Giotto Biotech® synthesized the recombinant version of Phα1β via pression . The pepNext 50 μL of recombinant Phα1β, morphine (positive control for antinociceptive effect), or PBS was administered intravenously with a needle (30 G) into the caudal veins of the rats . The anilog in milligrams (mg) and was evaluated several times after i.v. administration of the recombinant Phα1β, morphine, or PBS. Mechanical sensitivity in rats was evaluated by using von Frey filaments and the up-down paradigm , 22. AftA chronic constriction injury (CCI) of the sciatic nerve was examined by using a previously described method, with minor modifications . Rats weTo evaluate paclitaxel-induced pain syndrome, rats were initially divided into two groups. The first group received a single injection of paclitaxel  to induce acute painful syndrome, and the mechanical sensitivity was measured before and at 24 h after injection. The second group received four consecutive paclitaxel injections  on alternate days. The acute pain was evaluated before and at 15 days after the first of the four paclitaxel injections . The mice were anaesthetized with isoflurane (2% for induction and 1% for maintenance). The left carotid was exposed for implantation of a radio telemeter transmitter . Next, behavioral and cardiovascular side effects were analyzed. Seven days after the surgical procedure, the mice were treated with the recombinant Phα1β toxin . Locomotor activity (cpm), mean arterial pressure (mmHg), and heart rate  were continuously recorded for 24 h after treatment. Serpentine-like tail movements, body shaking, and dynamic allodynia were also evaluated using a 7-point scale, as previously described , 27. CarTo reinforce and confirm the locomotor data from animals in the box, locomotor activity was also evaluated using the open-field paradigm. Briefly, at 2 h after treatment with vehicle, morphine , or recombinant Phα1β , mice were introduced to individual boxes (50 × 50 × 25 cm) to which they had not been previously exposed. Spontaneous activities were measured  for over® colorimetric assay according to the manufacturer's instructions. After 24 h of recombinant Phα1β treatment , the mice were sacrificed. Blood samples were collected, and serum was isolated and frozen (-10ºC) until analysis. Serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were evaluated using Bioclin2test was used. The 50% effective dose (ED50) values were calculated by nonlinear regression. These values were attained by using a dose-response equation that was adjusted to provide the best description of the values obtained from the individual experiments. Values of p< 0.05 were considered significant. The results are presented as mean ± standard error , median ± interquartile range (adverse effect scores), or geometric means accompanied by their respective 95% confidence limits (for ED50).Statistical analyses were performed using the software Graph Pad Prism, Version 5.01. The data were analyzed using one-way or two-way analyses of variance followed by the Bonferroni or Student-Newman-Keuls test, when appropriate. Adverse effects were assessed by the Kruskal-Wallis test followed by Dunn's test, when applicable. For analyses of proportions, the χmax = 100%) at a dose of 0.2 mg/kg (i.v.), with an ED50 of 0.02 (0.01- 0.03) mg/kg (max = 100%). Intravenous (i.v.) administration of recombinant Phα1β toxin at 0.2 mg/kg significantly reduced CCI-induced mechanical hypersensitivity of neuropathic nociception. The analgesic effects of the recombinant Phα1β lasted for 4 h , reachin3) mg/kg . Adminis3) mg/kg  and chro3) mg/kg  mechanicTo evaluate whether i.v. administration of Phα1β recombinant toxin induces the development of acute toxicities, we evaluated some possible side effects that had been predicted for the calcium channel blockers when administered by this route. Thus, Balb-C mice treated with morphine (20 mg/kg i.v. - positive control) or different doses recombinant Phα1β  were observed at different time points after each treatment. Morphine increased motor activity at 2 and 4 h after administration, and tended to decrease the number of rearing-behavior instances and the latency time to escape the central square in the open-field test, showing motor changes and anxiety behavior . In addiSimilarly, morphine also induced significant changes in heart rate at 2 and 4 h after administration, as well as a 14 mmHg increase in blood pressure at 4 h after administration; these events were not observed in mice treated with recombinant Phα1β toxin  . MorphinAdministration of an analgesic drug by the i.t. route is invasive and often painful; moreover, it poses a risk of infection and requires surgical skills and expensive specialized equipment. Thus, it is preferable to use this route as a last resort, giving preference to other routes, such as intravenous administration. In the present study, we described the effectiveness of recombinant Phα1β administration via i.v. injection in two distinct animal models of neuropathic pain. It is currently unknown whether Phα1β or its recombinant form can penetrate the blood-brain barrier to reach the spinal VSCCs, or whether Phα1β can target calcium channels outside the central nervous system (CNS). The effect of Phα1β outside the CNS would open the way for its use in accessing VSCC blockers, thereby helping patients that cannot tolerate an intrathecal catheter. Future studies that investigate the mechanism and factors, such as bioavailability, distribution, and tissue-specific effects, of the i.v. administration of the peptide are necessary.Phα1β (0.2 mg/kg) administration via the i.v. route caused pronounced analgesia (anti-hypersensitivity) in two distinct rat models: chronic constriction injury and the acute and chronic paclitaxel pain models. The native Phα1β is one of the high-voltage calcium channel blockers (HVCCs), whose functional role in neuropathic pain mechanisms has been described . Current-1) caused a similar decrease in blood pressure and tachycardia that rapidly reached the maximum [Ziconotide, either as a monotherapy or in combination with other i.t. drugs, is a potential therapeutic option for patients with refractory neuropathic pain . However maximum . In thisin vivo [  a broad channel with a well-defined role in inflammation-associated pain, may account for the action of the toxin.The data in the present study suggest the possibility of a non-spinal route for Phα1β recombinant delivery. The analgesic effects of i.v. administration of the recombinant toxin in neuropathic pain models presented efficacy and time-course effect similar to those of i.t. peptide administration , 15, 16.in vivo  . On the in vivo  . Thus, tin vivo   and TRPAin vivo  , a broadThe results have advanced the clinical trials and development of i.v. recombinant Phα1β for management of persistent pain conditions. This is especially important when spinal-route administration is not possible.50: effective dose; i.p.: intraperitoneal; i.t.: intrathecal; i.v.: intravenous; VSCCs: voltage-sensitive calcium channels.CCI: chronic constriction injury; ED