Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension. Vascular tone, mediated by smooth muscle cells in the tunica media, is finely balanced among endothelium-derived vasoactive substances including vasodilators and vasoconstrictors. For example, endothelial nitric oxide synthase (eNOS) can be activated by shear stress or neurotransmitter acetylcholine to produce vasodilating nitric oxide (NO) ,2. OtherEmploying in silico simulations and molecular models, preliminary studies indicated several molecules can possibly be associated with the target protein TSP1 ,15,16, wCommercially available myograph systems such as multiwire myograph and pressure myograph by Danish Myo Technology A/S , combined with a digital data acquisition device and a data recording and analysis software, enable to access the vasomotor function under different conditions and/or treatments with high sensitivity and efficiency. The protocol described herein used a two-pin multiwire automated myograph to evaluate responsiveness in isolated murine thoracic aorta, with a focus on the potential efficacy and potency of screened/selected candidate molecules as direct antagonists of/against TSP1-blunted endothelium-dependent vasorelaxation. C57BL/6J mouse, a common animal model used for the creation of transgenic or treatment-induced vascular disease murine in vivo models, was utilized in the present study. In spite of a distinct mechanism in regulating the vasoreactivity in the conduit vessel such as thoracic aorta from that in the resistance vessels like renal arterioles, others revealed that TSP1 signaling leads to resistance vessel dysfunction in the same manner as the systemic vasculature, altogether by debilitating vasodilation and enhancing vasoconstriction ,18. TherNative TSP1 protein, human CD47 antibody, clone mIAP301 SIRPα antibody, clone C20 Dextrose Calcium chloride dehydrate Sodium bicarbonate Potassium phosphate Magnesium phosphate Potassium chloride KCl Sodium chloride EDTA calcium Disodium salt Phenylephrine hydrochloride Acetylcholine chloride Forskolin DMSO Meclofenamate sodium 0.9% saline Isoflurane 2/5% CO2 95% OAutomated Multi Myograph System—620M PowerLab 8/35 Force Calibration Kit Dissecting scope Dissecting dish Dumont #5 forceps Dumont #7 forceps Tissue forceps Moria Miniature Vannas—curved Vannas scissors—straight Surgical scissors straight sharp/blunt Surgical scissors straight sharp Surgical tape (Various suppliers)30G × 1 in needle 3 mL syringe Water bath with immersion circulator Alternate option to water bath: automatic buffer filler system—625FS Microfuge tube LabChart 8 (ADInstruments Version 8)GraphPad Prism version 8.0 Optional: Myography CalibrationWeight calibration is recommended at least once a month for wire myography by the manufacturer. A step-by-step procedure is included in the FORCE CALIBRATION sub-menu under SETTING and detailed in the manual . FollowiOn the day of experiment, power on PowerLab and myograph with HEAT OFF, and leave them on standby. Open the most recent calibration file in LabChart.KCl Stock2O, to make a stock solution with a concentration of 223.65 g/L or 3 M, and store at room temperature.Dissolve 22.365 g of KCl in 100 mL of ddHFoskolin StockDissolve 5 mg of forskolin in 1 mL of DMSO, to make a stock solution with a concentration of 5 mg/mL or 12.18 mM, which is stable at room temperature for at least 6 months.Optional stock solutions for fresh Krebs–Henseleit solution preparation.Krebs Stock Salts2O, dissolve 101.3 g of NaCl, 4.67 g of KCl, 1.88 g of MgSO4, and 2.37 g of CaCl2. Transfer to a storage container and store at 4 °C.Fill a 2 L Erlenmeyer flask or beaker with 1 L of ddH4·7H2O or 3.137 g of CaCl2·2H2O can be used.Alternately, 3.845 g of MgSOKrebs Stock Phosphate2PO4 (monobasic) in 500 mL of ddH2O, to make a stock solution with a concentration of 13.38 g/L or 1.18 mM, and store at 4 °C.Dissolve 6.69 g of KHKrebs Stock EDTA2Ca EDTA·nH2O in 100 mL of ddH2O, to make a stock solution with a concentration of 9.73 g/L or 26 mM, and store at 4 °C.Dissolve 0.973 g of NaFresh Meclofenamate working stock2O/ethanol (50%/50% v/v), to make a stock solution with a concentration of 1 × 10−2 M, and keep at room temperature until use. Leftover is disposed.On the day of the experiment, dissolve 31.81 mg of meclofenamate in 10 mL of ddHFresh phenylephrine hydrochloride (PE) and acetylcholine chloride (Ach) working stocks2O to make 1 × 10−2 M PE or 1 × 10−2 M Ach stock solutions, respectively.Dissolve 20.37 mg of PE or 18.17 mg of Ach into 1 mL of ddH−1 M PE is mixed with 180 µL of Krebs buffer to make 200 µL of 1 × 10−2 M PE.Make 10× serial dilution of PE and Ach working stocks by mixing 20 µL of PE or Ach and 180 µL of Krebs buffer. For example, 20 µL of 1 × 10Fresh Krebs buffer can be prepared with Krebs stock salts, Krebs stock phosphate, and Krebs stock EDTA.2O, dissolve 1.25 g of NaHCO3 and 0.99 g of dextrose, followed by 12 mL of KH2PO4 stock and 1 mL of EDTA stock, while stirring.To prepare 1 L of fresh Krebs buffer, fill a calibrated Erlenmeyer flask with 750 mL of ddH2/5% CO2, gradually add 75 mL of stock salts.Gas the solution with 95% OCRITICAL STEP Slow addition of stock salts while stirring and bubbling is required to prevent precipitation.2O to make a final volume of 1 L, and add 310 µL of meclofenamate stock. Continue bubbling for around 5 min, seal the flask with parafilm, and store at 4 °C until use.Add the remaining volume of 162 mL of ddHOPTIONAL STEP Fresh Krebs–Henseleit Solution can be alternatively prepared as follows, without using stock solutions mentioned in step 1 and 2. Fill a calibrated Erlenmeyer flask with 1 L of ddH2O, dissolve 1.25 g of NaHCO3 and 0.99 g of dextrose, 7.6 g of NaCl, 350.4 mg of KCl, 140.8 mg of MgSO4 (or 288.4 mg of MgSO4·7H2O), 177.6 mg of CaCl2 (or 235.2 mg of CaCl2·2H2O), 160.6 mg of KH2PO4 (monobasic), and 9.7 mg of Na2Ca EDTA·nH2O. Add 310 µL of freshly prepared meclofenamate stock, seal the flask with parafilm, and store at 4 °C until use.2, 1.6; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 14.9; dextrose, 5.5; Na2Ca EDTA, 0.026; meclofenamic acid, 0.0031; pH 7.4).Both preparations concordantly produce a standard Krebs buffer  followed by a Bonferroni post hoc test. The logarithms of EC50 values were compared using a two-tailed Student’s TM-TSP1 and Novus-TSP1, respectively) were purified from human platelets, while TSP1 protein obtained from Athens Research & Technology (Athens-TSP1) was purified from human blood/plasma.Native TSP1 is widely expressed in human tissues with low tissue specificity and is marginally detected in normal subjects. However, upregulation of tissue TSP1 and/or circulating TSP1 can be found to be associated with various organ injuries and chronic clinical conditions ,24,25,26TM—TSP1 enhanced vasoconstriction by inhibiting acetylcholine-induced endothelium-dependent vasorelaxation over a wide concentration range, starting at 3 × 10−8 M until 1 × 10−5 M. The concentration–response curve of vasorelaxation normalized to 1 × 10−6 M phenylephrine-induced vasocontraction in the presence of three different concentrations of PierceTM—TSP1, specifically, 2.2, 4.4, and 6.6 nM, was first compared to determine if TSP1-inhibited vasorelaxation was indeed dose-dependent . Likewise, the same pattern of concentration effect of TSP1 was also observed in Novus—TSP1 prepared from human platelet (data not shown). Furthermore, a comparison of the dose–response curves in the presence of the same concentration, e.g., 4.4 nM of TSP1, based on the three different TSP1 vendors, showed that PierceTM—and Novus—TSP1 significantly impaired vasorelaxation up to almost the same extent. Both PierceTM—TSP1 and Novus—TSP1 products had a significantly greater inhibition of vasorelaxation induced by 1 × 10−7 and 3 × 10−7 M acetylcholine, respectively, in comparison with Athens—TSP1 (TM—TSP1 at 4.4 nM (TM—TSP1 at 4.4 nM was applied hereafter.As evident from tension traces demonstrated in ependent B. Piercelaxation C (p ≥ 0.ens—TSP1 D. Compart 4.4 nM E. EquivaTM—TSP1). An evaluation of the two anti-TSP1 drug candidates was conducted against vehicle control and positive control with anti-mouse monoclonal CD47 antibody as well. Tension trace demonstrated that the vessel rings treated with a proposed CD47-based drug candidate  for another 1 h, was applied to investigate whether these pharmacological approaches were able to protect aortic vessels from TSP1 modulation. The CD47-based drug prototype substantially blocked the modulating effect by TSP1, as demonstrated through significant improvement in vasorelaxation in the presence of TSP1, while showing no significant difference from the cohort that was pre-treated with CD47 blocking antibody; meanwhile, SIRPα antibody failed in preserving appreciable vasorelaxation after TSP1 addition.The initial screening assay was conducted to determine the modulating effect on vasorelaxation by several commercial TSP1 protein products. TSP1 from three vendors replicated a significant inhibition of endothelium-dependent vasorelaxation, as published by others ,10. A maAs TSP1 was demonstrated to impair endothelium-dependent vasorelaxation in as short as 15 min .Adapted from the standard ex vivo myograph protocols, our protocol presents several practical benefits, especially regarding TSP1-related applications. The pre-treatment protocol in the presence of TSP1 only, utilizing TSP1 as the primary vasodilation antagonist, would be considered a more cost-effective and efficient approach/alternative for pharmacological baseline screening of vasodilators and/or vasoconstrictors, compared with utilizing expensive and complex transgenic animal models of TSP1-related pathologies. Owing to multiple TSP1-related vascular interactions with endothelial and smooth muscle cells, this pre-treatment protocol—utilizing healthy murine vessels—would also be very useful to pharmacologically evaluate several drug molecules and proteins that influence TSP1-modulation of vascular effects on native/normal tissue, to be compared later under pathological conditions. In addition to the relatively standard pre-treatment scenario, we have adopted a post-TSP1 treatment set-up of this ex vivo myograph-based protocol. In this new variation, an acute challenge by TSP1 for 15 min is introduced, followed by treatment with the candidate drug for 45 min. This post-treatment scenario has been useful in therapeutically screening anti-TSP1 drug candidates at various dose levels .The flexible and facile pharmacological protocol presented here can be modified to any other type of tubular structure suitable for multiwire myograph receiving different soluble treatments and/or under diverse conditions ,11,29. FA notable limitation of the current protocol is the viability time frame for mounted vessels, particularly concerning their endothelial responsiveness. Different from in vitro assays, which generally can be paused at certain step(s) and stored for later processing, this ex vivo myograph-based protocol must be completed all in one experimental session, right after initial set-up. It is recommended that test vessels be freshly excised, which is quite important when endothelium-based proteins/functions are being investigated. With only a single layer of delicate endothelial cells, this experimental protocol should not continue to use test vessels, if mounted for longer than 6 h overall. Our laboratory observed somewhat reduced responsiveness to acetylcholine-induced endothelium-dependent vasorelaxation of isolated vessel (such as mouse thoracic aorta) when mounted for longer than 6 h on myograph. Observing this time window would merit superior consistency in response data. In general, prepared vessels can remain viable for about 6–9 h after mounting into the myograph, which is applicable for studying endothelium-independent vasoreactivity, owing to the relative robustness of multiple vascular smooth muscle cell layers.


MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31  were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we knocked-down the ZFC3H1 expression and found that the inhibition of ZFC3H1 significantly inhibited PRAD cell migration and invasion. Furthermore, ZFC3H1 siRNA treatment could reduce cell viability and increase the number of apoptotic cells in PRAD cells. Taken together, ZFC3H1 could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.The present study was aimed to evaluate the expression profile of Zinc finger C3H1 domain-containing protein (ZFC3H1) using bioinformatic analysis of public datasets from The Cancer Genome Atlas database (TCGA). The results showed that the expression levels of ZFC3H1 were notably lower than the corresponding non-cancerous tissues in prostate adenocarcinoma (PRAD), and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. We predicted the regulatory target and protein interaction partner network of ZFC3H1, and identified a PPI network composed of 26 node genes in PRAD. Furthermore, we found that the expression levels of  Cancer is the second most common cause of death worldwide (8.97 million deaths) after ischemic heart disease . PRAD isZinc finger C3H1 domain-containing protein (ZFC3H1), also designated as CCDC131 (coiled-coil domain containing protein), or PSRC2 (proline/serine-rich coiled-coil protein), is a large protein (1989 amino acids), with a C3H1- type zinc finger motif in its center, and contains five tetratricopeptide (TRP) repeats and six half-tetratricopeptide (HAT) repeats at the C-terminal region. ZFC3H1 is a central factor in the retention and degradation of polyadenylated RNA, and is involved in the processing of a wide range of RNAs, playing a crucial role in the degradation of nuclear RNAs . GarlandZFC3H1 and its target genes in PRAD, explore the role of ZFC3H1 in PRAD, and finally determine whether ZFC3H1 could provide a reference to evaluate PRAD prognosis and treatment by detecting its influence on the migration and invasion of PRAD cells.In the present study, based on The Cancer Genome Atlas (TCGA) database, we aimed to analyze the expression levels of ZFC3H1 in different human cancers and corresponding non-cancerous tissues (http://gepia.cancer-pku.cn/), which is based on the TCGA database [The TCGA is a central repository of multidimensional experimental cancer data, comprised of data pertaining to more than 30 types of human tumors. We obtained the expression profile of  tissues , includidatabase . Enterinhttps://string-db.org/), BioGRID (https://thebiogrid.org/), and IntAct (https://www.ebi.ac.uk/intact/) [http://bioinformatics.psb.ugent.be/webtools/Venn/).The target genes of ZFC3H1 were predicted using three databases: STRING (intact/) . To imprhttp://metascape.org/gp/index.html)[Enrichment analysis of overlapping target genes in the ZFC3H1 signaling pathway, was performed using Metascape (dex.html). We analdex.html).PPI analysis was performed using overlapping target genes of ZFC3H1 through Metascape, which identified 26 node degree genes . Moleculhttp://starbase.sysu.edu.cn/index.php) tool to observe whether their expression levels varied between PRAD and corresponding non-cancerous tissues, and whether these genes had any influence on the prognosis of PRAD [To prevent any errors resulting from the use of a single database, the 26 node degree genes were analyzed using the starbase  were obtained from American Type Culture Collection . 22RV1 and DU145 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium , supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . All cells were incubated at 37°C in a humidified atmosphere containing 5% COSmall interfering RNAs (siRNAs) against ZFC3H1, along with RNAi negative controls, were synthesized by Genepharm Technologies . The PRAD cells’ transfection was performed by/using Lipofectamine 2000  according to the manufacturer’s protocol. The siRNA sequences were as follows: ZFC3H1-homo-2234: 5´- CCAAGAAGCAAUCUAUCAATT-3´ and 5´-UUGAUAGAUUGCUUCUUGGTT-3´; ZFC3H1-homo-3986: 5´- GGAGUAAACAAAGAUCGAATT-3´ and 5´- UUCGAUCUUUGUUUACUCCTT-3´; ZFC3H1-homo-5975: 5´-GCUGCUGAGAUUGUUCUAATT-3´ and 5´- UUAGAACAAUCUCAGCAGCTT −3´; Negative control: 5´-UUCUCCGAACGUGUCACGUTT-3´ and 5´-ACGUGACACGUUCGGAGAATT-3´.5 cells/well. A 200-μl micropipette tip was used to create a wound scratch across a confluent monolayer of cells. After saline washing, capsules were visualized at 0 and 24 h using an inverted light microscope . Wound closure rate /initial wound area) was calculated, and the wound area was quantified using ImagePro Plus v. 6.0 .Cell migration ability was measured by the wound healing assay as described. For transfection with ZFC3H1 siRNA or negative siRNA, cells were plated in six-well plates at 3 × 104) in a serum-free medium, were seeded in the top chamber, while 700 μL of medium containing 10% FBS was added to the lower chamber. After a 24 h incubation, the cells were fixed with methanol for 10 min, and stained with 1% crystal violet at room temperature for 10 min. The number of migrated cells was counted and photographed in five randomly selected fields under an inverted microscope .The cell invasion assay was conducted using 24-well transwell chambers . For invasion assays, the upper chambers of inserts were coated with 70 μl of Matrigel . Transfected PRAD cells , after which the protein concentration was quantified using a Bicinchoninic Acid (BCA) kit . 40 μg of proteins in each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes . After the transfer, the membranes were blocked with 5% nonfat milk in Tris-buffered saline, and then incubated with the primary antibody against E-cadherin, vimentin, and Zinc finger C3H1 domain-containing protein (ZFC3H1) , diluted 1:1000 in TBS-T overnight at 4°C. After washing them in TBS-T three times, with 10 min intervals, the membranes were incubated with the appropriate horseradish peroxidase–labeled secondary antibody , diluted 1:2000 in TBS-T for 1.5 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression was used as the internal control. Protein signals were detected using enhanced chemiluminescence reagents  and the immunoreactive protein’s final signals were detected by Image Lab Software Version 5.0 .−∆∆Ct was applied to analyze the data. Primer sequence of ZFC3H1: forward primer: 5´-TGTCTCAGTGTCATACCCATCT-3´, reverse primer: 5´-CCTTCTGGGGTCTGAAAGAACTT-3´. Primer sequence of GADPH: forward primer: 5´-AGAAGGCTGGGGCTCATTTG-3´, reverse primer: 5´-AGGGGCCATCCACAGTCTTC-3´.Total RNA was extracted from 22RV1 cells and DU145 cells by TRIzol reagent (Invitrogen), and the primers were synthesized by Genepharm Technologies . The target gene and internal reference gene of each sample were amplified simultaneously. 23 cells/well, planted in a 96-well plate. The CCK-8 reagent was added to each well for 2 h, respectively, and then the cells were cultured at 37°C for another 1 h.After 48 h of transfection, cells were collected and detected by CCK-8 kit . The cell density of 22RV1 and DU145 was adjusted to 5 × 105 cells. Then, the supernatant was discarded in the dark, and a 5 μl/sample of FITC-labeled Annexin-V was added and incubated for 30 min. Following this, a 5 μl/sample of PI was added and allowed to react for 5 min. The number of apoptotic cells was counted using flow cytometry.According to the manufacturer’s protocol, we used the Annexin V-FITC cell apoptosis detection kit to determine the number of apoptotic cells. Briefly, the PRAD cells were transfected with ZFC3H1 siRNA later, prepared with a single-cell suspension using 2 × 10Immediately after harvesting PRAD tissues, they were rinsed in PBS, fixed in buffered paraformaldehyde solution (4%) and embedded in paraffin. After cutting paraffin samples of PRAD tissues into 3 μm thick slices, they were dried in an oven at 60°C for 1 h, then dewaxed twice in xylene, rinsed with ethanol, and rehydrated in alcohol solutions with decreasing gradient. Paraffin sections were incubated with 2% H2O2 for 15 min after heat-induced antigen retrieval in a 10 mM citrate buffer (pH 6.0), then washed 3 times and incubated in 0.3% Triton X-100 PBS for 2 h. The paraffin sections were then blocked by incubation with 5% BSA for 1 h. The slides were incubated with an anti–ZFC3H1 antibody , overnight at 4°C. The corresponding second antibody incubated for 20 min at 37°C and a diaminobenzidine (DAB) DAB substrate kit (Abcam) were used as detection reagents. At last, the sections were dehydrated, mounted and observed under light microscopy . The positive rates were measured using Image-Pro Plus v. 6.0 software .As shown in Analysis using the STRING, BioGRID, and IntAct databases, led to the identification of 71, 103, and 98 genes, respectively . We seleGO enrichment analyses was performed to determine the functions of the 81 target genes in tumor biological processes . The putAPP, C1D, CDC5L, CSNK2A1, CUL7, EXOSC1, EXOSC5, EXOSC7, HNRNPK, MOV10, MPHOSPH6, MRPS11, MRPS14, MRPS23, MRPS24, MRPS25, MRPS31, MRPS35, MRPS5, MRPS7, MTREX, PTCD3, RALY, SRRT, UPF1, and ZC3H3.To explore the interaction between the 81 overlapping target genes, a PPI network was constructed using Metascape . From thAPP, CDC5L, MPHOSPH6, MRPS31, and MTREX in PRAD tissues were observed to be lower than those in corresponding non-cancerous tissues  pathway; therefore, treatments that target and inhibit androgen biosynthesis are widely used. While these therapies are initially effective, resistance and progression of PRAD often occurs, underscoring the need to develop new treatments . ZFC3H1,First, we found that ZFC3H1 expression levels in many cancers, including PRAD, were significantly lower compared with those in corresponding non-cancerous tissues. In addition, the survival time of patients with PRAD and/containing high ZFC3H1 expression groups was (was becomes ‘were’ if you use the word ‘and’) shorter than that in patients with low ZFC3H1 expression.APP, CDC5L, MPHOSPH6, MRPS31, and MTREX in PRAD tissues were notably lower than those in corresponding non-cancerous tissues, and the patients in the MPHOSPH6 and MRPS31 high-expression groups showed much shorter overall survival than patients in the respective low-expression groups. Furthermore, we further analyzed the correlation of ZFC3H1 and these five genes. Transfection with ZFC3H1 siRNA could down-regulate APP, CDC5L, MPHOSPH6, MRPS31, and MTREX mRNA expression. It is interesting that APP is the downstream signaling of ZFC3H1 because APP is a representative prognostic factor and regulated by the androgen receptor [ZFC3H1 knockdown significantly inhibited PRAD cell migration and invasion abilities. CCK-8 and flow cytometry analysis showed that ZFC3H1 siRNA could reduce cell viability and increase the number of apoptotic cells in PRAD cells.Second, we predicted 81 target genes of ZFC3H1 and overlapping target genes were examined via GO enrichment analyses to provide clues as to the functional roles of these genes in the biological processes of PRAD. A PPI network was constructed, and the most significant node degree genes n = 26) were selected. We found that the expression levels of  were selIn summary, we reported that ZFC3H1 is closely related to the migration and invasion ability of PRAD. It is also associated with cell viability and apoptosis in PRAD. ZFC3H1 or its regulated genes might provide new biomarkers for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD. However, the low expression of ZFC3H1 in PRAD has not yet been explained and further studies are warranted.Click here for additional data file.


Recent studies have linked the activity of ER aminopeptidase 2 (ERAP2) to increased efficacy of immune-checkpoint inhibitor cancer immunotherapy, suggesting that pharmacological inhibition of ERAP2 could have important therapeutic implications. To explore the effects of ERAP2 inhibition on the immunopeptidome of cancer cells, we treated MOLT-4 T lymphoblast leukemia cells with a recently developed selective ERAP2 inhibitor, isolated Major Histocompatibility class I molecules (MHCI), and sequenced bound peptides by liquid chromatography tandem mass spectrometry. Inhibitor treatment induced significant shifts on the immunopeptidome so that more than 20% of detected peptides were either novel or significantly upregulated. Most of the inhibitor-induced peptides were 9mers and had sequence motifs and predicted affinity consistent with being optimal ligands for at least one of the MHCI alleles carried by MOLT-4 cells. Such inhibitor-induced peptides could serve as triggers for novel cytotoxic responses against cancer cells and synergize with the therapeutic effect of immune-checkpoint inhibitors. Cancer actively tries to evade the human immune response through the process of immunoediting . Thus, cCytotoxic T-lymphocytes can kill cancerous cells after recognizing small peptides derived from cancer-specific antigens. These small peptides, called antigenic peptides if they elicit an immune response, are presented as a complex with major histocompatibility complex class I molecules  on the cell surface and are produced by complex proteolytic cascades inside the cell . The sum6g in -4′-methylpentyl} phosphinic acid] was prepared according to the synthetic strategy shown below (2 and acidic deprotection (See Phosphinic inhibitor DG011 [((1wn below . Brieflytion See . MOLT-4 cells (5000 cells/well) were cultured as described above in the presence of varying concentrations of DG011 (0–100 μM) for 48 h. The culture medium was replenished with 100 μL of RPMI 1640 containing 2 mg/mL MTT reagent, to a final concentration of 1 mg/mL. After incubation for another 4 h the cell cultures were centrifuged at 1250 rpm for 5 min at room temperature. The resultant formazan crystals were dissolved in 100 μL DMSO and the absorbance intensity was measured on a TECAN infinite M200 microplate fluorescence reader  at 540 nm with reference at 620 nm. All experiments were performed at least three times and the relative cell viability (%) was expressed as a percentage relative to untreated control cells.2. After five days of incubation, cells were harvested for immunopeptidome isolation or for flow cytometry. For immunopeptidome analysis, cells were cultured at a large scale and collected from flasks using Centrifuge 5430 R . Cell pellets were stored at −80 °C until immunopeptidome isolation. For the flow cytometry analysis, cells were seeded in a 12-well plate and harvested with 10 mM EDTA pH 8.0, in PBS.MOLT-4 cells were treated with a DG011 inhibitor for a total of five days. The inhibitor was added to the complete culture medium at a final concentration 1 μM. The cell medium that contained the inhibitor was refreshed every two days. Cell cultures were incubated at 37 °C, 5% CO4 cells per sample were transferred in FACS tubes and washed twice with 1 mL FACS buffer . The cells were stained with 4 μL undiluted W6/32 antibody labeled with Fluorescein  for 30 min on ice. After incubation, the cells were washed with 0.5 mL FACS buffer, centrifuged at 200× g for 5 min at 4 °C and re-suspended in 300 μL FACS buffer. The samples were analyzed in a FACScalibur flow cytometer using the BD CellQuest™ Pro software . Approximately 20,000 events per sample were measured.Approximately 5 × 103 0.1 M, NaCl 0.5 M, pH 8.3) overnight. To generate one 1 mL bed volume of cyanogen bromide-activated Sepharose 4B , 0.285 g of dry beads was used. Sepharose was rehydrated with 1 mM HCl for 30 min and then washed thoroughly with a coupling buffer. The solution of the antibody was added to the beads and left for coupling overnight at 4 °C. After coupling, the beads were washed with coupling buffer and then with blocking buffer . After the washes the beads were transferred to a 50-mL tube with blocking buffer and were mixed for 3 h at room temperature. Finally, the beads were washed with three cycles of acidic buffer  and then basic buffer  solutions and then with 20 mM Tris–HCl, pH 7.5, 150 mM NaCl. For the pre-columns, the exact same procedure was followed except for the W6/32 coupling step. The columns and the pre-columns were stored at 4 °C until needed.W6/32 antibody (2 mg per column) was dialyzed in coupling buffer  for 1 h at 4 °C. The cell lysate was cleared with ultracentrifugation at 100,000× g for 1 h at 4 °C and then loaded onto a cyanogen bromide activated Sepharose pre-column, blocked as described above. The flow through from the pre-column was passed through W6/32-coupled beads three times and then washed with 20 bed volumes 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 20 bed volumes 20 mM Tris–HCl, pH 8.0, 400 mM NaCl, 20 bed volumes 20 mM Tris–HCl, pH 8.0, 150 mM NaCl and finally with 40 bed volumes 20 mM Tris–HCl, pH 8.0. The MHC-I–peptide complexes were eluted from the immunoaffinity column with 1% TFA. The peptides were separated from the MHC-I molecules using reversed-phase C18 disposable spin columns . The fraction containing peptides was dried prior to LC-MS/MS analysis.For the isolation of the immunopeptidome, 0.5 × 106 and max IT of 45 ms, followed by MS/MS scans of the 12 most abundant ions, using 15K resolving power with an AGC of 1 × 105 and max IT of 22 ms and an NCE of 28.The purified peptidic samples were pre-concentrated on a pepmap LC trapping column  at a rate of 5 μL of Buffer A (0.1% Formic acid in water) for 5 min. Then the samples were injected onto a 50 cm long pepmap column  placed in an oven set to 55 °C, using a gradient of 100% Buffer A (0.1% Formic acid in water) to 27.5% Buffer B (0.1% Formic acid in acetonitrile) in 35 min followed by an increase to 40% buffer B in 2.5 min and a second increase to 99% Buffer B in 0.5 min and then kept constant for 1 min. Finally, the column was equilibrated for 15 min prior to the subsequent injection. A full MS was acquired using a Q Exactive HF-X Hybrid Quadropole-Orbitrap mass spectrometer  in the scan range of 350–1500 m/z using 60 K resolving power with an AGC of 3 × 10https://www.maxquant.org/ accessed on 27 December 2021) , using “unspecific” search against the human uniprot protein database (downloaded 19/9/2019). Search parameters included a molecular weight ranging from 350 to 5000 Da, a precursor mass tolerance of 20 ppm, an MS/MS fragment tolerance of 0.5 Da and methionine oxidation, deamidation of asparagine and glutamine and protein N-terminal acetylation were set as variable modifications. The protein and peptide false discovery rate (FDR) was set to 1%. The match-between-run function was enabled. The raw files were searched and the identified peptides were quantified in MaxQuant (


Depending on the water content, metalions in the isostructural Co2+ and Ni2+ derivativesare octahedrally coordinated (n = 3) or consist ofoctahedral together with dimeric trigonal bipyramidal (n = 1) or square pyramidal (n = 0) environments.The imidazole molecule acts as a ligand  or charge-compensating protonated species (Zn2+ derivative). For the latter, the proton conductivity is determinedto be ∼6 × 10–4 S·cm–1 at 80 °C and 95% relative humidity (RH). By pyrolyzing in 5%H2–Ar at 700–850 °C, core–shell electrocatalystsconsisting of Co2+-, Ni2+-phosphides or Co2+/Ni2+-phosphide solid solution particles embeddedin a N-doped carbon graphitic matrix are obtained, which exhibit improvedcatalytic performances compared to the non-N-doped carbon materials.Co2+ phosphides consist of CoP and Co2P in variableproportions according to the used precursor and pyrolytic conditions.However, the Ni2+ phosphide is composed of Ni2P exclusively at high temperatures. Exploration of the electrochemicalactivity of these metal phosphides toward the oxygen evolution reaction(OER), oxygen reduction reaction (ORR), and hydrogen evolution reaction(HER) reveals that the anhydrous Co2(ETID)(Im)3 pyrolyzed at 800 °C (CoP/Co2P = 80/20 wt %) is themost active trifunctional electrocatalyst, with good integrated capabilitiesas an anode for overall water splitting (cell voltage of 1.61 V) andpotential application in Zn–air batteries. This solid alsodisplays a moderate activity for the HER with an overpotential of156 mV and a Tafel slope of 79.7 mV·dec–1 in0.5 M H2SO4. Ni2+- and Co2+/Ni2+-phosphide solid solutions show lower electrochemicalperformances, which are correlated with the formation of less activecrystalline phases.This work deals withthe synthesis and characterization of one-dimensional(1D) imidazole-containing etidronates, [M By combining(poly)phosphonic acid linkers13 and a variety of syntheticmethodologies, including mechanochemical15 and high-throughput17 procedures, numerous metal phosphonateshave been prepared for targeted applications, as they are amenableto establish appropriate structure/property relationships, assistedby advanced characterization techniques, such as synchrotron radiation18 or electron diffraction tomography.19Metalphosphonates represent a rich area of coordination and materialschemistry that has seen impressive growth in the last few decades20 The incorporation of the SAL can serve several purposes: (a) alterationsin framework dimensionality, (b) creation of hydrophobic domains,(c) changes in the coordination mode of the metal centers, (d) framework“decoration”, and, in certain cases, (d) rupture ofthe “coordination polymer” architecture. Apart fromthe common N-heterocyclic ligands, such as 2,2′-bpy, 1,10-phen,and 4,4′-bpy , otherN-heterocycles have drawn attention because these may participatein proton transfer pathways, to increase the proton concentrationin the material. The basic character of the N atom can act as a strongproton acceptor, thus forming protonic charge carriers.22Recently, novel metal phosphonate compoundscontaining a secondaryauxiliary ligand (SAL) have been synthesized.25 Due to the uniform dispersion of the metal sitesin metal phosphonates, only a one-step pyrolytic treatment is neededfor the construction of TMPs, thus making them very attractive precursorsof nonprecious metal catalysts (NPMCs) with interesting electrocatalyticproperties. Moreover, heteroatom-doped carbon/TMP composites may alsobe generated by pyrolysis of phosphonate compounds with appropriateorganic linkers. These heteroatoms, such as N, P, and S, could finelytune the electronic structure correspondingly.26 Taking this into consideration, the incorporation of aspecific SAL on metal phosphonates can induce a modulation on theelectronic density of the resulting pyrolyzed derivative and, therefore,improve the electrocatalytic performance.26On the other hand, phosphorus-based materials, specificallytransitionmetal phosphides (TMPs), have received considerable attention as promisingelectrocatalysts owing to their potential application in energy conversionand storage systems.Based on the aforementioned arguments, herein, we investigatetheformation of metal phosphonates as precursors of TMPs with the ligandhydroxyethylidene-1,1-diphosphonic acid  andimidazole as SAL, as part of a direct N-doping carbon strategy. Arepresentative illustration of the synthetic strategy is shown in 27 Τhe N-doping strategy influences the carbon shell on thesematerials.28 However, N-doped TMPs generallyexhibit poor electrochemical activity toward the oxygen evolutionreaction (OER) and oxygen reduction reaction (ORR) due to the lackof active sites,29 presumably in the carbonshell, which precludes the access to multifunctional TMP electrocatalysts.Recently, electrocatalysts derived from ETID have been investigatedtoward the OER, ORR, and HER. For example, Maiti et al. obtained cobaltphosphosulfide nanoparticles dispersed into N,S,P-doped graphene withhigh ORR activity.30 Furthermore, the pyrolyticreduction of a Co-ETID at 750 °C yielded a mixture of cobaltphosphides (Co2P and CoP) with remarkable overpotentialsin acidic, neutral, and basic media for the HER.31 On the other hand, bimetallic phosphonates may offer additionaladvantages in the electrochemical reactions due to the synergisticeffect of the metals used.32 For instance,Fe/Ni phosphides dispersed into N,P-doped carbon nanosheets were obtainedby one-step pyrolysis under an inert atmosphere and exhibited exceptionalresults toward the OER, ORR, and HER.33To date, only a few TMPs derived from metalphosphonates have beenreported in the literature, and most of those have been selectivelystudied for the hydrogen evolution reaction (HER) due to their highelectroactivity toward this reaction.2(ETID)(Im)3]·3H2O}n (CoLIm-3), {[Co2(ETID)(Im)3]·H2O}n (CoLIm-1), {[Co2(ETID)(Im)3]}n (CoLIm-0), {[Ni2(ETID)(Im)3]·3H2O}n (NiLIm-3), and {[Zn2(ETID)2(H2O)2](Im)2}n (ZnLIm-2) (L = ETID). Their syntheses, crystal structures,and proton conductivities are studied and discussed in detail. Moreover,the electrocatalytic behavior of diverse TMPs, obtained from Co2+, Ni2+, and their bimetallic phosphonate derivatives,are evaluated toward the OER, ORR, and HER.In this paper, we report the syntheses, structures, and electrochemicalbehavior of several three-component metal–ETID–Im  compounds. In thesesolids, the imidazole plays the role of either a terminal ligand ora counterion (protonated Im) to an anionic metal phosphonate framework.Five compounds with novel structural features are presented: {, C10–OH [1.40(1)Å], P–OH [2.68(2) Å], C11–OH [2.40(2) Å];and imidazole ring/C–C [1.40(1) Å], C–N [1.40(1)Å], Cring···Cring [2.20(2)Å], Cring···Nring [2.20(2)Å]. Furthermore, the structural disorder in the atomic positionof the metal ions and imidazole rings of MLIm-0  solids was modeled. No attempts to locate the H atoms werecarried out due to the limited quality of the PXRD data. Final Rietveldplots are given in Figure S1. All crystallographicdata are given in Powder samples were analyzed bylaboratory powder X-ray diffraction (PXRD) on a PANalytical EMPYREANdiffractometer in Bragg–Brentano or transmission configurationusing Cu KαNiLIm-3, CoLIm-1, and ZnLIm-2 were obtained on samples loaded on anAnton Paar HTK1200N Camera, under an inert atmosphere, on a PANalyticalX’Pert Pro automated diffractometer with Cu Kα1 and the X’Celerator detector. Data were collected from roomtemperature up to 900 °C with a heating rate of 5 °C·min–1 and a delay time of 5 min to ensure thermal stabilization.Thermodiffractometric data for –1) from 25 to 80 °C and 95% relative humidity(RH) to ensure equilibrium with the atmosphere, and EIS data wererecorded on cooling using a stabilization time of 3–5 h ateach measurement temperature. Water condensation on samples was avoidedby first reducing the relative humidity before decreasing the temperature.The measurements were automatically controlled with winDETA41 or NOVA 2.1.542 software.For all compounds, the total pellet resistance (RT) was obtained from the analysis of the spectra usingthe ZView43 program.Proton conductivity ofthe samples was determined by electrochemical impedance spectroscopy(EIS) on cylindrical pellets (diameter of ∼5 mm and thicknessof ∼1.02–1.20 mm) obtained by pressing ∼38 mgof sample at 250 MPa for 1 min. The pellets were coated on both faceswith a silver conductive paste (Sigma-Aldrich) and placed inside atemperature- and humidity-controlled chamber (Espec SH-222). EIS datawere acquired with an HP4284A impedance analyzer or AUTOLAB PGSTAT302Nequipped with a frequency response analyzer (FRA 32M) module overthe frequency range from 20 Hz to 1 MHz with an AC applied voltagebetween 0.35 and 0.5 V. The pellets were first preheated (0.2 °C·min2) as a working electrode and a platinumrod and Ag/AgCl (3 M KCl) as counter and reference electrodes, respectively.The working electrode was prepared by mixing 4 mg of catalysts and1.3 mg of carbon black  into470 μL of ethanol/H2O (v/v = 1/1) with 30 μLof 5 wt % Nafion solutions, followed by sonication for 30 min to obtaina homogeneous ink. Then, 5 μL of the catalyst ink was drop-coatedon the GCE and dried at room temperature to achieve a catalyst loadingof 0.20 mg·cm–2. For comparison purposes, commercialRuO2  and 20 wt % Pt/C  were prepared and characterized under identicalconditions. For the stability tests, a hydrophobic carbon paper  with a catalyst loading of1 mg·cm–2 was employed as the working electrode.The electrochemicalactivity of the catalysts was performed using a BioLogic VSP-128 potentiostat/galvanostat/impedanceanalyzer. The electrochemical tests were measured in a typical three-electrodeconfiguration using a 5 mm diameter glassy carbon rotating disc electrode = E(Ag/AgCl) + 0.205 + 0.059 pH. Polarization curves were iR compensated (95%) with respect to the ohmic resistance of the solution.Before each measurement, the corresponding electrolyte was previouslybubbled for 30 min with N2 gas for the OER and HER, andO2 in the case of the ORR, and maintained over the wholemeasurement to ensure the gas saturation in the solution. The optimalflow was found to be 20 mL·min–1 to obtainoptimal OER/ORR/HER electrocatalytic performance (Table S1).All current densities were normalized to the geometrical surfacearea of the electrodes. The measured potentials vs Ag/AgCl were convertedand referenced to the reversible hydrogen electrode (RHE) accordingto the Nernst equation: –1, respectively, in the potential window of 1.0–1.7 V in a1.0 M KOH electrolyte with the RDE rotate at 1600 rpm. The overpotentialwas determined from the equation η = E(RHE)– 1.23. HER measurements were conducted in a 0.5 M H2SO4 electrolyte with RDE rotation at 2000 rpm. CV experimentswere performed at a scan rate of 20 mV·s–1 inthe potential window of −0.6 to 0 V, while LSV curves weremeasured with a scan rate of 10 mV·s–1 in thepotential range from −0.7 to 0 V.For the OER, cyclic voltammograms (CVs) andlinear sweep voltammograms(LSVs) were measured at a scan rate of 20 and 10 mV·sb, and j correspond tothe overpotential, Tafel slope, and thecurrent density, respectively. EIS measurements were carried out from100 kHz to 0.1 Hz with an amplitude of 5 mV at a determined potential.The stability measurements were carried out by a continuous cyclingtest and a chronoamperometric (CA) response at their correspondingoverpotential value.The Tafel slope wascalculated according to the equation–1 in the potential window from 0.2 to 1.2 V. LSVs wereconducted at different rotation speeds (400–2025 rpm) undera scan rate of 10 mV·s–1 in the same potentialrange. The electron transfer number (n) for the ORRwas determined according to the Koutecky–Levich (K–L)equationJ represents the currentdensity (A·cm–2), JL and Jk are the diffusion-limiting andkinetic current density (A·cm–2), ω isthe electrode rotation rate (rad·s–1), F is the Faraday constant (96 485 C·mol–1), CO is the bulk concentrationof O2 in 0.1 M KOH (1.2 × 10–6 mol·cm–3), DO is the diffusioncoefficient of O2 in 0.1 M KOH (1.9 × 10–5 cm2·s–1), and v is the kinematic viscosity of the electrolyte (1.09 × 10–2 cm2·s–1). Notethat the constant 0.62 is employed with the rotation speed expressedin rad·s–1. The stability measurements werecarried out by CA at 0.5 V, and the response was also evaluated underthe addition of methanol.ORR tests were conducted in a 0.1 M KOHelectrolyte with the RDErotating at 1600 rpm. CVs were measured at a scan rate of 20 mV·sCoLIm-0@800 as symmetrical electrodematerials and as an anodic electrode combined with a Pt/C cathodicelectrode. Nafion 117 was used as a proton exchange membrane. Thecatalyst inks were drop-dried on carbon paper (1 × 1 cm2) with a loading mass of 1.0 mg·cm–2.Overall, water splittingwas carriedout on an AUTOLAB PGSTAT302N electrochemical workstation in 1.0 MKOH. A two-electrode homemade water-splitting electrochemical cellwas assembled using 2 gas was calculated from Faraday’slawn is the number ofmoles, I is the current, t is thetime, z is the transfer of electrons (for O2z = 4), and F is the Faraday constant.The theoretical amount of O2 gas was evaluated fromthe water displacement method by Dalton’s lawV2O isthe volume of O2 produced, T is the temperature,and R is the gas constant.Finally, the Faradaic efficiency was obtained using the followingrelationThe experimental amount of OKa values of ETID,45 when conducting the syntheses at ∼pH 7.0, the phosphonicgroups of the ETID ligand are completely deprotonated, and ETID carriesa “4–” charge, requiring two coordinated divalentmetal ions per ligand to obtain a neutral framework. Thus, the useof Co2+ and Ni2+ resulted in the formation of MLIm-3  and CoLIm-1, with differentwater contents. In addition, an anhydrous phase of Co2+ (CoLIm-0) could be directly prepared by refluxing thereactants without stirring. In the case of ZnLIm-2, synthesizedat pH = 2.5, the phosphonate ligand is tris-deprotonated and the imidazoleis present as a positively charged imidazolinium cation. The purityof the bulk polycrystalline samples was confirmed by PXRD and elementalanalysis .All syntheses were carriedout in aqueoussolutions, and the products were isolated as crystalline precipitateswithout the addition of any secondary precipitating solvent. Accordingto the reported pTable S3 and Figure S3), indicate thatthese compositions correspond to solid solutions, (CoxNix2–n)LIm- . Depending on the Co2+/Ni2+ molar ratio, the resulting bimetallic solids crystallize with variablewater contents and, thus, different crystal structures. Solids with x ≤ 1.2 are isostructural with MLIm-3 ; while for x > 1.2, the compounddisplaysthe crystal structure of CoLIm-1.Three monophasic bimetallic compositions were also isolated and laterused to evaluate the electrochemical properties of their pyrolyzedderivatives. Their crystal structures, as determined by Le Bail fit polymeric structure. Eachasymmetric unit contains two kinds of Co2+ ions, one coordinatedETID molecule, three Co-coordinated imidazole molecules, two coordinatedwater molecules, and one lattice water molecule. Both metal ions arefound in octahedral coordination geometry but surrounded by differentligand atoms.The Co49 It is augmented by the chelating effect induced by the two adjacentphosphonate groups. Two neighboring Co1 centers are bridged by twophosphonate moieties (P1), which utilize the O1 and O3 oxygens, thuscreating two Co–O–P–O–Co bridges. Eachbridge is unsymmetrical as the Co1–O1 (2.064 Å) and Co1–O3(2.202 Å) are not equal.Specifically, the Co1ion is coordinated with two nitrogen atoms from imidazole, three oxygen atoms  from thephosphonic groups  of the ETID ligand, and one oxygen atom(O4) from the hydroxyl group of the ETID ligand. Metal coordinationof the −OH of the ETID ligand is uncommon but not unprecedented.2O2 rhomb is asymmetric, with Co–O bond lengths of 2.152 and2.446 Å.The Co2 ion is coordinated withone nitrogen atom (N5) from theimidazole, two oxygen atoms  from the phosphonic groups  of the ETID ligand, one oxygen atom (O9) from a water molecule,and two oxygen atoms (O8) from other water molecules, which bridgeanother Co2 atom. Two neighboring Co2 centers are bridged symmetricallyby two water molecules (O8), but the formed CoCoLIm-1, presents the distinctive structural features shownin 2O2 are still maintained but now formedby penta-coordinated edge-sharing polyhedra of Co2, which are interconnectedthrough the octahedra of Co1. In this case, the oxygens of Co2O2 moieties belong to the phosphonate group P1.The monohydrate phase, CoLIm-3. However, the Co2 dimeric units haveundergone the following structural changes: (a) the Co centers aretrigonal bipyramidal, (b) no bridging waters are present, (c) oneterminal water is present, and (d) the trigonal bipyramidal unitsare now edge-sharing .The coordination environment of Co1 remains the same as in thecompound NiLIm-0, obtained bydehydration of the trihydrated phase at 220 °C , is isostructural to the “as-synthesized” CoLIm-0. The crystal structure of the anhydrous NiLIm-0 was solved from PXRD data. Here, the chains are formed by alternatingsquare pyramidal M2+ polyhedral and M2+ octahedra,linked to each other via phosphonate bridging oxygens . Each asymmetricunit contains two types of Zn2+ centers , two ETID ligands, two coordinated water molecules,and two protonated imidazole cations as counter ions. Zn1 is foundin a tetrahedral coordination environment, and it is bound by fouroxygens  from two different ETID ligands . The metal center Zn2 is found in an octahedral coordinationenvironment. Specifically, Zn2 is coordinated by four phosphonateoxygens  from two different ETID ligands and two oxygen atoms  from the bound water molecules.The structure of 2+ cations. Their combination createsan anionic coordination polymer, with the negative charge of eachunit compensated by the two imidazolium cations.The ETID ligand is tris-deprotonated. This means that one phosphonicacid group (P1) is singly deprotonated, whereas the other (P2) isdoubly deprotonated. The “6–” total negativecharge from the two ETID ligands is partially offset by the “4+”charge of the two Zn2+ coordinationpolymer containing ETID and imidazolium cations, Zn2(ETID)(Im)2, has been recently reported in the literature.50 This three-dimensional (3D) compound only containstetrahedrally coordinated Zn2+ and terminally bound imidazole.A similar,albeit structurally different, Zn2+, Ni2+, and Zn2+ derivativesare shown in Figure S6. The TG curves for MLIm-3  display a slightly different thermal behavior,with the first weight-loss step of CoLIm-3 starting ata lower temperature (100 °C). This weight loss corresponds tothe loss of all water molecules . The second weight-loss step (∼28 wt %) starts at ∼220°C, and it is due to the decomposition of the ETID and imidazolemolecules. The monohydrate phase CoLIm-1 presents a similarweight-loss profile for dehydration/decomposition, with the firstweight loss of 3.9 wt % , in accordance with the lossof a water molecule. Compound ZnLIm-2 shows a uniquebehavior, displaying three weight-loss processes. The first occursin the 25–200 °C range, with 5.5 wt % weight loss . It is ascribed to the removal of two water molecules producinga crystalline anhydrous phase; however, its PXRD pattern could notbe indexed . The subsequentweight-loss steps, ∼20.5 wt % between 250 and 600 °C,may be attributed to the decomposition of the organic components.Thermal decomposition (in N2) of NiLIm-3 and CoLIm-n leads to Ni2P2O7 (PDF01-074-1604) and a mixture of Co2P2O7 (PDF 01-070-1491) and Co2P (PDF 01-089-3030), respectively.The thermogravimetric analyses forCo51 However, the synthesized solids present low proton conductivities(S8), except for ZnLIm-2 (∼6× 10–4 S·cm–1 at 80°C and 95% RH). This can be related to the low pH used in thesynthesis, facilitating the protonation of the phosphonate groups.The presence of phosphonic acid groups (−PO3H– and/or −PO3H2) is a keyfactor for the creation of more extended H-bonding networks (Tables S5–S7) in metal phosphonate chemistryand, therefore, higher proton mobility. The conductivity values arecomparable to other metal etidronates53 and the activationenergy values (Ea), which range from 0.55to 0.59 eV, are indicative of a vehicle proton transport mechanism.54As reported for other compounds,the presence of imidazole molecules may assist in the creation ofextended and well-defined proton-conducting pathways via H-bonding.tivities3 and S8,2 thermal reduction ofthe corresponding metal phosphonate precursors in the temperaturerange of 550–850 °C . In the case of NiLIm-3, the reduction at 550 and600 °C led to a mixture of crystalline and amorphous compounds,where two nickel phosphides were identified: Ni3P (PDF01-074-1384) and Ni12P5 (PDF 01-074-1381). Athigher temperatures, from 700 to 850 °C, only the hexagonal phaseNi2P (PDF 01-089-2742) was observed. Comparatively, higherpyrolytic temperatures were needed to obtain the cobalt phosphides;otherwise, phases with lower electrocatalytic activity, such as M2P2O7, would be present. For CoLIm-3, a reduction at 825 °C is required to achieve a complete conversionto cobalt phosphides, namely, CoP (PDF 98-004-3249) and orthorhombicCo2P (PDF 01-089-3030). In addition, pyrolysis of CoLIm-1 and CoLIm-0 led to a mixture of CoP andCo2P, albeit at 800 °C. Pyrolysis of the bimetallicsolids resulted in phosphide mixtures, the hexagonal phase NiCoP (PDF01-071-2336), and/or single monophosphide phases, depending on theCo2+/Ni2+ molar ratios and pyrolysis conditions.For non-N-doped carbon control materials, CoL@800 and Co1.4Ni0.6L@700, the crystalline fractions were identifiedto be a mixture of phases Co2P (47 wt %) and CoP (53 wt%), and NiCoP (∼96 wt %), respectively.The preparation ofTMPs was conducted by one-step HTable S8) indicatesthat the amount of carbonaceous material decreases considerably attemperatures higher than 800 °C. The Raman spectra  in the D and G band region showedtwo nitid signals at 1352 and 1583 cm–1 with practicallyidentical D/G intensity ratios, suggesting that the extent of defectsin the carbonaceous residue55 of thesematerials is quite similar. SEM images showed that thin rectangularmicrometric particles, characteristic of the precursor metal etidronates,transform gradually into quasi-spherical nanometric particles featuringmetal phosphide phases . As expected, increasing the temperature of the reduction processgave rise to the size increase of the metal phosphide particles , which is detrimental to the electrochemicalproperties. Thus, we focused on materials pyrolyzed in the range of700–800 °C. In addition, TEM images show heterogeneityin size, with the smaller nanosized metal phosphide particles beingembedded into a graphitic carbon matrix, CoLIm-0@800, determined by the HRTEM image in the [212̅]zone axis . Element mapping images, S15, confirma uniform distribution of C and N in the graphitic substrate, whileCo/Ni and P are located exclusively in the metal phosphide nanoparticles.In addition, the homogeneous distribution of the metal ions indicatesthe formation of solid solutions for the bimetallic compositions . This metal ion distributiondiffers from that reported elsewhere,29 in which doping metal ions are dispersed throughout both the metalphosphide nanoparticles and the graphitic substrate.Elemental analysisof the pyrolyzed samples  and phosphate (782.2 and 784.6 eV) environments, respectively.31 Spectra at different etching levels revealed more clearly the phosphidecore in agreement with the X-ray diffraction data . In the P 2p spectrum, the peaks at 129.7 and 130.7eV are assigned to Co–P bonds, while those shown at 134.3 eVare attributed to P–O bonds.56 For the caseof NiLIm-3@700, similar metal–P and P–Osignals were observed in the Ni 2p3/2 and P 2p regions,respectively .57 As shown elsewhere, the N 1s spectra  can be fitted into three N contributions: pyridinic-N(398.7 eV), pyrrolic-N (400.5 eV), and graphitic-N (402.9 eV),58 suggesting the formation of a N-doped carbonmatrix surrounding the metal phosphide core, XPS spectra for ion data S9. In th2 (S19). Ascan be seen, the anhydrous phase CoLIm-0 pyrolyzed at800 °C, CoLIm-0@800, showed better performance (η10 = 298 mV) than the hydrated precursor phases  led to the mostactive electrocatalyst. The overpotential displayed by CoLIm-0 is close to other reported metal phosphides (Table S9) and slightly lower than that determined for commercialRuO2 (308 mV). Comparatively, CoL@800 displayedlower performance, which may be attributed to a lower concentrationof the CoP phase , probablydue to the smaller particles of Ni2P as well as the absenceof other low activity nickel phosphides upon pyrolyzing at 700 °C.At higher pyrolysis temperatures, particle sintering gives rise tolower electrocatalytic activity .In the case of nickelphosphides, Co0.74Ni1.26LIm@700, Co1.2Ni0.8LIm@700, and Co1.64Ni0.36LIm@800, displayed higher η10 values (Table S10) compared to the monometallicphases.This trend was also observed for the control electrocatalysts. Theabsence of a Co2+/Ni2+ synergistic effect, asreported elsewhere,59 may be due to theformation of the less catalytically active phase, NiCoP (∼60–95%),together with CoP (∼5–40%).The bimetallic derivatives, CoLIm-0@800, CoL@800, and CoLIm-3@825 were 64.9, 60.1, and 72.3 mV·dec–1, respectively.The two first are around the theoreticalvalue of 60 mV·dec–1, in which the rate-determiningstep is the chemical reaction after one-electron transfer reaction59 and are indicative of favorable reaction kineticstoward the OER upon comparing with the reference catalyst RuO2. The EIS data elements, where R is a resistance in parallel with a constant phaseelement CPE, are needed to describe the electrode response adequately.The high-frequency process is usually related to surface porosityand exhibits a similar resistance value for the different catalysts(RHF = 3 Ω).60 This contribution appears at a relaxation frequency of∼100 Hz and has a capacitance value of 0.3 mF. The low-frequencysemicircle, assigned to charge transfer, is the main contributionto the electrode polarization at a relaxation frequency of ∼10Hz and has a capacitance similar to that of the high-frequency process.It is also worth noting that the charge-transfer resistance (Rct) of CoLIm-0@800 (15.9 Ω)is comparable to that of commercial RuO2 (18.1 Ω)and significantly lower than that of CoL@800 (32.2 Ω).In addition, CoLIm-0@800 exhibits an improved durabilitycompared to that of RuO2 for 15 h, with a current decayof only 7.6% for CoLIm-0@800 against 23.8% for RuO2  and is indicative ofa high electrocatalytic ORR capability.As observedfor selectedelectrocatalysts, the CV curves 6a show aS20 and CoLIm-0@800 achieves the most positive onset potential (Eonset) and half-wave potential (E1/2) with values of 0.86 and 0.80 V, respectively, close to those obtainedfor the Pt/C catalyst . Newly, theanhydrous precursor, CoLIm-0, led to better performancethan that of the trihydrate (CoLIm-3) upon pyrolyzingat 800 °C. The catalysts derived from imidazole-containing precursorsexhibited a significant enhancement of the ORR activity with respectto the control precursor, which can be related to a beneficial effectof a N-doped graphitic matrix formed when imidazole was used as SAL.Furthermore, N-doped carbon shell cobalt electrocatalysts displayedlimiting current density (jlim) values,close to the value of the Pt/C benchmark electrocatalyst (∼−5mA·cm–2).For this reaction, the catalyst performance trend was like thatobserved for the OER S20 and 2. Thus, NiLIm-3@750, which showed slightly better propertiesthan NiLIm-3@700, attributed to a higher reduction degreewith an increase of the temperature. By increasing the cobalt contentin the Ni2P framework, an enhancement of the ORR activitywas observed up to a Co2+/Ni2+ molar ratio of1.5 (Co1.2Ni0.8LIm-3@750). Higher cobalt molarratios in the bimetallic compounds gave rise to the formation of amixture of phases NiCoP (60 wt %) and CoP (40 wt %) in the solid Co1.64Ni0.36LIm-2@800, which resulted in a considerable improvementof the ORR activity, very close to that of the end member CoLIm-1@800, which may be ascribable to the presence of the highly active phaseCoP (Table S10).Regarding the nickel derivatives,the highest activity was achievedfor n) per oxygen moleculewas determined by applying the Koutecky–Levich (K–L)equation at different potentials , the most activeelectrocatalyst was CoLIm-0@800 (η10 = 156 mV) followed by CoLIm-1@800 (η10 = 162 mV), CoL@800 (η10 = 170 mV), CoLIm-3@825 (η10 = 185 mV) and NiLIm-3@700 (η10 = 188 mV), values that are far away from thatobserved for Pt/C (34 mV). Although the reactions were conducted inacidic conditions, the repetitive trend observed points to the conclusionthat having a high concentration of the CoP phase and low sinteringof the particles are the most beneficial features for high performance.The overpotential values for CoLIm-0@800 and NiLIm-3@700 fall within the range obtained with monophasic cobalt (CoP) andnickel (Ni2P) phosphides, respectively, embedded into N,P-codopedcarbon backbones (Table S9).25TheHER activity trendof the electrocatalysts is similar to that previously observed forthe OER 2.Accord62 The overpotentialsand Tafel slopes found are similar to those previously reported formetal phosphide electrodes (Table S9).The Tafel slope values, CoLIm-0@800 (Rct = 22.7 Ω),determined from the low-frequency semicircle of the Nyquist plots. CoLIm-0@800 also exhibited remarkable durability  varied between 77:23and 83:17, indicating small deviations from the initial electrocatalystcomposition  revealedno significant changes in morphology and particle size after durabilitytests. XPS showed only marginal changes in the surface compositionof CoLIm-0@800 after the HER test , while postreaction materials exposed to alkalineconditions (OER and ORR) experienced changes mainly in the Co 2p3/2 region due to the appearance of a new signal at 780.8 eVascribable to CoO(OH).63 Interestingly,the signals in the P 2p region remain practically unchanged, pointingto the fact that the catalyst integrity is maintained after the electrochemicaltests.To evaluate the stabilityof position 2. Also, CoLIm-0@800 wasselected as the working electrode owing to its remarkable electrocatalyticproperties toward the OER, ORR, and HER, comparable to other high-performancecobalt materials.66 Its bifunctional activity for electrochemical oxygencycle  in alkaline electrolytes ishighlighted, as the potential difference (ΔE = Ej=10 – E1/2)29 between OERand ORR calculated for this electrode material, 0.74 V, is comparableto the values for the Pt/C (ΔE = 0.83 V) andthe RuO2 (ΔE = 1.04 V) benchmarkelectrocatalysts.To evaluate the electrochemicalcapability of the prepared catalysts, CoLIm-0@800, experiments were carried out in an assembledalkaline water electrolyzer to test its capability for overall watersplitting (S24). Although CoLIm-0@800 exhibits a modest behavior as a bifunctional catalyst, the cellvoltage of 1.77 V at a current density of 10 mA·cm–2 is comparable to those reported for other nanosized cobalt phosphides.68 However, the system CoLIm-0@800||20 wt % Pt/C needsa cell voltage of 1.61 V, which is slightly higher than that of theRuO2||20 wt % Pt/C (1.56 V) and exhibits considerable durability,with a current density loss of 11.3% for over 24 h. The generatedamounts of oxygen agree well with the theoretical value  electrocatalystshas beenprepared and tested as electrode materials for the OER, ORR, and HER.The electrochemical behavior was dependent on the metal phosphidecomposition, pyrolysis temperature, and particle size. The coordinationenvironment of the metal ions in metal phosphonate precursors, whichin turn is conditioned by the hydration degree, is a key factor indetermining the TMP phase composition. We have also demonstrated thatinserting a N-containing SAL, such as imidazole, is a convenient wayto enhance the electrocatalytic properties. In addition, the presenceof protonated imidazole in [Zn2(ETID)2(H2O)2](Im)2 gives rise to a moderate protonconductivity with a proton transfer vehicle mechanism. For cobaltelectrocatalysts, a CoP/Co2P molar ratio of ∼4 (CoLIm-0@800) was found as the most active composition withcharacteristic η10 values of 298 mV (OER) and 156mV (HER), and relatively high ORR performance (Eonset = 0.86 V and E1/2 = 0.80V), as well as favorable kinetics and adequate stability. Furthermore,this trifunctional electrocatalyst exhibited a remarkable integratedcapability as an anode for overall water splitting (cell voltage =1.61 V). Although less active than the cobalt derivatives, the obtainednickel phosphide-based electrocatalysts displayed the best performancewhen the crystalline phase Ni2P was present. For theseelectrocatalyst systems, bimetallic Co2+/Ni2+ solid solutions could be prepared. Increasing the Co2+ content in the structure of Ni2P, up to a Co2+/Ni2+ molar ratio of 1.5, did not significantly improvethe electrocatalytic behavior. However, a higher Co2+ enrichmentin the bimetallic composition led to a mixture of CoNiP (60 wt %)and CoP (40 wt %) with a noticeable improvement of electrocatalyticactivity attributed to the high proportion of the CoP phase.A family of N-doped carbon/TMP (Co


A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.Human α Proteases constitute the largest enzyme family in humans with 641 currently identified members , 3, 4. T2-macroglobulin (A2M) is a 720 kDa homotetrameric protein that is abundant in plasma and is archetypical for the alpha-macroglobulin (αM) protease inhibitor family, whose human members also include A2M-like protein 1 (A2ML1) and pregnancy zone protein (PZP). A2M has a unique mechanism of inhibition where it collapses around proteases and irreversibly sequesters them from protein substrates  and also has an MMP cleavage site (PEGL) (A). In vitro, A2M has been shown to inhibit proteases involved in coagulation and fibrinolysis, e.g., plasma kallikrein, thrombin, FXa, and plasmin  proteases , and HIV protease 1, and delivers these potential antigens to LRP1-expressing antigen-presenting cells such as macrophages and dendritic cells  A. In vit plasmin , 17, 18, chymase , 20, androteases , 15, 21.ic cells , 24, 25.ic cells , 27, 28.B and C) ) (D). The bait regions occupy the interior space of native A2M and may interact with each other, as suggested by the formation of intersubunit disulfides upon the introduction of cysteine residues into the bait region  . CrystalB and C) , 36, 37,ne (MA)) , 39, andne (MA)) . We havene (MA)) . This mone (MA)) D. The bat region .e.g., lysine side chain ε-amino groups on the surface of proteases) takes place during or after its conformational change .The bait region is both ground zero for A2M’s proteolytically induced conformational change and the major determiner of which proteases are inhibited by A2M. However, the low conservation of the bait region sequence across species  suggestsFig. 2tabula rasa bait region could be incorporated into recombinant A2M, resulting in a tabula rasa A2M protein that was predominantly tetrameric and in its native conformation, with an intact thiol ester apparent from the formation of characteristic heat-induced autolysis products in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (B and C). Upon aminolysis of its thiol ester with methylamine, tabula rasa A2M underwent a conformational collapse indistinguishable from that of wild-type A2M, as determined by pore-limited native PAGE; however, tabula rasa A2M was not cleaved in its bait region by either trypsin or LysC and remained in its native conformation despite proteolysis by these proteases outside of its bait region (B and C). While the majority of tabula rasa A2M assembled into the normal homotetramer, dimeric tabula rasa A2M that had failed to tetramerize was slightly more abundant than wild-type A2M; dimers were removed during purification prior to all characterization . Before using these proteins in quantitative protease inhibition assays, nonnative A2M was depleted using LRP1-conjugated resin (The resulting DS-PAGE) , B and Ct region , B and Crization . Similartype A2M B. Beforeed resin .A–C); as the wild-type bait region and the initial tabula rasa bait region contain no lysine residues, LysC cleaved these A2Ms in locations other than the bait region without inducing a conformational change, as has been previously reported (C). Altogether, these results showed that while the wild-type bait region may make minor contributions toward A2M’s tetrameric assembly and the assumption of its native conformation, no part of its sequence is structurally or functionally essential. The majority of tabula rasa A2M is tetrameric and in a native conformation and can undergo a methylamine- or proteolysis-induced conformational change (if protease substrate sites are incorporated into the bait region).The TR K704 bait region added a lysine residue at position 704 and was cleaved by trypsin and LysC, which caused A2M to undergo a conformational collapse , A–C; asreported . The proreported C. AltogeC). The bait region is spatially close to the TE domain when A2M is proteolytically activated (B–D), and we hypothesized that the nucleophilic ε-amine group of Lys704’s side chain might attack the thiol ester following proteolysis, conjugating the N- and C-terminal bait region cleavage fragments together into an aberrantly migrating ∼180 kDa product. This proposed product of Lys704 autoconjugation disappeared when 3-aminopropanenitrile (BAPN), a small nucleophile that reacts with the thiol ester after it is proteolytically exposed and out-competes conjugation to other nucleophiles (A). After cleavage of A2M TR K704 with Cy5-labeled trypsin, this band was not fluorescent and therefore does not contain trypsin (A). In fact, the product band became more intense upon cleaving A2M TR K704 with acetylated trypsin, which is not efficiently conjugated as it lacks primary amine groups (A). LC-MS/MS analysis of the pepsin-digested product band identified a cross-linked peptide containing an isopeptide cross-link between the thiol ester Gln975 and the bait region Lys704 (B). Furthermore, the band was not produced upon tryptic cleavage of tabula rasa A2M with a bait region arginine residue instead of a lysine residue (A and B). These results conclusively show that this band represents an autoconjugation product of the thiol ester and a bait region lysine residue.Bait region cleavage of A2M TR K704 by both trypsin and LysC produced an intense band migrating as ∼250 kDa in reducing SDS-PAGE that was not produced by trypsin-cleaved wild-type A2M C. The bactivated , B–D, aneophiles , was inceophiles A. After  trypsin A. In face groups A. LC-MS/n Lys704 B. Furthe residue , A and Btabula rasa-based A2Ms using a fluorescently labeled gelatin substrate. In addition to A2M TR K704, bait regions incorporating an Arg704, Lys710, or Arg710 residue were produced (A). These A2M proteins appeared similar to A2M TR K704 during their initial characterization by electrophoresis (A and B). In order to quantitatively determine the inhibitory capacity of the native tabula rasa A2Ms, nonnative A2M was depleted from these proteins using LRP1-conjugated resin before all inhibitory assays . However, all four tabula rasa A2M with basic residues incorporated into their bait regions showed a decreased inhibitory capacity relative to recombinant wild-type A2M or plasma-purified A2M. Tabula rasa A2M with an arginine residue at position 704 or 710 possessed approximately 80% and 60% of the wild-type inhibitory capacity, respectively, while a lysine residue at these positions conveyed approximately 30% and 50% inhibition (B).We proceeded to investigate the trypsin-inhibiting ability of produced A. These phoresis , A and By assays . A2M wit trypsin B. Howevehibition B.Figure C). These results show that trypsin inhibition by tabula rasa A2Ms is dependent on covalent protease conjugation, in contrast to wild-type A2M.To investigate the mechanistic explanation for these decreased inhibitory capacities, trypsin inhibition assays were repeated for plasma-purified A2M, A2M TR K710, and A2M TR R704 with and without BAPN, which out-competes covalent conjugation of other nucleophiles by A2M’s proteolytically activated thiol ester . The inhFig. 4tabula rasa A2Ms retained the ability to inhibit proteases using the model protease trypsin, we designed four bait regions incorporating substrate sequences for human MMP2 (A), a potential therapeutic target in several types of cancer including ovarian cancer and pancreatic adenocarcinoma . Despite the rapid formation of A2M-MMP2 complexes (A), A2M was never fully bait region cleaved even by a large excess of MMP2 (B), and A2M:MMP2 complexes migrated with an intermediate electrophoretic mobility (C). These results resemble A2M’s reaction with plasmin . The incorporation of any of the four MMP2 substrate sequences into the tabula rasa bait region conveyed cleavage by all tested MMPs, but they were differently cleaved by non-MMP proteases; for example, the C9 substrate was the only one containing an arginine residue and was the only A2M to be cleaved by plasmin (A and B). Notably, the S1 substrate was only cleaved by MMPs (A and B), and the A2M TR S1 protein was therefore selected for further optimization as an MMP inhibitor with improved specificity relative to wild-type A2M.We then tested whether the four MMP2 substrate bait regions were cleaved by nine additional human proteases  using reducing SDS-PAGE to assess bait region cleavage and the formation of high-MW conjugation products . All assula rasa B. The in plasmin , A and B by MMPs , A and Btabula rasa A2M proteins were expressed with an increased amount of nonnative A2M compared with wild-type A2M. While this was not a major issue during their characterization in this study, as native A2M could be purified using LRP1-based depletion prior to inhibitory assays, such nonnative A2M constitutes a decrease in yield and its depletion would be difficult to scale up. In order to resolve this issue, we tested two altered tabula rasa bait regions (A). The first, TRΔ7, was shortened by seven residues to a total length of 32 residues, to compensate for the tabula rasa bait region possibly being effectively longer than the wild-type bait region due to altered dynamics, interactions, or secondary structure. The second, TR QRT4, reintroduced the C-terminal quarter of the wild-type bait region; while the bait region sequence in generally is not conserved, there is a tendency toward acidic and hydrophobic residues in its C-terminal quarter preceding the α’NT region (B). TRΔ7 is the preferable sequence for bait region design as it does not reintroduce any protease cleavage sites, in contrast to TR QRT4 . While the TRΔ7 S1 I703 bait region conveyed inhibition that was equivalent to that of plasma-purified or wild-type recombinant A2M, the TRΔ7 S1 I710 bait region, which was identical apart from the cleavage site position, inhibited with approximately 40% efficiency (C). The two 39-residue long tabula rasa bait regions TR S1 and TR S1 QRT4, which both incorporate the S1 cleavage site at position 710, had approximately 70% of the wild-type inhibitory capacity (C). These results demonstrate that tabula rasa A2Ms are capable of inhibiting MMP2 with the same efficiency as wild-type A2M, but that the bait region length and cleavage site position can affect the efficiency of protease inhibition and requires optimization.MMP2 inhibition by A2M was assessed using the same fluorescent gelatin substrate used in the trypsin inhibition assays. While all ficiency C. While ficiency C. The twcapacity C. These tabula rasa bait region substitution and demonstrate an approach to developing new protease inhibitors, which selectively inhibit a target protease. Wild-type A2M is an efficient protease inhibitor with an extensive inhibitory repertoire, and it is well established that A2M can inhibit new proteases when an appropriate substrate sequence is added into the bait region  independent of covalent conjugation  in αM complement factors disrupts interactions between the anaphylactic, MG3, and MG8 domains, thus triggering the overall conformational change . Our reshttps://clinicaltrials.gov/ct2/show/NCT03656575)  , where rA pcDNA3.1(+) plasmid with the gene for wild-type A2M under control of a CMV promoter  was usedAnother pcDNA3.1(+) plasmid encoding human proMMP2 with two StrepII tags at the N-terminal end of its activation peptide was prepared by gene synthesis and cloning into pcDNA3.1(+), by Genscript.g and adding pH 7.4 HEPES to a final concentration of 50 mM.All recombinant A2Ms were expressed in HEK293 FreeStyle cells using a standard transient transfection protocol. Briefly, 25 kDa linear polyethyleneimine (Polysciences) and plasmid DNA were incubated for 10 min in antibiotic-free FreeStyle medium (Thermo Fisher Scientific) at a 4:1 w/w PEI:DNA ratio, then slowly dripped into a culture of cells at a density of 1 million cells per ml, to a final DNA concentration of 1 μg per ml culture. After 4 days, the supernatant was harvested by spinning down the cells at 15002+-loaded Chelating HiTrap column  and eluted with 50 mM EDTA, 150 mM NaCl, 100 mM sodium acetate, pH 7.4. The EDTA eluate was dialyzed against 20 mM HEPES at pH 7.4, then loaded onto a HiTrap Q column , and eluted by a gradient of 0 to 400 mM NaCl (with a constant 20 mM HEPES at pH 7.4). Fractions containing A2M were pooled, concentrated by ultrafiltration, and purified by size-exclusion chromatography on a Sephacryl S-300 HR , using a 20 mM HEPES, 150 mM NaCl, pH 7.4 running buffer . Endogenous human A2M was purified from plasma provided by a healthy volunteer using this same protocol; prior to the first chromatography step, 4% polyethylene glycol (PEG) was used to precipitate and remove contaminant plasma proteins, after which 16% PEG was used to precipitate and isolate A2M for further purification.Purification of recombinant A2M was performed using an established protocol , 59. Sup2 using a PD-10 column .N-terminally StrepII-tagged proMMP2 was expressed using the same transient transfection protocol as A2M. Supernatants were treated with streptavidin to remove free biotin. ProMMP2 was then purified by StrepTactin affinity chromatography (IBA Lifesciences), followed by size-exclusion chromatography on a Superdex 200 Increase . ProMMP2 was activated using 1 mM APMA by incubating for 15 min at 37 °C, followed by desalting into HBS with 10 mM CaClRecombinant expression, purification, and activation of MMP-1, -3, -8, and -13, as well as ADAMTS-4 lacking the C-terminal spacer domain and ADAMTS-5 lacking the C-terminal thrombospondin domain, were performed as previously described , 64, 65.2 to a 6:1 mol/mol ratio of MMP2:A2M, incubated for 15 min at 37 °C, and then inhibited using 20 mM EDTA. When cleaving A2M using other human proteases, incubation lasted 1 h at 37 °C in HBS with 10 mM CaCl2, and PMSF or EDTA was used to inhibit serine proteases and metalloproteases, respectively.To aminolyze A2M’s thiol ester, methylamine (pH 8) was added to 250 mM and incubated for at least 45 min at 37 °C. To assess the cleavage of A2M by trypsin and LysC, proteases were added to a 2.2:1 mol/mol ratio of protease:A2M and incubated for 5 min at 37 °C. The digestion was then inhibited using the serine protease inhibitor PMSF . To assess the cleavage of A2M by MMP2, MMP2 was added to A2M in HBS with 10 mM CaClThe suspected autoconjugation product observed upon proteolytic activation of A2M TR K704 mutant was investigated by digesting the autoconjugation product separated by reducing SDS-PAGE with pepsin and subsequent LC-MS/MS analysis. Gel bands were excised, shrunk with acetonitrile, and then swelled with 0.1% v/v acetic acid, pH 3. Shrinking and swelling were repeated twice to wash the gel bands. The gel bands were shrunk a final time, dried, and then swelled in 0.1% v/v acetic acid, pH 3 with pepsin added to a final 1:20 w/w ratio of pepsin:sample. Digestion with pepsin was carried out overnight at 37 °C. Peptides from the digested samples were then purified using pipette tips packed with POROS 50 R2 C18 resin (PerSeptive Biosystems).CGEQN peptide, precursors were selected for a second MS2 scan using electron transfer dissociation (ETD), which resulted in the flagging of relevant precursor peptides with ETD scans.Approximately 250 ng of peptide was analyzed by LC-MS/MS with an EASY-nLC 1200 (Thermo Fisher Scientific) and an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). A data-dependent acquisition method selected peptides for fragmentation by high-energy collision dissociation (HCD) and MS2; upon detection of the b2 and b3 fragment ions from the thiol-ester-covering LQMPYGQN and FTNSKIRKPKMCGGSGGSGGSGGSGGK peptides cross-linked by an isopeptide bond. Both cysteines were propionamidylated as they had been reduced prior to SDS-PAGE; there were no other amino acid modifications. Fragment ions in this precursor’s HCD MS2 spectrum were manually annotated with a mass tolerance of 10 ppm. Manual annotation identified ions resulting from fragmentation in both peptides, in addition to b- and y-type single-fragmented ions. The raw data file containing the annotated spectrum has been deposited to the ProteomeXchange Consortium via the PRIDE  was added to a final concentration of 0.1 mg/ml. The fluorescence (excitation at 485 nm and emission at 520 nm) of the unquenched digestion products of DQ gelatin after 2 min at 37 °C was measured in a FLUOstar Omega plate reader (BMG LABTECH). Where noted, 50 mM BAPN was included in the initial reaction buffer with trypsin and A2M. All reactions were performed in triplicates.The inhibition of trypsin by A2M was investigated using a fluorescently labeled gelatin substrate. In total, 2.1 pmol (11.9 nM) of active trypsin was reacted with 0 to 8.6 pmol (0–47.7 nM) of A2M in 50 mM HEPES, 100 mM NaCl, 5 mM CaClSimilar reactions were carried out to determine the inhibition of MMP2 by recombinant wild-type A2M and the mutants TR+ S1, TRd7 S1, TR S1, and TR d7 I703. The only difference in the experimental setup was that 1.4 pmol (7.5 nM) of MMP2 was reacted with 0 to 2.7 pmol (0–15 nM) of A2M, and fluorescence was measured after 10 min of incubating the gelatin with the A2M/MMP2 reactions.via the PRIDE (The raw data file containing the spectrum identifying A2M TR K704’s autoconjugation product peptide has been deposited to the ProteomeXchange Consortium he PRIDE  partner Full protein sequences for all recombinant A2M proteins are included in the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K+ current, IK1. The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of IK1 using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic IK1 was inhibited with a low concentration (50 µM) of Ba2+ ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic IK1 through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) IK1 with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD90) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD90 and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant IK1 to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations.The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the  TO (Kr and IKs) contribute to repolarization over AP plateau voltages  as well as extra-cardiac abnormalities  repolarization depends on the dynamic interplay between multiple ionic currents, including those carried by a number of key potassium channels . Thus, eTO , whilst voltages . The terent, IK1 . Early aotential . From wog2+ ions . The relrization . Functiosubunits . KCNJ2 intricles . The impmalities . Conversmalities . Moreoveillation  and the illation .KCNJ2 missense mutations thus far identified impair Kir2.1 current rectification, with the consequence that outward IK1 is augmented and thereby makes a greater contribution to AP repolarization  and asymmetric T waves  tall upright T waves and increased susceptibility to ventricular arrhythmias . The SQTrization . The firrization . Both fa T waves . The D17fication . The D17fication . Simulathythmias . The M30hythmias .KCNH2 mutation linked SQT1 variants of the SQTS  and of increased IK1 due to the effects of the E299V SQT3 mutation  IK1, using WT and mutant formulations for IK1 previously derived from experimental data and used for in silico cell and tissue models . First, basic solution containing 750 μM of CaCl2 was perfused for 2 min, then Ca2+-free basic perfusion solution was supplied for 5 min, followed by low Ca2+ (150 μM) containing basic solution that also contained collagenase  and protease  for 4–7 min. Cells were then released from left ventricle by mechanical dispersion. Isolated myocytes were stored in low calcium solution (150 μM) at room temperature. Aliquots of the cell suspension were transferred into a chamber mounted on an inverted microscope and left to settle for several minutes, before being exposed to normal Tyrode solution. Only cells with a clear rod-shape and striated appearance were chosen for recording. The mean cell capacitance of guinea-pig ventricular myocytes used in this study was 98.3 ± 12.5 pF (12 myocytes from 8 hearts).Adult male guinea-pigs (300–600 g) were killed in accordance with UK Home Office legislation. Left ventricular myocytes were then isolated by enzymatic and mechanical dispersion as described previously . Briefly4 cells/ml) on gelatin (0.1%) coated glass shards in 12-well plates. After incubation at 37°C in 5% CO2 for 3 h, the culture medium was added to the plates. The cells were kept in a 5% CO2 incubator at 37°C with culture medium changed twice a week. Whole-cell patch clamp was conducted in weeks 2–3 of cell culture. The mean cell capacitance of hiPSC-CMs in this study was 56.8 ± 4.8 pF (n = 14 cells).Cor.4U hiPSC-CMs were obtained from Axiogenesis . The cells were cultured according to the manufacturer’s instructions for manual patch clamp experiments (MPC Protocol V1.1). Briefly, cells were thawed and suspended in culture medium (Axiogenesis) and seeded . For guinea-pig myocyte experiments, glass-pipettes  were pulled with a horizontal micropipe puller  to give a resistance of 1–2 MΩ. For stem cell experiments, glass-pipettes were pulled with a vertical puller  and polished  to give a final resistance of 2–3 MΩ. The pipette dialysis solution contained (in mM): 110 KCl, 10 NaCl, 5 MgATP, 0.4 MgCl2, 10 HEPES, 5 Glucose. Pipette pH (and thereby bulk intracellular pH) was buffered to pH 7.2 with 10 mM HEPES . The theoretical potassium equilibrium potential (EK) for these external and pipette solutions was −90.85 mV at 37°C. Series resistance values  were compensated by >70%. Transmembrane currents and action potentials were recorded in the whole-cell mode using an Axopatch 1D amplifier (Molecular Devices) and a CV-4 1/100 head stage. Data were recorded via a Digidata 1200B interface and stored on the hard-disk of a Viglen computer. Data digitization rates were 10 kHz during all protocols and an appropriate bandwidth of 2 kHz was set on the amplifier. Data acquisition (Clampex 8.2) and analysis (Clampfit 10.7) were performed using pCLAMP (Molecular Devices), Excel 365, Origin (2018 b) and GraphPad Prism (8) Software.Aliquots of adult guinea-pig left ventricular myocytes or glass shards with hiPSC-CMs were placed on the glass bottom of the chamber mounted on the microscope and the cells were continuously superfused at 37 ± 1°C with a standard Tyrode’s solution containing (in mM): 140 NaCl, 4 KCl, 2 CaClDynamic clamp was carried out using a commercially available Cybercyte dynamic clamp system (Cybercyte V10) . The simK1 were based on the formulations of The model formulations for IWTWT-D172ND172NK1 is the maximal conductance of IK1; the values given above are exemplar starting values. In practice, the WT amplitude was scaled to produce an APD90 in Ba2+-conditions that matched that in control Tyrode’s solutions and the values for WT-D172N and D172N were then scaled accordingly (see next section). XK1∞ is the time-independent inward rectification factor that is a function of voltage; o dependence of the current.GK1 formulations to ventricular myocytes, an experiment was conducted in which the IK1 formulations to be used were applied in voltage clamp mode to a model cell . K1 for D172N-WT was set to be 1.72 the WT current, whilst that for D172N was set to be 2.24 that for the WT current , GraphPad PRISM 8.4.3 or GraphPad Instat 3.10. K1 with virtual IK1 via dynamic clamp, it was necessary to utilize an inhibitor of IK1. In early experiments we attempted to use the pentamidine analogue PA-6  ions was used to inhibit IK1. Native IK1 has been reported to be inhibited by Ba2+ with an IC50 of 4.7 µM  , but invelop cf making if 4.7 µM  and the   . Figure protocol .2+ ion application on the guinea-pig ventricular AP configuration and resting potential. For these experiments brief (2–10) ms suprathreshold depolarizing currents were applied to elicit APs . 2+. Control and +Ba2+ APs were closely superimposed early during repolarization, but they diverged later in the AP plateau phase and terminal repolarization. The Poincaré plot in 90 in control solution and in the presence of IK1 inhibition by Ba2+: variability appeared to be greater in the latter condition. 2+ by ∼ 6 mV . APD90 was significantly increased . BVR in APD was quantified as shown in 2+. To summarise: IK1 inhibition with Ba2+ ions depolarized resting potential, increased APD90 and AP triangulation and augmented BVR.ncreased  and the gulation ) was als2+-inhibited IK1 was replaced by WT virtual IK1, scaling the magnitude of IK1 to restore APD90. In 12 cells from 8 animals, APD90 in control Tyrode was 177.3 ± 8.7 ms; once endogenous IK1 had been inhibited by Ba2+ and replaced in dynamic clamp, APD90 was 183.9 ± 9.3 ms (p > 0.05 (p = 0.09) vs control Tyrode). The resting potential was also restored ). For these experiments, we also measured the peak outward IK1 inhibited by Ba2+ ions during a voltage ramp protocol  and compared this with the peak outward virtual IK1 supplied in dynamic clamp to restore APD90 . Although there was a trend for the latter to be greater, this was not statistically significant .For experiments investigating the effects of the D172N mutation, for baseline APs the BaK1. The brief depolarizing current pulses supplied to elicit APs were altered as required when the WT IK1 formulation was replaced with WT-D712N and D172N formulations. Both heterozygotic and homozygotic D172N IK1 formulations led to APD abbreviation, associated with increased IK1 amplitude during late repolarization. The mean charge (product of current amplitude and duration) supplied to elicit APs was 8.7 ± 1.2 pC, 13.3 ± 2.1 pC and 19.0 ± 3.7 pC for WT, WT-D172N and D172N formulations respectively . K1 during the AP repolarization phase, which closely resembled those seen with the model cell in K1 transient was seen during the AP that was coincident with the AP upstroke and that differed between WT and mutant conditions , this was not statistically significantly different from WT-D172N (p > 0.05). Switching from WT to WT-D172N IK1 led to a marked reduction in APD triangulation  with WT and mutant formulations; there was a modest but significant reduction in Vmax compared to WT with both heterozygotic and homozygotic D172N formulations. As the current stimuli used to elicit APs were set beyond threshold levels, it was not possible to ascertain whether or not the reduction in Vmax was accompanied by any significant change in AP threshold voltage. However, peak overshoot potential values were analysed and found to be: 58.2 ± 3.5 mV, 51.7 ± 2.4 mV and 54.0 ± 3.2 mV (n = 5) for WT, WT-D172N and D172N formulations respectively. Although there was a trend towards a reduction in overshoot amplitude with the mutant IK1 formulations, this did not reach statistical significance .The upper panel of nditions . This rarotocols  and is vobserved . Figure p > 0.05, one-way ANOVA test followed by Tukey’s multiple comparisons test; n = 5 cells from 4 hearts).K1 formulations in turn  APs shortened and APD90 was accordingly reduced. Switching from WT IK1 to either heterozygotic (WT-D172N) or homozygotic (D172N) mutant conditions led to a marked abbreviation of APD90 at all rates tested  with the 3 different IK1 on APs from adult guinea-pig ventricular myocytes, for comparative purposes a limited series of experiments were conducted at 1 Hz on human iPSC-CMs. COR-4U cardiomyocytes have only low KCNJ2 expression . In 14 cells APs with the WT IK1 formulation were elicited by brief depolarizing current injections . The peak outward WT IK1 supplied by dynamic clamp during repolarization was 164.4 ± 10.2 pA (3.0 ± 0.1 pA/pF). For each cell, WT-D172N and D172N formulations were then applied at the same fixed ratios as used in guinea-pig myocyte experiments. Changes in the magnitude of the stimulus required to elicit APs were not required when IK1 formulations were changed. K1 also shown (lower traces). For completeness, also superimposed are membrane potential recordings from each cell prior to activation of dynamic clamp. The cell in K1 under dynamic clamp. The IK1 profiles under dynamic clamp were qualitatively similar to those seen in guinea-pig myocyte experiments with the clear difference that proportionately greater current was evident during the resting potential . Furthecurrent; . Such trK1). In the initial report of SQT3 arising from the D172N mutation, simulations using the Priebe-Beuckelmann human ventricular AP model , and C terminus (E244 and E299) of the channel . The D17AP model  predicterization . Our ownrization  using morization  showed Arization . An inderization . Steepenrization . Our dynrization , in showK1 over a greater membrane potential range than does D172N  hyperpolarization of resting membrane potential together with an (up to ∼20 V/s) increase in AP maximal upstroke velocity  cell preparation. Further, spontaneously active myocytes typically have highly labile membrane potentials over the diastolic potential range  in guinea-pig ventricular AP maximal upstroke velocity seen with the WT-D172N and D172N Iysiology , prominemutation  and a demutation . Howevervelocity . Althougal range  and so iK1 involve other Kir2.x isoforms in addition to Kir2.1  over other isoforms  the experimental results observed here are concordant with major AP changes predicted from computational modelling odelling . This hibe valid . Anotherbe valid , whilst