Journal of the American Society for Mass Spectrometry | Year: 2015
Mass selective axial ejection (MSAE) from a low pressure linear ion trap (LIT) is investigated in the presence of added auxiliary nonlinear radio frequency (rf) fields. Nonlinear rf fields allow ions to be ejected with high sensitivity at large excitation amplitudes and reduced deleterious effects of space charge. These permit the operation of the LIT at ion populations considerably larger than the space charge limit usually observed in the absence of the nonlinear fields while maintaining good spectral resolution and mass accuracy. Experimental data show that the greater the strength of the nonlinear field, the less the effects of space charge on mass assignment and peak width. The only deleterious effect is a slight broadening of the mass spectral peaks at the highest values of added nonlinear fields used. © 2015 American Society for Mass Spectrometry.
Diao X.,U.S. National Institute on Drug Abuse |
Wohlfarth A.,U.S. National Institute on Drug Abuse |
Pang S.,SCIEX |
Scheidweiler K.B.,U.S. National Institute on Drug Abuse |
Huestis M.A.,U.S. National Institute on Drug Abuse
Clinical Chemistry | Year: 2016
BACKGROUND: Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. METHODS: We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600+) to identify optimal metabolite markers after incubating 10 μmol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. RESULTS: Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. CONCLUSIONS: With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids. © 2015 American Association for Clinical Chemistry.
Journal of the Institute of Brewing | Year: 2015
Following recent studies that showed that the agrochemical mepiquat (1,1-dimethylpiperidinium) forms during the roasting of coffee beans and barley, this work investigates the presence of mepiquat in malted barley and commercially available beers. Liquid chromatography-tandem mass spectrometry was used to develop a sensitive and precise analytical method, with detection limits of 0.031 ng/g in malted barley and 0.014 ng/g in beer. Mepiquat was detected in nine out of 10 malted barley samples, with all results under the Canadian maximum residue limit (100 ng/g). The data suggest a relationship between perceived malted barley colour and mepiquat concentration. The concentration of mepiquat in the beers analysed was also below the maximum residue limits in Canada (100 ng/g) and in the EU (600 ng/g), suggesting that mepiquat is not a regulatory concern in finished beers. © 2015 The Institute of Brewing & Distilling.
The complementary DNA of PALB2 was obtained from the Mammalian Gene Collection (MGC). Full-length PALB2 and BRCA1 were amplified by PCR, subcloned into pDONR221 and delivered into the pDEST-GFP, pDEST-Flag and the mCherry-LacR vectors using Gateway cloning technology (Invitrogen). Similarly, the coiled-coil domain of BRCA1 (residues 1363–1437) was amplified by PCR, subcloned into the pDONR221 vector and delivered into both mCherryLacR and pDEST-GFP vectors. The N-terminal domain of PALB2 was amplified by PCR and introduced into the GST expression vector pET30-2-His-GST-TEV29 using the EcoRI/XhoI sites. The coiled-coil domain of BRCA1 was cloned into pMAL-c2 using the BamHI/SalI sites. Truncated forms of PALB2 were obtained by introducing stop codons or deletions through site-directed mutagenesis. Full-length CtIP was amplified by PCR, subcloned into the pDONR221 and delivered into the lentiviral construct pCW57.1 (a gift from D. Root; Addgene plasmid #41393) using Gateway cloning technology (Invitrogen). The USP11 cDNA was a gift from D. Cortez and was amplified by PCR and cloned into the pDsRed2-C1 vector using the EcoRI/SalI sites. The bacterial codon-optimized coding sequence of pig USP11 was subcloned into the 6×His–GST vector pETM-30-Htb using the BamHI/EcoRI sites. siRNA-resistant versions of PALB2, BRCA1 and USP11 constructs were generated as previously described11. Full-length CUL3 and RBX1 were amplified by PCR from a human pancreas cDNA library (Invitrogen) as previously described30 and cloned into the dual expression pFBDM vector using NheI/XmaI and BssHII/NotI respectively. The NEDD8 cDNA was a gift from D. Xirodimas and was fused to a double StrepII tag at its C terminus in the pET17b vector (Millipore). Human DEN1 was amplified from a vector supplied by A. Echalier and fused to a non-cleavable N-terminal StrepII2× tag by PCR and inserted into a pET17b vector. The pCOOL-mKEAP1 plasmid was a gift from F. Shao. The pcDNA3-HA2-KEAP1 and pcDNA3-HA2-KEAP1ΔBTB were gifts from Y. Xiong (Addgene plasmids #21556 and 21593). gRNAs were synthesized and processed as described previously31. Annealed gRNAs were cloned into the Cas9-expressing vectors pSpCas9(BB)-2A-Puro (PX459) or pX330-U6-Chimeric_BB-CBh-hSpCas9, a gift from F. Zhang (Addgene plasmids #48139 and 42230). The gRNAs targeting the LMNA or the PML locus and the mClover-tagged LMNA or PML are described previously28. The lentiviral packaging vector psPAX2 and the envelope vector VSV-G were a gift from D. Trono (Addgene plasmids #12260 and 12259). His -Ub was cloned into the pcDNA5-FRT/TO backbone using the XhoI/HindIII sites. All mutations were introduced by site-directed mutagenesis using QuikChange (Stratagene) and all plasmids were sequence-verified. All culture media were supplemented with 10% fetal bovine serum (FBS). U-2-OS (U2OS) cells were cultured in McCoy’s medium (Gibco). 293T cells were cultured in DMEM (Gibco). Parental cells were tested for mycoplasma contamination and authenticated by STR DNA profiling. Plasmid transfections were carried out using Lipofectamine 2000 Transfection Reagent (Invitrogen) following the manufacturer’s protocol. Lentiviral infection was carried out as previously described15. U2OS and 293T cells were purchased from ATCC. U2OS 256 cells were a gift from R. Greenberg. We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07–434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 (ref. 32), rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a gift from M. Tyers), rabbit anti-ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem). The following antibodies were used as secondary antibodies in immunofluorescence microscopy: Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 555 donkey anti-mouse IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-mouse IgG, Alexa Fluor 647 donkey anti-human IgG, Alexa Fluor 647 donkey anti-goat IgG (Molecular Probes). All siRNAs employed in this study were single duplex siRNAs purchased from ThermoFisher. RNA interference (RNAi) transfections were performed using Lipofectamine RNAiMax (Invitrogen) in a forward transfection mode. The individual siRNA duplexes used were: BRCA1 (D-003461-05), PALB2 (D-012928-04), USP11 (D-006063-01), CUL1 (M-004086-01), CUL2 (M-007277-00), CUL3 (M-010224-02), CUL4A (M-012610-01), CUL4B (M-017965-01), CUL5 (M-019553-01), KEAP1 (D-12453-02), RAD51 (M-003530-04), CtIP/RBBP8 (M-001376-00), BRCA2 (D-003462-04), 53BP1 (D-003549-01) and non-targeting control siRNA (D-001210-02). Except when stated otherwise, siRNAs were transfected 48 h before cell processing. We employed the following drugs at the indicated concentrations: cycloheximide (CHX; Sigma) at 100 ng ml−1, camptothecin (CPT; Sigma) at 0.2 μM, ATM inhibitor (KU55933; Selleck Chemicals) at 10 μM, ATR inhibitor (VE-821; a gift from P. Reaper) at 10 μM, DNA-PKcs inhibitor (NU7441; Genetex) at 10 μM, proteasome inhibitor MG132 (Sigma) at 2 μM, lovastatin (S2061; Selleck Chemicals) at 40 μM, doxycycline (#8634-1; Clontech), Nedd8-activating enzyme inhibitor (MLN4929; Active Biochem) at 5 μM and olaparib (Selleck) at the indicated concentrations. In most cases, cells were grown on glass coverslips, fixed with 2% (w/v) paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.3% (v/v) Triton X-100 for 20 min at room temperature and blocked with 5% BSA in PBS for 30 min at room temperature. Alternatively, cells were fixed with 100% cold methanol for 10 min at −20 °C and subsequently washed with PBS for 5 min at room temperature before PBS-BSA blocking. Cells were then incubated with the primary antibody diluted in PBS-BSA for 2 h at room temperature. Cells were next washed with PBS and then incubated with secondary antibodies diluted in PBS-BSA supplemented with 0.8 μg ml−1 of DAPI to stain DNA for 1 h at room temperature. The coverslips were mounted onto glass slides with Prolong Gold mounting agent (Invitrogen). Confocal images were taken using a Zeiss LSM780 laser-scanning microscope. For G1 versus S/G2 analysis of the BRCA1–PALB2–BRCA2 axis, cells were first synchronized with a double-thymidine block, released to allow entry into S phase and exposed to 2 or 20 Gy of X-irradiation at 5 h and 12 h post-release and fixed at 1 to 5 h post-treatment (where indicated). For the examination of DNA replication, cells were pre-incubated with 30 μM BrdU for 30 min before irradiation and processed as previously described. 293T and U2OS cells were transiently transfected with three distinct sgRNAs targeting either 53BP1, USP11 or KEAP1 and expressed from the pX459 vector containing Cas9 followed by the 2A-Puromycin cassette. The next day, cells were selected with puromycin for 2 days and subcloned to form single colonies or subpopulations. Clones were screened by immunoblot and/or immunofluorescence to verify the loss of 53BP1, USP11 or KEAP1 expression and subsequently characterized by PCR and sequencing. The genomic region targeted by the CRISPR–Cas9 was amplified by PCR using Turbo Pfu polymerase (Agilent) and the PCR product was cloned into the pCR2.1 TOPO vector (Invitrogen) before sequencing. 293T cells were incubated with the indicated doses of olaparib (Selleck Chemicals) for 24 h, washed once with PBS and counted by trypan blue staining. Five-hundred cells were then plated in duplicate for each condition. The cell survival assay was performed as previously described33. GST and MBP fusions proteins were produced as previously described34, 35. Briefly, MBP proteins expressed in Escherichia coli were purified on amylose resin (New England Biolabs) according to the batch method described by the manufacturer and stored in 1× PBS, 5% glycerol. GST proteins expressed in E. coli were purified on glutathione sepharose 4B (GE Healthcare) resin in 50 mM Tris HCl pH 7.5, 300 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM EDTA, 15 μg ml−1 AEBSF and 1× complete protease inhibitor cocktail (Roche). Upon elution from the resin using 50 mM glutathione in 50 mM Tris HCl pH 8, 2 mM DTT, the His –GST tag was cleaved off using His-tagged TEV protease (provided by F. Sicheri) in 50 mM Tris HCl pH 7.5, 150 mM NaCl, 10 mM glutathione, 10% glycerol, 2 mM sodium citrate and 2 mM β-mercaptoethanol. His -tagged proteins were depleted using Ni-NTA-agarose beads (Qiagen) in 50 mM Tris HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 5 mM glutathione, 10% glycerol, 1 mM sodium citrate and 2 mM β-mercaptoethanol followed by centrifugal concentration (Amicon centrifugal filters, Millipore). GST–mKEAP1 was purified as described previously36, with an additional anion exchange step on a HiTrap Q HP column (GE Healthcare). The GST tag was left on the protein for in vitro experiments. Purification of CUL3 and RBX1 was performed as previously described30. NEDD8 (gift from D. Xirodimas) and DEN1 were expressed in E. coli BL21 grown in Terrific broth media and induced overnight with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 16 °C. Cells were harvested and resuspended in wash buffer (400 mM NaCl, 50 mM Tris-HCl, pH 8, 5% glycerol, 2 mM DTT), supplemented with lysozyme, universal nuclease (Pierce), benzamidine, leupeptin, pepstatin, PMSF and complete protease inhibitor cocktail (Roche), except for DEN1-expressing cells where the protease inhibitors were omitted. Cells were lysed by sonication and the lysate was cleared by centrifugation at 20,000 r.p.m. for 50 min. The soluble supernatant was bound to a 5 ml Strep-Tactin Superflow Cartridge with a flow rate of 3 ml min−1 using a peristaltic pump. The column was washed with 20 column volumes (CV) of washing buffer and eluted with 5 CV washing buffer, diluted 1:2 in water to reduce the final salt concentration, and supplemented with 2.5 mM desthiobiotin. The elution fractions were pooled and concentrated to a total volume of 4 ml using a 3 kDa cut-off Amicon concentrator. DEN1 was further purified over a Superdex 75 size-exclusion column, buffer exchanged into 150 mM NaCl, HEPES, pH 7.6, 2% glycerol and 1 mM DTT. The C-terminal pro-peptide and StrepII2×-tag were removed by incubation with StrepII2×–DEN1 in a 1:20 molar ratio for 1 h at room temperature. The DEN1 cleavage reaction was buffer exchanged on a Zeba MWCO desalting column (Pierce), to remove the desthiobiotin, and passed through a Strep-Tactin Cartridge, which retains the C-terminal pro-peptide and DEN1. The GST-tagged Sus scrofa (pig) USP11 proteins were expressed in E. coli as described37. Cells were lysed by lysozyme treatment and sonication in 50 mM Tris pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM AEBSF, 1× Protease Inhibitor mix (284 ng ml−1 leupeptin, 1.37 μg ml−1 pepstatin A, 170 μg ml−1 PMSF and 330 μg ml−1 benzamidine) and 5% glycerol. Cleared lysate was applied to a column packed with glutathione sepharose 4B (GE Healthcare), washed extensively with lysis buffer before elution in 50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol and 25 mM reduced glutathione. DUB activity was assayed on fluorogenic ubiquitin-AMC (Enzo life sciences), measured using a Synergy Neo microplate reader (Biotek). His -TEV-ubiquitin-G76C was purified on chelating HiTrap resin, following the manufacturer’s instructions, followed by size-exclusion chromatography on a S-75 column (GE healthcare). The protein was extensively dialysed in 1 mM acetic acid and lyophilized. HA-tagged N-terminal fragments of PALB2 (1–103) (1 μM) were in vitro ubiquitylated using 50 μM wild-type (Ubi WT, Boston Biochem) or a lysine-less ubiquitin (Ub-K0, Boston Biochem), 100 nM human UBA1 (E1), 500 nM CDC34 (provided by F. Sicheri and D. Ceccarelli), 250 nM neddylated CUL3/RBX1, 375 nM GST–mKEAP1 and 1.5 mM ATP in a buffer containing 50 mM Tris HCl pH 7.5, 20 mM NaCl, 10 mM MgCl and 0.5 mM DTT. Ubiquitylation reactions were carried out at 37 °C for 1 h, unless stated otherwise. For USP11-mediated deubiquitylation assays, HA–PALB2 (1–103) was first ubiquitylated using lysine-less ubiquitin with enzyme concentrations as described earlier in 50 μl reactions in a buffer containing 25 mM HEPES pH 8, 150 mM NaCl, 10 mM MgCl , 0.5 mM DTT and 1.5 mM ATP for 1.5 h at 37 °C. Reactions were stopped by the addition of 1 unit Apyrase (New England Biolabs). Reaction products were mixed at a 1:1 ratio with wild-type or catalytically inactive (C270S) USP11, or USP2 (provided by F. Sicheri and E. Zeqiraj) using final concentrations of 100 nM, 500 nM and 2,500 nM (USP11) and 500 nM (USP2) and incubated for 2 h at 30 °C in a buffer containing 25 mM HEPES pH 8, 150 mM NaCl, 2 mM DTT, 0.1 mg ml−1 BSA, 0.03% Brij-35, 5 mM MgCl , 0.375 mM ATP. PALB2 in vitro ubiquitylation reaction products were diluted in a buffer at final concentration of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl , 0.25 mM DTT and 0.1% NP-40. Twenty micrograms MBP or MBP–BRCA1-CC was coupled to amylose resin (New England Biolabs) in the above buffer supplemented with 0.1% BSA before addition of the ubiquitylation products. Pulldown reactions were performed at 4 °C for 2 h, followed by extensive washing. Cells were collected by trypsinization, washed once with PBS and lysed in 500 μl of lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 1% NP-40, complete protease inhibitor cocktail (Roche), cocktail of phosphatase inhibitors (Sigma) and N-ethylmaleimide to inhibit deubiquitylation) on ice. Lysates were centrifuged at 15,000g for 10 min at 4 °C and protein concentration was evaluated using absorbance at 280 nm. Equivalent amounts of proteins (∼0.5–1 mg) were incubated with 2 μg of rabbit anti-PALB2, rabbit anti-USP11 antibody, rabbit anti-GFP antibody or normal rabbit IgG for 5 h at 4 °C. A mix of protein A/protein G-Sepharose beads (Thermo Scientific) was added for an additional hour. Beads were collected by centrifugation, washed twice with lysis buffer and once with PBS, and eluted by boiling in 2× Laemmli buffer before analysis by SDS–PAGE and immunoblotting. For mass spectrometry analysis of Flag–PALB2, 150 × 106 transiently transfected HEK293T cells were lysed in high-salt lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 3 mM MgCl , 3 mM CaCl ), supplemented with complete protease inhibitor cocktail (Roche), 4 mM 1,10-Phenantroline, 50 U benzonase and 50 U micrococcal nuclease. Cleared lysates were incubated with Flag-M2 agarose (Sigma), followed by extensive washing in lysis buffer and 50 mM ammoniumbicarbonate. After immunoprecipitation of transiently transfected Flag–PALB2 from siCTRL-transfected or USP11 siRNA-depleted 293T cells, cysteine residues were reduced and alkylated on beads using 10 mM DTT (30 min at 56 °C) and 15 mM 2-chloroacetamide (1 h at room temperature), respectively. Proteins were digested using limited trypsin digestion on beads (1 μg trypsin; Worthington) per sample, 20 min at 37 °C), and dried to completeness. For LC-MS/MS analysis, peptides were reconstituted in 5% formic acid and loaded onto a 12 cm fused silica column with pulled tip packed in-house with 3.5 μm Zorbax C18 (Agilent Technologies). Samples were analysed using an Orbitrap Velos (Thermo Scientific) coupled to an Eksigent nanoLC ultra (AB SCIEX). Peptides were eluted from the column using a 90 min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid. Tandem MS spectra were acquired in a data-dependent mode for the top two most abundant multiply charged peptides and included targeted scans for five specific N-terminal PALB2 tryptic digest peptides (charge state 1+, 2+, 3+), either in non-modified form or including a diGly-ubiquitin trypsin digestion remnant. Tandem MS spectra were acquired using collision-induced dissociation. Spectra were searched against the human Refseq_V53 database using Mascot, allowing up to four missed cleavages and including carbamidomethyl (C), deamidation (NQ), oxidation (M), GlyGly (K) and LeuArgGlyGly (K) as variable modifications. In vitro ubiquitylated HA–PALB2 (1–103) (50 μl total reaction mix) was run briefly onto an SDS–PAGE gel, followed by total lane excision, in-gel reduction using 10 mM DTT (30 min at 56 °C), alkylation using 50 mM 2-chloroacetamide and trypsin digestion for 16 h at 37 °C. Digested peptides were mixed with 20 μl of a mix of 10 unique heavy isotope-labelled N-terminal PALB2 (AQUA) peptides (covering full or partial tryptic digests of regions surrounding Lys 16, 25, 30 or 43, either in non-modified or diG-modified form; 80–1,200 fmol μl−1 per peptide, based on individual peptide sensitivity testing) before loading 6 μl onto a 12 cm fused silica column with pulled tip packed in-house with 3.5 μm Zorbax C18. Samples were measured on an Orbitrap ELITE (Thermo Scientific) coupled to an Eksigent nanoLC ultra (AB SCIEX). Peptides were eluted from the column using a 180 min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid. Tandem MS spectra were acquired in a data-dependent mode for the top two most abundant multiply charged ions and included targeted scans for the ten specific N-terminal PALB2 tryptic digest peptides (charge states 1+, 2+, 3+), either in light or heavy isotope-labelled form. Tandem MS spectra were acquired using collision induced dissociation. Spectra were searched against the human Refseq_V53 database using Mascot, allowing up to two missed cleavages and including carbamidomethyl (C), deamidation (NQ), oxidation (M), GlyGly (K) and LeuArgGlyGly (K) as variable modifications, after which spectra were manually validated. 293 FLIP-IN cells stably expressing His –Ub were transfected with the indicated siRNA and treated with doxycycline (DOX) for 24 h to induce His –Ub expression. Cells were pre-treated with 10 mM N-ethylmaleimide for 30 min and lysed in denaturating lysis buffer (6 M guanidinium-HCl, 0.1 M Na HPO /NaH PO , 10 mM Tris-HCl, 5 mM imidazole, 0.01 M β-mercaptoethanol, complete protease inhibitor cocktail). Lysates were sonicated on ice twice for 10 s with 1 min break and centrifuged at 15,000g for 10 min at 4 °C. The supernatant was incubated with Ni-NTA-agarose beads (Qiagen) for 4 h at 4 °C. Beads were collected by centrifugation, washed once with denaturating lysis buffer, once with wash buffer (8 M urea, 0.1 M Na HPO /NaH PO , 10 mM Tris-HCl, 5 mM imidazole, 0.01 M β-mercaptoethanol, complete protease inhibitor cocktail), and twice with wash buffer supplemented with 0.1% Triton X-100, and eluted in elution buffer (0.2 M imidazole, 0.15 M Tris-HCl, 30% glycerol, 0.72 M β-mercaptoethanol, 5% SDS) before analysis by SDS–PAGE and immunoblotting. Parental U2OS cells and U2OS cells stably expressing wild-type CtIP or CtIP(T847E) mutant were transfected with the indicated siRNA and the PALB2-KR construct, synchronized with a single thymidine block, treated with doxycycline to induce CtIP expression and subsequently blocked in G1 phase by adding 40 μM lovastatin. Cells were collected by trypsinization, washed once with PBS and electroporated with 2.5 μg of sgRNA plasmid and 2.5 μg of donor template using the Nucleofector technology (Lonza; protocol X-001). Cells were plated in medium supplemented with 40 μM lovastatin and grown for 24 h before flow cytometry analysis. PALB2 (1–103) polypeptides, engineered with only one cross-linkable cysteine, were ubiquitylated by cross-linking alkylation, as previously described38, 39, with the following modifications. Purified PALB2 cysteine mutant (final concentration of 600 μM) was mixed with His -TEV-ubiquitin G76C (350 μM) in 300 mM Tris pH 8.8, 120 mM NaCl and 5% glycerol. Tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich) reducing agent was added to a final concentration of 6 mM to the mixture and incubated for 30 min at room temperature. The bi-reactive cysteine cross-linker, 1,3-dichloroacetone (Sigma-Aldrich), was dissolved in dimethylformamide and added to the protein mix to a final concentration of 5.25 mM. The reaction was allowed to proceed on ice for 1 h, before being quenched by the addition of 5 mM β-mercaptoethanol. His -TEV-ubiquitin-conjugated PALB2 was enriched by passing over Ni-NTA-agarose beads (Qiagen). No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
The use and care of animals complied with the guideline of the Biomedical Research Ethics Committee at the Shanghai Institutes for Biological Science (CAS), which approved the application entitled ‘Reproductive physiology of cynomolgus monkey and establishment transgenic monkey’ (#ER-SIBS-221106P). Laparoscopy was used for oocyte collection. Oocytes were aspirated from follicles 2–8 mm in diameter, about 32–36 h after hCG stimulation31. The collected oocytes were cultured in the pre-equilibrated maturation medium32. Metaphase II arrested oocytes were selected for perivitelline space injection32 of lentiviruses and ICSI. The lentivirus concentration for injection was 1 × 1010 viral genome (vg) per ml. After microinjection, the oocytes were cultured in the maturation medium at 37 °C (in 5% CO ) for about 1 h, until fertilization by ICSI. Monkey semen was collected by penile electro-ejaculation. For ICSI, a single sperm was immobilized and aspirated with the tail first. A single oocyte was fixed by the holding pipette, and the injection pipette was pushed through the zona pellucida and subsequently through the oolemma to release the spermatozoon32. After ICSI, the oocytes were cultured in pre-equilibrated Hamster Embryo Culture Medium 9 (HECM-9) at 37 °C (in 5% CO ) until the next morning33, 34. Menstrual cycles of females were recorded daily. To synchronize the developmental stage of embryos with the recipient, monkeys were chosen for tubal embryo transfer at 0–3 days after ovulation, and a stigma or a new corpus luteum on the ovary could be observed by laparoscopy. About 2–3 pronuclear-stage embryos were selected for tubal transfer to each surrogate female31. Hair-root samples collected from newborn monkey pups were used to extract DNA. Samples were digested by proteinase K overnight at 65 °C and precipitated for DNA and PCR with specific primers again GFP and mCherry were used for initial genotyping analysis as follows: mCherry-R: 5′-TGCTTGATCTCGCCCTTCAG-3′, mCherry-F: 5′-GCCATCATCAAGGAGTTCATGC-3′; GFP-F: 5′-AAGTTCATCTGCACCACCG-3′, GFP-R: 5′-TCCTTGAAGAAGATGGTGCG-3′. A total of 15 μg of genomic DNA was prepared and digested with BamHI and EcoRI, which released transgenes. Genomic DNAs were separated with 1% agarose gel and transferred to Nippon N+ membrane (GE). DNA probes from hMECP2-2a-GFP was prepared using ready-to-go DNA label kit (279240D-20, GE Life Sciences). 32P-labelled probes were hybridized with blots of genomic DNAs and exposed to phosphor-imager after extensively washing. Decisions of whether euthanasia procedures would be carried out for sick or aborted newborn monkeys are made by veterinarians, after consulting with principal investigators and followed the approved protocol (#ER-SIBS-221106P). Aborted or sick MECP2 TG and WT monkeys were deeply anaesthetized with ketamine hydrochloride (5–10 mg kg−1) to avoid possible pain and then perfused with 0.9% saline with 2–4% paraformaldehyde (PFA) for further immunohistochemistry experiments. The procedure is approved by the Biomedical Research Ethics Committee at the Shanghai Institutes for Biological Science (CAS), described in the protocol entitled ‘Reproductive physiology of cynomolgus monkey and establishment transgenic monkey’ (#ER-SIBS-221106P). After perfusion, the hemispheres of the brain were dissected, cut in to small blocks, fixed with 4% PFA in phosphate buffer, and equilibrated in 30% sucrose. Fixed and equilibrated brain tissue blocks were cut into 30-μm cortical sections with a Microm HM525 cryostat. Sections were washed for 5 min in PBS containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100, and incubated with primary antibodies (in PBS with 3% BSA and 0.3% Triton X-100) overnight at 4 °C and subsequently with corresponding secondary antibodies (Alexa-Fluor-conjugated, Invitrogen, at 1:1,000). DAPI was used to label the nuclei and sections were mounted with 75% glycerol. Other antibodies used: HA antibody (Covance, MMS-101R), NeuN antibody (Millipore, MAB377), MeCP2 antibody (Cell Signaling, 3456S) and GFP antibody (Abcam, ab6673). Four sets of primers targeted to MECP2 were designed. One set (mecp2_1) was a cross-intron primer targeted to transgenic cDNA fragments representing the copy number of transgenic DNA; the second (mecp2_2) was targeted to one exon of transgenic cDNA fragments representing the total MECP2 copy number; and the other two primer sets (mecp2IN_1 and mecp2IN_2) were targeted to introns of monkey MECP2 gene representing the endogenous MECP2 copy number. Two sets of EGFP primers (EGFP_1 and EGFP_2) were designed to verify the copy number of the transgene, and one set of mCherry primers was designed as negative control. The copy number of these DNA fragments was measured using custom-designed Multiplex AccuCopyTM Kit (Geneskies Biotechnologies, CN0105). The copy number of these target DNA fragments was measured using custom-designed Multiplex AccuCopy kit (Geneskies Biotechnologies, CN0105). For each DNA fragment amplified, a piece of synthesized competitive double-stranded DNA of known concentration and with insertions or deletions of a few base pairs was added to the PCR reaction mix. Each PCR reaction was carried out by mixing the synthesized competitive double-stranded DNAs for target and reference genes (POP1, RPP14 and POLR2A) together with a defined amount of sample DNAs. A multiplex competitive PCR was then performed to simultaneously amplify all reference and target genes from both sample and competitive DNAs using multiple fluorescence-labelled primer pairs. In brief, the 20-μl PCR reaction for each sample contained 1× AccuCopy PCR Master Mix, 1× Fluorescence Primer Mix, 1× Competitive DNA mix and ~10 ng sample DNA. The PCR program used was: 95 °C for 10 min; 11 cycles of 94 °C for 20 s, 65 °C–0.5 °C/cycle 40 s, 72 °C for 1.5 min; 24 cycles of 94 °C for 20 s, 59 °C for 30 s, 72 °C for 1.5 min; 60 °C for 60 min. PCR products were diluted 20-fold before loaded on ABI3730XL sequencer (Applied Biosystems) to separate amplicons of different sizes by capillary electrophoresis. Raw data were analysed using GeneMapper4.0, and the peak ratios of sample DNA to competitive DNA (S/C ratio) for all target and reference fragments were exported to Excel. The S/C ratio of each target fragment was first normalized to the S/C ratio of the reference genes, and then further normalized to the median copy number of the entire data set. The final normalized ratio was averaged for each MECP2 primer and EGFP primer, and the similarity between the two ratio further confirmed the copy number of the transgene. Lentiviruses were produced by standard protocols and provided at a titre of 1010 vg ml−1 by the Shanghai SBO Medical Biotechnology Co. Ltd. A total of 2 μg genomic DNA was used to construct a DNA library for each case35, 36, 37, 38. Sequencing linkers were further added onto genomic segments (length around 500–700 base pairs (bp)) (Extended Data Fig. 1b). After end repairing and 3′ A-adding, the fragmented DNAs were ligated with Y-shape adaptor. Amplification was performed with the adaptor primers. Asymmetry-primer PCR (APP) was used to enrich the viral integration sites in each library. The APP method includes two PCR systems. The first PCR system includes only LTR specific primer. After 12 cycles of linear amplification, adaptor specific primer was added in the PCR system followed by 12 cycles of exponential amplification. PCR products were purified using 0.7 × AMPure beads (Beckman, A63882). The second PCR system uses a pair of primers nest the primers in the first PCR system. After 12 cycles of linear amplification and 15 cycles of exponential amplification, the PCR products of 500–700 bp in size were isolated by agarose gel electrophoresis before being used to construct libraries with Illumina paired-end adapters according to the manufacturer protocol and sequenced by Illumina MiSeq V3 (2 × 300 base paired ends). Only the paired-end reads showing the fusions of viral sequences and the cynomolgus (Macaca fascicularis) genome segments were selected, in which two mismatches were allowed. The reads showing the same integration position were merged and treated as a unique integration site. Experiments were repeated three times independently with different sequencing linkers. Determination of insertion sites is under the following criteria: (1) total insert numbers are greater than 100 times after three experiments; (2) being detected at least twice after three experiments. Cynomolgus monkey genome is used in the following database: http://www.ncbi.nlm.nih.gov/genome/?term=crab+eating+monkey. Target sequences containing LTR of transgene cassettes and genomic segments flanking the transgenes were analysed (Supplementary Tables 2 and 6). Monkey brain tissues were homogenized in RIPA buffer (containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, protease inhibitor cocktail and phosphatase inhibitor cocktail) on ice and then centrifuged at 1,000g for 10 min at 4 °C. The supernatant was stored at −80 °C until use. Protein concentration was measured with BCA method. Approximately 30 μg protein of each sample was loaded in on 10% SDS–PAGE and run at 120 V constant voltage. A constant current of 0.36 mA was used for transblotting. Blots were probed with primary antibodies (1:1,000) overnight at 4 °C. After washing three times, blots were then incubated with goat anti-rabbit secondary antibody (1:3,000) at room temperature for 2 h. Chemiluminescence was used to visualize protein bands. Antibodies used: HA antibody (Abcam, ab9110), MeCP2 antibody (Cell Signaling, 3456S) and GFP antibody (Invitrogen, A11122). Fresh whole blood from upper arms of monkeys was taken by a professional veterinarian in the morning before feeding. Whole blood (200–400 μl) was dropped onto filter paper immediately. After air drying, filter papers were store at −20 °C before mass spectrometry analysis. API2000 from AB SCIEX were used for analysing fatty acid and amino acids. Data were obtained from three rounds of blood collections independently. Behaviour observation and analysis were performed by two independent trained observers, with demonstrated inter-observer reliability of at least 80%. All observers were blinded to the genotypes of the monkeys. The dimension of cages using for living and behaviour monitoring is 1.5 × 1 × 1.1 m. Monkeys individually, were observed alone in an observation cage (1.5 × 1 × 1.1 m) after they had been accustomed to community living following weaning. The observation cage was similar to their home cage. All locomotion behaviours were video-record without interruption for 20 min each day for 5 days. Data from 5 days were pooled. Social behaviours of TG and WT monkeys with familiar and unfamiliar monkeys were studied by examining the interactions of monkey from the same and different home cage, respectively. To study the interaction with familiar monkeys, we housed three groups of monkeys, each consisting three WT and two TG monkey of the same age, in three separate cages for 6 months before the observation (at about 1.5 years old). In this analysis, the observer followed the time each monkey spent sitting together with another monkey for a duration of 1 h each day for 5 consecutive days. We defined that two monkeys sat together by obvious interactions between the two for more than 3 s, during which the monkeys may exhibit touching and grooming behaviours or lean against each other. To study interaction with unfamiliar monkeys, we regrouped the females from same cohorts after the above observation for another 8 months in four separate cages (see Supplementary Table 4a, b). (Males were kept together separately owing to their proximity to sexual maturity, thus not used for observation). For each observation of social interaction, we paired two monkeys from different group and observation was made in the same manner as that described above for the interaction between familiar monkeys. To study the interaction with F TG monkeys, we housed two groups of monkeys (group info see Supplementary Table 7), each consisting of three WT and two TG F monkeys of similar age (at 10–11 months old), in two separate cages before the observation. In this analysis, the observer followed the time each monkey spent sitting together with another monkey for a duration of 1 h each day for 5 consecutive days. The TAD behavioural model was used to assay the monkey’s response to human gaze (Extended Data Fig. 5a). In each session of observation, an individual monkey from either the transgenic or WT group was placed in an observation cage (1.5 × 1 × 1.1 m), and allowed to adapt to the cage alone for 9 min. An observer then sat in front of the cage at a distance of 2 m, showing the face profile to the monkey without eye contact for 9 min (‘non-gaze period’). This was followed by the relaxation period (3 min) without the human presence, and the ‘gaze period’ (9 min) in which the observer sat in front of the cage and gazed at monkey with a neutral face. Behaviour and vocalizations were recorded on videotape39, 40, 41. WGTA tests were performed on 8 TG and 6 WT monkeys at the age of 1.5 years, in accordance to WGTA protocol25, 26, by trained technicians. The WGTA apparatus includes a testing box that for observing subject’s activity, a presentation board with food wells for reward placing, a trial door and an access door connected by pulley cord to separate the subject and presentation board, and a camera for recording. All tests were carried out in a quiet and standard lighted room. This test includes three stages: adaptation, discrimination and reversal. For the adaptation step, each monkey was tested for the ability to take the food reward on the presentation board that was placed by experimenter. Before the adaptation step, the monkey needs to pass several pre-test steps: the reward was placed in front of the food well, in the food well, in the food well next to the adaptation block, and in the food well with half covered by the adaptation block. Finally for adaptation step, the monkey had to take the food in the food well with the block covered completely. Each monkey received a maximum of 25 trials per day, and was considered to be passed when showing correct responses on 23 out of 25 trials. During the discrimination step, each monkey needed to choose the only reward in the food well that was covered by either a black or white block with an empty well covered the opposite colour. The same monkey was always rewarded with either black or white but with random location, with assignment of monkeys by the Gellerman order. Each monkey received 25 trials per day and was considered to be passed when showing correct responses on 23 out of 25 trials. For the reversal step, the procedure was the same as discrimination step, except that the monkey was rewarded black if white was rewarded during the discrimination step, and vice versa. This test includes four steps: adaptation, Hamilton search, Hamilton search set-breaking, and Hamilton search forced set-breaking. The adaptation step was similar to that for black/white test, with the same criterion for passing. For the Hamilton search step, four little boxes that represented the different positions from experimenter’s left to right on the presentation board were used for testing. The only reward was randomly placed in one of the four closed boxes in each trial. Monkey was allowed to find the reward from these four closed boxes. One trial was terminated when the monkey open the correct box. Each monkey performed 25 trials per day for 5 consecutive days. For the Hamilton search set-breaking step, the box that was the least preferred was first determined from the above step, and was always rewarded when chosen by the monkey. One trial was terminated when the subject open the correct box. Each monkey performed 25 trials per day for 5 days. For the Hamilton search forced set-breaking step, the procedure was the same as the set-breaking test, except that the monkey was allowed to make only one choice for finding the reward that placed in the least preferred box. The monkey was scored for the rate of correct choice over 25 trials each day for 5 consecutive days. The monkeys were tested for the ability to distinguish 240 pairs of toys. The toys in each pair were labelled A or B to cover the two food wells, one of which had food. For each monkey, either A or B was always rewarded. Each pair of toys was presented for 6 trials and 6 pairs were tested each day. Six different pairs were used for different days, with the test lasting 8 weeks until all 240 pairs were used. The monkey was scored for the rate of correct choice, averaged over 180 trials (5 days). Total RNA was extracted from three independent pieces of cortical tissues from brains of T05, T07, T09 and T14 and four WT monkeys by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at −80 °C. The RNA with RIN (RNA integrity number) > 8.0 is acceptable for cDNA library construction. RNA-seq and bioinformatic data analysis were performed by Shanghai Novelbio Ltd. The cDNA libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer’s instructions. The cDNA libraries were then processed for the proton sequencing process according to the commercially available protocols. Samples were diluted and mixed, the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI Ion Sphere Particles (Life Technologies) according to Ion PI Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI Ion Sphere Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies). Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20% of bases with qualities of <13. The clean reads were then aligned to crab eating macaque genome (version: Mfa5.0) using the MapSplice program (v2.1.6). In alignment, preliminary experiments were performed to optimize the alignment parameters (-s 22 -p 15–ins 6–del 6–non-canonical) to provide the largest information on the AS events42. Dif-Gene-Find er-t. We applied DEseq algorithm to filter the differentially expressed genes, after the significant analysis and false discovery rate (FDR) analysis under the following criteria: (1) fold change > 1.5 or < 0.667; (2) FDR < 0.05 (ref. 43). A Volcano plot was drawn by P value based on the differential gene analysis, and the colour was determined by the filtering criteria (red, log (P value) > 1.5; blue, log (P value) < 1.5; black, log (FC(TG/WT)) < ±0.5). The F offspring was generated by ICSI using sperms obtained from testicular tissue xenografts of the T07 monkey. The method of testicular xenografting greatly shortened the time required for sexual maturation of TG monkey44.