The viral ribonucleoprotein (vRNP) of influenza A virus is formed by virion RNA (vRNA), viral polymerase complex, and nucleoprotein (NP). The NP plays an important role in facilitating the replication and stabilization of viral RNA. To explore host factors that may be involved in the regulation of viral replication through interactions with NP, we conducted an immunoprecipitation experiment followed by mass spectrometry to identify NP-associated cellular proteins. Here, we demonstrate that NP can interact and colocalize with heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in mammalian cells and that the interaction may occur via direct binding to the glycine-rich domain (GRD) of hnRNP A2/B1. In addition, two residues in the tail loop of NP, F412 and R422, are required for the interaction of hnRNP A2/B1. Because the knockdown of hnRNP A2/B1 expression reduces viral RNP activity, hnRNP A2/B1 may act as a positive regulator in viral RNA synthesis of influenza A virus. More importantly, the findings in this research demonstrate that host proteins can regulate the replication of influenza A virus by interacting with NP. Influenza A virus causes respiratory diseases in humans and leads to annual epidemics and pandemics worldwide. The virion of influenza A virus consists of eight segments of genomic RNA with negative polarity. Each of the virion RNA segments is associated with a viral polymerase complex and bound with viral nucleoprotein (NP) to form ribonucleoprotein (RNP). The viral polymerase complex comprises three subunits, PB1, PB2 and PA, and functions as a transcriptase and replicase to generate viral mRNA and virion RNA [NP from influenza A virus has been demonstrated to interact directly with the viral polymerase complex and enhance unprimed viral RNA replication , 4. ThisN6-methyladenosine marks of primary miRNA transcripts and associates with the pri-miRNA microprocessor complex. This finding demonstrates that hnRNP A2/B1 is involved in pri-miRNA processing [Heterogeneous nuclear ribonucleoproteins (hnRNPs), which include more than 20 protein members, are RNA-binding proteins bound to pre-mRNA to form hnRNP particles in eukaryotic cells. These proteins typically contain an RNA recognition motif (RRM) and an arginine/glycine-rich domain (GRD) and play roles in several biological processes, such as transcription, RNA processing, and RNA trafficking and localization , 12. Theocessing . In addiocessing , 20.In the present study, we revealed, using co-immunoprecipitation and mass spectrometry (MS), that the NP encoded by influenza A virus could interact with cellular hnRNP A2/B1. The interaction of NP and hnRNP A2/B1 was found to be a direct protein–protein interaction between the GRD of hnRNP A2/B1. In addition, two residues, F412 and R422, on the tail loop of NP play a critical role in the interaction. Furthermore, we provide evidence suggesting that hnRNP A2/B1 could be a positive regulator of influenza A viral RNP activity and replication.2. Influenza A/WSN/33/H1N1 virus (WSN/H1N1) was amplified in MDCK cells and titrated by plaque formation assay with MDCK monolayers, as previously described [A549, 293T, HeLa, and MDCK cells were cultivated with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. The cells were maintained in an incubator at 37°C with 5% COescribed . The opeThe 293T cells that were transfected with a plasmid expressing FLAG-tagged NP (or co-transfected with the indicated NP construct and HA-tagged hnRNP A2 expression plasmid) were washed with PBS and then lysed in a lysis buffer containing 50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100. After centrifugation at 12,000 X g for 30 minutes, the supernatants were collected and mixed with ANTI-FLAG M2 affinity gel (Sigma-Aldrich). The mixture was rotated at 4°C for 2 h, and the affinity gel was then collected after centrifugation at 8,000 X g for 30 seconds. The affinity gel was thrice washed with a buffer containing 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The FLAG-tagged NP and the associated proteins on the affinity gel were eluted with 3X FLAG peptide (Sigma-Aldrich) in wash buffer. The eluted proteins were then separated on SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were reacted with anti-FLAG , anti-HA , anti-hnRNP A1 , anti-hnRNP A2/B1  or anti-β-actin antibodies  and then detected with an HRP-conjugated anti-mouse or anti-rabbit IgG antibody .4HCO3 (Sigma-Aldrich) containing 10 mM dithiothreitol  at 60°C for 30 min and alkylated with 25 mM NH4HCO3 containing 55 mM iodoacetamide  at room temperature for 30 min. After reduction and alkylation, the proteins were digested with sequencing-grade modified porcine trypsin  at 37°C for 16 h.Proteins were separated on a 10% SDS gel and then subjected to silver staining. Protein bands were excised, destained, and subjected to in-gel digestion with trypsin, as previously described , 23. BriTM 600/384 TF . Peptide mass was determined using an UltraflexTM MALDI-TOF mass spectrometer  controlled by the FlexControl 2.2 software package . Peptide mass fingerprints (PMFs) were acquired in reflectron mode (26.7 kV accelerating voltage) with 300 laser shots per spectrum.The in-gel tryptic peptides were extracted with acetonitrile  containing 0.5% trifluoroacetic acid (Sigma-Aldrich) and then loaded onto an MTP AnchorChipm/z; maximal number of peaks = 200; and quality factor threshold = 50. The acquired masses were internally calibrated to a mass accuracy within 50 ppm, using a peptide mixture of ACTH (m/z 2465.19), human angiotensin II (m/z 1046.54), and bovine serum albumin (m/z 927.49). Annotated and calibrated masses were searched with the Mascot search engine  in BioTools 2.2 software  against the Swiss-Prot human sequence database  of the European Bioinformatics Institute. The mass tolerance of the ions was set to 50 ppm, with trypsin as the digestion enzyme. Up to one missed cleavage was allowed, and searches were performed with the parameters of variable oxidation on methionine (+15.99 Da) and fixed carbamidomethylation on cysteine (+57 Da). Protein with statistically significant search scores  were treated as positive identifications. MS spectra with multiple matches were manually inspected to ensure the correct PMF assignment.Peptide masses were annotated using FlexAnalysis software ; the settings were signal-to-noise threshold = 2; peak detection algorithm = SNAP; peak width = 0.75 A549 cells grown on coverslips were infected with influenza A WSN virus at a multiplicity of infection (MOI) 2. At the indicated time points, the cells were fixed with 4% paraformaldehyde. After treating the fixed cells with 0.3% Triton X-100, the cells were subjected to immunofluorescence staining, as previously described . BrieflyEscherichia coli BL21 cells, and they were expressed under induction by isopropyl-β-D-thiogalactopyranoside. The E. coli cells were harvested by centrifugation and then disrupted by sonication. The supernatants of the cell lysates were subjected to a glutathione Sepharose 4B affinity column  to purify the full-length, and RRM1, RRM2, and GRD regions of GST-fused hnRNP A2. The purified 6X His-tagged NP was incubated with the purified full-length or different regions of GST-fused hnRNP A2 at 4°C for 60 min. A glutathione Sepharose 4B affinity column was used to pull down the GST-tagged proteins. The 6X His-tagged NP associated with the GST-fused proteins was detected by SDS-PAGE and immunoblotting.The 6X His-tagged NP was expressed and purified, as described previously . The GST5’-CAGAAGAAAGUUUGAGGAACUACUA- 3’ & 5’-UAGUAGUUCCUCAAACUUUCUUCUG-3’; #2: 5’-GAAGCUGUUUGUUGGCGGAAUUAAA-3’ & 5’-UUUAAUUCCGCCAACAAACAGCUUC-3’), siRNA against hnRNP A1 , or control siRNA  with Lipofectamine 2000  for 24 h and then co-transfected with plasmids that expressed PB1, PB2, PA, and NP from influenza A/WSN/H1N1 virus and a polymerase I promoter-driven plasmid expressing a negative-sensed ORF of firefly luciferase bearing 5’ and 3’ untranslated regions of NS vRNA. A Renilla luciferase reporter under polymerase II promoter control was also co-transfected to monitor transfection efficiency. At 24 h post-transfection, the lysates of the transfected cells were collected and the relative RNP activity was measured using a Dual-Luciferase Reporter Assay (Promega), as described previously [HeLa cells were transfected with siRNA against hnRNP A2/B1  of the NP protein plays important roles in oligomerization and interactions with other cellular factors –28. We fTo further identify the residues that are required for the interaction, we examined the interaction of hnRNP A2/B1 and NP with single residue mutation: I408A, F412A, R422A, or D421A, in the tail loop. As shown in Because NP is crucial in facilitating influenza viral polymerase in viral genome replication, our previous results prompted us to investigate the role of hnRNP A2/B1 in regulating the RNP activity of influenza A virus. To this end, influenza viral RNA synthesis was analyzed in HeLa cells with knockdown of hnRNP A2/B1 expression by siRNA transfection. The hnRNP A2/B1 knockdown cells were then co-transfected with expression plasmids that express NP, PA, PB1, PB2, and a vRNA-like reporter containing an antisense firefly luciferase ORF flanked by noncoding regions of the NS segment. At 24 h post-transfection, total RNA of the transfected cells was collected and subjected to RT and real-time PCR to determine the levels of vRNA and mRNA transcribed by the RNP complex, which represents the activity of the influenza polymerase complex . The results demonstrated that knockdown of hnRNP A2/B1 expression could reduce the RNP activity determined by the luciferase activity assay , as wellBecause hnRNP A1 is highly homologous to hnRNP A2/B1, we also examined whether hnRNP A1 could interact with influenza NP and have a regulatory function in viral RNA synthesis. The lysates of 293T cells transfected with a FLAG-tagged NP expressing plasmid were subjected to anti-FLAG immunoprecipitation. Interestingly, hnRNP A1 was detected in the precipitates by immunoblotting with an anti-hnRNP A1 antibody . We furtPrevious results have demonstrated that the interaction of influenza NP and host hnRNP A2/B1 impacts viral RNP activity. We then investigated the role of hnRNP A2/B1 in influenza virus replication. A549 cells were transfected with siRNA against hnRNP A2/B1 expression for 24 h and then reseeded for 24 h. The transfectants were infected with influenza A/WSN/H1N1 virus at an MOI of 0.001. At 36 and 48 h post-infection, the supernatants and lysates of the infected cells were collected to determine virus titers and hnRNP A2/B1 expression. The result showed that knockdown of hnRNP A2/B1 significantly increased the replication of influenza A WSN virus in A549 cells . These dThe NP encoded by influenza A viruses plays a critical role in viral RNA replication. Previous studies have revealed that NP can interact with the polymerase complex of the influenza A virus to facilitate viral RNA synthesis , 4. In aNP forms oligomers, and the tail loop of NP is crucial for the inter-subunit interactions for oligomerization . AlthougBecause NP is involved in influenza viral RNA synthesis, hnRNP A2/B1 might contribute to viral RNA synthesis by regulating the activity of the viral polymerase complex. We used a mini genome reporter assay to determine the ability of influenza polymerase to perform viral RNA synthesis, and we observed a reduced level of the viral-model RNAs derived from the reporter in hnRNP A2/B1 knockdown cells. Consistently, we found that a mutation at R422 of the NP, which was demonstrated to be a key residue for the interaction with hnRNP A2/B1, decreased the ability to support influenza polymerase activity. This result was consistent with previous research that examined the mutation in the NP of influenza H5N1 virus . Neverth6-methyladenosine-dependent miRNA maturation processing [hnRNP A2/B1 may be involved in pre-mRNA processing in cells . It remaocessing , the intAs previously reported, hnRNP A2/B1 could interact with the NS1 protein of influenza A PR/8/H1N1 virus, and the reduction of hnRNP A2/B1 expression could contribute to the replication of the virus as a result of increasing levels of NS1 mRNA in the cytosol . HoweverS1 Fig(A) 293T cells were transfected with a plasmid that expresses FLAG-tagged WSN NP encoded or an empty vector. The lysates of transfected cells were subjected to anti-FLAG immunoprecipitation. (B) HeLa cells were transfected with siRNA against hnRNP A1, and then co-transfected with plasmids that express NP, PA, PB1, PB2, renilla luciferase, and the vRNA-like firefly luciferase reporter as described previously. At 24 h post-transfection, lysates from the transfected cells were collected and analyzed with the Dual-Luciferase Reporter Assay and by immunoblotting with anti-hnRNP A2/B1 and anti-β actin antibodies. *** P < 0.005.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Here, we provide direct evidence that using recombinant proteins expressed in eukaryotic cells as antigen is a practical way to generate monoclonal antibodies (mAbs) against heavily glycosylated proteins. Heavily glycosylated proteins are typically difficult targets for mAb generation, being limited by unsatisfactory affinity and low specificity. Using the heavily glycosylated CD45 protein as an example, we demonstrate the entire process of expressing the protein in eukaryotic cells and using it as an antigen to generate CD45-targeting mAbs in mice. The mAbs generated showed robust affinity and specificity, which are crucial factors for differentiate circulating tumor cells from white blood cells in human breast cancer patient samples. Only 1 cell fusion and 2 cyclic sub-cloning steps were necessary before mAbs with satisfactory performance were obtained. Purchasing commercial mAbs is commonly done in biological laboratories to avoid the process of preparing mAbs, which is considered time- and labor-intensive , 2. HoweIn this study, we demonstrated the generation of a mAb recognizing the heavily glycosylated human CD45 protein and its application oappn identifying CTCs. Instead of utilizing a prokaryotic protein or peptide, we used the eukaryotic protein as the antigen. Antibody preparation, including mouse immunization, hybridoma screening, and mAb characterization was completed in 3 months, as only 1 time hybridoma fusion and 2 cyclic sub-cloning steps were performed to acquire the anti-CD45 mAb, which showed greater affinity and consistency than commercial mAbs. This study provides direct evidence that using eukaryotic proteins to generate mAbs could become a practical solution for biological laboratories encountering affinity and specificity issues with commercial mAbs. The efforts of plasmid construction and eukaryotic protein preparation paid off in terms of less time consumption and enhanced binding affinity.A mouse anti-his tag mAb (B1169), a commercial Alexa647-conjugated anti-CD45 mAb (BDLS0479), an Alexa647 mouse IgG1 isotype control (BDLS0031), rProtein G Beads (BDTL0003-1) and a mouse IgG control  were obtained from Beijing Biodragon Immunotechnologies Co., Ltd. A horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (M21001S) was purchased from Abmart Shanghai Co., Ltd. Complete Freund's adjuvant (F5881), incomplete Freund's adjuvant (F5506), PEG 1500 (807489) were purchased from Sigma–Aldrich (USA). Fetal bovine serum , Phosphate-buffered saline (PBS), Roswell Park Memorial Institute (RPMI) 1640 medium (SH30809.01B) and Dulbecco's modified Eagle's medium  were obtained from Thermo Scientific, USA.2 humidified atmosphere.To express the recombinant human CD45-his (rhCD45-his) protein, human embryonic kidney HEK-293T cells were cultured in DMEM supplemented with 10% FBS, and 50 μg/mL penicillin and streptomycin. HL60 (human promyelocytic leukemia cells), SKBR-3 (human breast cancer cells), and Sp2/0-Ag14 (a mouse myeloma cells) cells were obtained from National Infrastructure of Cell Line Resource (China) and cultured in RPMI 1640 medium supplemented with 10% FBS, 50 μg/mL penicillin, and streptomycin. All cells were incubated at 37°C in a 5% COWe obtained the necessary licenses and approvals from the Institutional Animal Care and Use Committee of Peking University (Permit Number: SYXK [Beijing] 2016–0028), who approved all of our animal-related experiments and protocols. BALB/C mice  were purchased from Vital River Laboratory Animal Technology (China). Animals were maintained in a sterile environment and feed sterilized food. At the end of the experiments, the animals were euthanized by carbon dioxide.We had received ethical approval from the Ethics Committee of Beijing Cancer Hospital. Informed consent was provided by 5 patients (who donated blood samples) and was approved by Institutional Review Board and Beijing Cancer Hospital and signed by 5 patients. All procedures were performed in accordance with relevant guidelines and regulations. Vacutainer tubes containing the anticoagulant ethylene-diaminetetraacetic acid were used to collect blood samples . The samples were placed at room temperature and processed within 24 h.cDNA encoding human CD45 was amplified by reverse transcriptase-PCR from total RNA extracted from HL60 cells and then cloned into the pMD19-T vector. Using the pMD19-T vector , as the template, the fragment encoding extracellular CD45 was amplified and inserted into the pCDNA3.1 vector  to generate the recombinant vector pCDNA3.1-CD45-his. cDNA encoding the CD45 ectodomain was amplified with the primers:5′-GGAATTGTACCCGCGGGCCCCGCCACCATGCAAAGCCCAACACCTTCCC-3′ (forward)5′ -GATCTCGGTCGACCGAATTCTCAATGATGATGATGATGATGCTTAGAATTATAAG ATGTTG-3′ (reverse).and The inserted nucleotide sequence was verified by DNA sequencing. The recombinant vector pCDNA3.1-CD45-his was transfected into HEK293T cells using Lipofectamine 2000. The recombinant protein was purified using Ni-NTA agarose . The protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its concentration was measured using the BCA Assay Kit . Finally, 2 mg protein was obtained from 200 mL cell culture supernatants and stored at –20°C.4HCO3 at room temperature and alkylated for 40 min with 15 mM iodoacetamide in 25 mM NH4HCO3 in the dark. After being washed and dehydrated, the alkylated sample was digested overnight at 37°C with trypsin , using a 1: 50 enzyme: substrate ratio. After digestion, the peptide mixtures were extracted with 50% acetonitrile and 0.1% trifluoroacetic acid for 2 h at 37°C. The peptide sample was dried, dissolved in 10 μL 0.1% formic acid, and subjected to nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.For glycosylation analysis, the rhCD45-his protein was reduced for 40 min with 5 mM dithiothreitol in 25 mM NHFor western blot assays, the rhCD45-his protein was reduced with loading buffer and boiled for 5 min. Two micrograms rhCD45-his protein and 2 μg PD-L1-his protein were added to separate lanes. After electrophoresis and blotting, the blots were blocked in PBS containing 5% skim milk and 0.1% Tween-20 (mPBST) at room temperature and then incubated with a primary antibody  in mPBST overnight at 4°C. The blots were washed in PBS with 0.1% Tween-20 (PBST) and incubated with an HRP-conjugated goat anti-mouse IgG secondary antibody in mPBST for 1 h at room temperature. The blots were washed again before the bands were detected.For the first mouse immunization, a 500-μL sample (50 μg rhCD45-his protein dissolved in 250 μL PBS plus 250 μL complete Freund’s adjuvant) was subcutaneously injected into each mouse. For the following 2 immunizations, complete Freund’s adjuvant was replaced by incomplete Freund’s adjuvant. The interval between each immunization was 2 weeks.8 splenocytes were mixed with 2.4 x 107 myeloma Sp2/0-Ag14 cells, and 1 mL 50% (v/v) PEG 1500 was added to the mixture to promote fusion. The mixture was evenly split into four 96-well plates. Thus, approximately 3 x 107 splenocytes were seeded into each plate. All cells were cultured for 5 days in RPMI medium with HAT reagent to exclude unfused cells. Each well contained at least 1 hybridoma clone.The mouse which exhibited the highest antiserum titer was sacrificed to retrieve its splenocytes. For the cell fusion, approximately 1.2 x 102SO4 was used to stop the reaction. The absorbance signal was measured at 450 nm.Enzyme-linked immunosorbent assay (ELISA) and flow cytometry were used to screen for hybridomas that secreted anti-CD45 antibodies. Hybridoma cell lines were established after 2 cycles of sub-cloning. ELISA plates were coated with rhCD45-his protein (1 μg/mL) or betaine homocysteine s-methyltransferase (BHMT)-his protein (1μg/mL) in carbonate buffer and incubated overnight at 4°C. Then, the plates were blocked with PBS containing 5% milk (mPBS) and incubated for 1 h at 4°C. The primary antibody consisted of antiserum collected from immunized mice (or the purified CD45 antibody) diluted in mPBS, which was incubated with the ELISA plate for 1 h at 37°C. After being washed 3 times with PBST, the plates were incubated for 1 h at 37°C with an HRP-conjugated goat anti-mouse IgG (1: 5000), which was diluted in mPBST. Tetramethylbenzidine was used as substrate to catalyze HRP reactions for 10 min, and 50 μL 2M HTo purify antibodies, ascites were diluted with 10-fold PBS and injected to Protein G affinity chromatography . Alexa647 dye was conjugated to antibody 4D3 by employing a Protein Labeling Kit .For surface plasmon resonance (SPR) assays, 2 μL rhCD45 protein (1 μg/μL) was immobilized on a SPR chip  surface modified with a gold-layer and incubated overnight at 4°C. The chip was blocked in 5% skim milk overnight at 4°C, washed 3 times in PBS, and dried under a stream of nitrogen. The Alexa647-4D3 and Alexa647-CmAb were diluted in PBST at different concentrations . SPR analysis was performed with the following cycle: running buffer ; sample (from low to high concentrations) for the binding assays (association phase); running buffer , and 0.5% (v/v) H3PO4 in deionized water (for chip regeneration). After 1 cycle was finished, the next sample was started on the same chip until all samples were completed. Real-time binding signals were recorded and analyzed using the PlexArray HT system . The dissociation constant was calculated by fitting the data to association–dissociation curves.For immunofluorescence analysis, cells were harvested, washed twice with PBS, and fixed with 4% paraformaldehyde. Cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) dye (1:100) for 10 min at room temperature to stain the cell nuclei, after which they were centrifuged for 3 min at 2000 rpm before the supernatant was removed. Five microliters of an Alexa647-labeled antibody (25 μg/mL) was added. After a 1-h incubation at room temperature, the cells were washed in PBS and fluorescently imaged.For flow cytometry assays, HL60 cells were collected and washed twice in PBS. Cells were incubated with 100uL culture supernatant or 5 μL of Alexa647-labeled anti-CD45 (25 μg/mL) for 1 h at room temperature, while mIgG1 was used as a negative control. In addition, lymphocytes were separated from blood using Lymphocyte Separation Medium .Two milliliters of peripheral blood from cancer patients was diluted with 5 mL PBS in a 15-mL tube and incubated directly with immunomagnetic beads  modified with a peptide targeting the epithelial cell adhesion molecule (EpCAM) for 1 h at room temperature . EpCAM-pThe overall experimental procedure used in this study is represented schematically in CD45 is a type-I transmembrane glycoprotein expressed abundantly on the surfaces of all leukocytes . The natWe then used LC-MS/MS to further investigate the post-translational modifications in detail. 39 glycopeptides were detected on 12 amino acids  rhCD45-his protein was injected per mouse. Seven days after the second immunization, the antiserum titers were determined by indirect ELISA. As shown in 6 hybridoma cells were injected intraperitoneally into a mouse that was pre-treated with liquid paraffin. After 7 days, ascites were collected, and antibodies were purified by protein G affinity chromatography. Then, the antibody subtype was determined. As shown in After establishing 4 hybridoma cell lines, which secreted antibodies that strongly reacted with the glycosylated CD45 protein, 1 x 10The purpose of producing an anti-CD45 mAb was to immunofluorescently detect glycosylated CD45 on leukocyte membranes. Thus, we fluorescently labeled mAb 4D3 with the fluorescent dye, Alexa647, and the resulting fluorescent labeled mAb was designated Alexa647-4D3. For comparison, we also purchased a commercial anti-CD45 mAb labeled with Alexa647, which was named as Alexa647-CmAb. To quantitatively investigate the binding affinity between Alexa647-4D3 and the glycosylated CD45 protein, we performed SPR assays to calculate the KD, which is widely accepted when characterizing antibody–antigen binding affinities . CompareEnumerating CTCs in the blood has been demonstrated to be helpful for selecting cancer treatment . AlthougOverall, Alexa647-4D3 and Alexa647-CmAb performed similarly in recognizing lymphocytes and CTC enumeration. However, before selecting Alexa647-CmAb, 2 commercial anti-CD45 mAbs which were purchased from Sino Biological (10086-MM05-F-25) and Abbiotec (251081) failed at CTC enumeration because of their weak binding with native CD45 on lymphocyte membranes .6 in 2 mL blood). Using our own experience as an example, we spent >8 months on testing 3 kinds of antibodies from 3 different manufacturers before obtaining an acceptable mAb. However, the next batch of mAbs failed to meet our requirements. While encountering such issues, tend to simply try other antibodies or query related databases, for instance, www.antibodypedia.com. The potential impact of glycosylation on antibody recognition has not been comprehensively investigated. Still, some studies [A stable supply of high-quality mAbs is crucial for conducting most protein-related studies in laboratories engaged in biological research. Commercial mAbs are normally considered easy to access and helpful for saving time and labor costs (versus producing mAbs in-house). However, for unusual or heavily glycosylated proteins, purchasing commercial mAbs may not be a good choice. First, there could be a long period before shipping orders, considering that providers need to prepare antibodies from the initial immunization stage. Second and more importantly, it is likely that the specificity and affinity of commercial mAbs are uncertain, especially for specifically glycosylated proteins. For instance, fluorescent staining of heavily glycosylated CD45 on leukocytes has become the most common method for differentiating CTCs from leukocytes. The method requires large quantities of mAbs with satisfactory affinity and high specificity, as the number of CTCs are quite low , compared with the number of leukocyte  was consistency with data from GenBank . The rhCD45-his vector was sequenced in Sangon Technology (Shanghai). The signal peptide is native.(DOCX)Click here for additional data file.S2 FigThe recombinant protein was detected by western blotting with an anti-his mAb. The protein was separated by 6% SDS-PAGE and transferred to a polyvinylidene fluoride membrane and probed with a mouse anti-his-tag mAb. His-tagged variant of the programmed cell death ligand 1 (PD-L1-his) protein was recognized with an anti-his-tag mAb, demonstrating the effectiveness of the mAb. The anti-his-tag mAb also recognized his-tag of recombinant rhCD45-his protein.(DOCX)Click here for additional data file.S3 FigThe experimental was performed as follow: The sample (4 μL) was loaded into reversed-phase C18-AQ resin ; Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. Data dependent on MS/MS: up to top 5 most intense peptide ions from the preview scan in the Orbitrap. The raw MS file were analyzed and searched against the new established protein sequence database based on the theoretical sequence of the target protein using Byonic software (Version 2.3.5). Only high confident identified peptides were chosen for downstream protein modification analysis. The result indicates that 39 peptides were modified by N- and O-glycosylation on 12 amino acid sites and an amino acid site was modified with different glycans as shown in S3A Fig. The sequence of amino acid were shown in S3B Fig.(DOCX)Click here for additional data file.S4 FigFor culturing hybridoma, four 96-well plates were used. Among 384 wells, 372 wells were occupied by at least one hybridoma clone. The reactions between supernatant from each wells and rhCD45-his protein were determined by ELISA. For ELISA assays, the procedure is as wells as describe in method, and 12 wells were used as control, as: 4 wells were filled with antiserum from immunized mice, considered as positive control; 4 wells were filled with antiserum from unimmunized mice, and 4 wells were filled with PBS, both considered as negative control. HRP conjugated goat anti-mouse IgG was used as second antibody, adding to every well. In this figure, each colored spot represent a well. The red boxes represent 4 wells which were filled with antiserum from immunized mice. Supernatants from 25 wells were with optical densities (450nm) larger than 0.2. The related 25 hybridomas were considered positive.(DOCX)Click here for additional data file.S5 FigCulture supernatants were reacted to 96 well plates coated with BHMT-his and CD45-his. As shown in S5 Fig, no supernatant was bound with BHMT-his.(DOCX)Click here for additional data file.S6 FigIndirect ELISA was used to confirm the subtype of 4 antibodies . The rhCD45-his protein (1 μg/mL) was coated on the ELISA plate. Antibodies were purified from mice ascites and added to the ELISA plate at 1 μg/mL. HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were added to separate wells as the second antibody. Data are presented as the mean ± SD of measurements derived from 2 independent assays.(DOCX)Click here for additional data file.S7 FigSize exclusion chromatography (SEC) and (SDS-PAGE) assays have been performed to determine the purity of antibody 4D3. (A) A peak indicating the existence of antibody 4D3 was detected at 9.43 min. (B) the purity of antibody 4D3 is 97.7%. The parameters of SEC are: Flow rate: 0.7ml/min; Temperature: 25°C; 40 μL antibody 4D3 is injected into the column which is purchased from Aglilent Technologies (Agilent AdvanceBio SEC). (C) 4D3 was separated with a reduced SDS-PAGE, and two clear bands indicates heavy chain (50kDa) and light chain (20kDa). No irrelevant band was observed.(DOCX)Click here for additional data file.S8 FigThe accurate molecular weight of 4D3 antibody was analyzed by LC-MS. The result exhibits 27 clear and independent peaks, without any irrelevant noise signal, demonstrating that monoclonal antibody is acquired.(DOCX)Click here for additional data file.S9 FigTo evaluate the specificities of Alexa647-4D3 and Alexa647-CmAb, SKBR3 and HL60 cells were stained by Alexa647-4D3 and Alexa647-CmAb, respectively. In this figure, blue spots represent cell nucleic stained by DAPI. As expected, neither Alexa647-4D3 nor Alexa647-CmAb bound to SKBR3 cells. As to the binding with HL60 cells, Alexa647-4D3 shows similar or slightly better performance with Alexa647-CmAb. Under the same optical conditional, Alexa647-4D3 is with higher fluorescent intensity than Alexa647-CmAb. Scale bar:50 μm.(DOCX)Click here for additional data file.S10 FigFlow cytometry was used to evaluate the binding between Alexa647-4D3 and lymphocyte subsets. CD3 (PECY5 labelled), CD4 (FITC labelled), CD8 (PE labelled), CD16 (PE labelled), CD19 (FITC labelled), and CD56 (FITC labelled) antibodies were introduced to differentiate lymphocyte subsets. Alexa647-4D3 was used to label all lymphocytes. S10A1, S10A2, S10B1 and S10B2 Fig demonstrated that T cells (CD3+CD4+ and CD3+CD8+) accounted for 61.83% of total lymphocyte amount while S10A3 and S10B3 Fig revealed that about 99.9% of T cells exhibited obvious binding with Alexa647-4D3. Similarly, B cells (CD3-CD19+) accounted for 14.57% of total lymphocyte amount  while about 99.9% of B cells exhibited obvious binding with Alexa647-4D3 ; NK cells (CD3-CD56+CD16+) accounted for 5.1% of total lymphocyte amount  while more than 96.4% of NK cells exhibited obvious binding with Alexa647-4D3 . S10F1 and S10F2 Fig were negative controls in which no antibody was used.(DOCX)Click here for additional data file.S11 FigBefore applying commercial mAbs on CTC identification, we tested the binding between commercial mAbs and HL60 cells. One of them  results in no binding at all, while another one (Abbiotec 251081) is with limited binding efficiencies. . The binding between PE-labelled Abbiotec 251081 antibody and HL60 cells shows that about 60% HL60 cells were recognized. Scale bar: 20 μm.(DOCX)Click here for additional data file.


Here we show that patients bearing DPYD c.1905+1G/A and c.2846A/T genotypes, together with UGT1A1*28 variant carriers, have an increased risk of experiencing clinically relevant toxicities, including hematological AEs and stomatitis. No carrier of the DPYD c.1679T>G minor allele was identified. Present results support the preemptive screening of mentioned DPYD and UGT1A1 variants to identify patients at risk of clinically relevant 5-fluoruracil- and irinotecan-related AEs, in order to improve treatments’ safety through a “genotype-guided” approach.Our study addresses the issue of the clinical reliability of three candidate DPYD and one UGT single nucleotide polymorphisms in predicting 5-fluorouracil- and irinotecan-related adverse events. To this purpose, we took advantage of a large cohort of metastatic colorectal cancer patients treated with first-line 5-fluorouracil- and irinotecan-based chemotherapy regimens  plus bevacizumab in the randomized clinical trial TRIBE by GONO ( Around A substantial interindividual difference in the occurrence and/or seriousness of 5-FU- and irinotecan-related toxicities may be partially due to clinical factors, including age, sex and performance status . NonetheMost cases of DPD and UGT1A1 deficiency are attributable to germline polymorphisms in their encoding genes, leading to partially non-functional enzymes , 14.DPYD . In . In DPYDAmong investigated clinical characteristics, age, sex and treatment arm had a significant impact on the occurrence of grade 3 or greater treatment-related overall AEs in univariate analysis. Associations between clinical variables and AEs are shown in Table DPYD variants  and 436 (98%) for UGT1A1*28 variant (Table Four hundred and thirty-nine (99%) out of 443 patients were successfully genotyped for investigated DPYD variants, minor alleles for DPYD c.1905+1G>A and DPYD c.2846 A>T were found only in heterozygosis in 5 (1.1%) and 5 (1.1%) patients, respectively; no carriers of DPYD c.1679T>G minor allele were identified . Out of 436 patients genotyped for UGT1A1, *1/*1, *1/*28 and *28/*28 genotypes were detected in 146 (33.5%), 251 (57.6%) and 39 (8.9%) cases, respectively  or DPYD c.2846A/T (n = 5) carriers experienced at least one grade ≥ 3 AE during the treatment. Seven (70%) out of 10 patients bearing a DPYD variant allele had a grade ≥ 3 AE within the first four cycles of induction therapy (DPYD c.1905+1G/G and DPYD c.2846A/A genotypes (P = 0.055). Most frequent AEs were neutropenia (70%), stomatitis (40%), diarrhea and febrile neutropenia (20%), and thrombocytopenia (10%).Eight (80%) out of 10  therapy  as compaDPYD c.1905+1G/A  and DPYD c.2846A/T  variants and grade ≥3 stomatitis . In the multivariate model, including age, sex, treatment arm and ECOG PS, both DPYD c.1905+1G/A  and DPYD c.2846A/T  variants retained their association with grade ≥ 3 stomatitis; the association between DPYD c.1905+1G/A and thrombocytopenia was also confirmed   (Table Significant associations were identified between both omatitis  and betwDPYD c.1905+1G/A or DPYD c.2846A/T variants (n = 10) had an increased risk of experiencing grade ≥ 3 overall hematological AEs , neutropenia , thrombocytopenia  and stomatitis , as compared to patients bearing DPYD c.1905+1G/G and DPYD c.2846A/A genotypes (n = 429) (P = 0.02) (Table Patients bearing n = 429) . These aUGT1A1*1/*28 or UGT1A1*28/*28 genotype, the incidence of grade ≥3 overall AEs was 136/251 (54%) and 24/39 (62%), respectively.Among patients carrying UGT1A1*28/*28 genotype experienced more frequently a grade ≥ 3 AE within the first four cycles of induction therapy  as compared to those carrying UGT1A1*1/*28  and UGT1A1*1/*1 genotypes  (P = 0.002).Patients bearing UGT1A1 variants and neutropenia (P = 0.001) with higher risk for UGT1A1*28/*28  than UGT1A1*1/*28 variants , both compared with the UGT1A1*1/*1 group. Consistent results were reported in the multivariable model (P = 0.001). UGT1A1 variants were also associated with the overall risk of developing hematological and overall AEs, both in the uni- and multivariable models.As shown in DPYD c.1905+1G/A or DPYD c.2846A/T and/or UGT1A1*28/*28 genotypes (n = 48) had a higher risk of experiencing grade ≥ 3 overall AEs , overall hematological AEs , stomatitis , neutropenia  and febrile neutropenia , compared to patients bearing DPYD c.1905+1G/G and DPYD c.2846A/A and UGT1A1*1/− genotypes (n = 387) (Patients carriers of n = 387) . These an = 387) .DPYD and UGT1A1 genotypes as predictor of overall grade ≥ 3 AEs were 14% and 92%, respectively.The sensitivity and specificity of the combined evaluation of DPYD and UGT1A1 variants as potential risk factors for toxicities in patients treated with fluoropyrimidines  and irinotecan, respectively [DPYD and UGT1A1 screening [DPYD genotyping [DPYD, slowed a widespread consensus on these tests and the definition of conclusive recommendations on the implementation in the clinical practice of a “genotype-guided” dosing of cytotoxic agents.In the last few years, a growing amount of mainly retrospective data suggested the role of ectively , 23. Nevcreening , 8, the notyping , 26. TheRecently, this controversy acquired growing remark in the precision medicine scenario, and in the perspective of individualizing therapies and improving patients’ safety and clinical benefit. Indeed, the preventive identification of patients deemed at risk of developing clinically relevant 5-FU- and irinotecan-related AEs, able to heavily affect treatment feasibility, may represent a useful and rationale tool to drive the therapeutic decision-making process, as well as treatment management.DPYD and one UGT1A1 variants in a large cohort of mCRC patients enrolled in the phase III TRIBE trial. With regard to DPYD, we focused on three SNPs whose relation with 5-FU-related toxicities is more robust [In the present work, we genotyped three e robust , as recoe robust .In our opinion, the main strength point of this analysis lies in the availability of a large cohort of patients enrolled in a clinical trial with homogeneous baseline characteristics, including type of cancer, stage of disease, and line of treatment, and with a careful and uniform assessment of treatment-related AEs, performed at every cycle of therapy and graded according to NCI-CTCAE, version 3.0.DPYD c.1905+1G/A and DPYD c.2846A/T genotypes and grade ≥ 3 hematological AEs and stomatitis, and between UGT1A1*28 variant and grade ≥ 3 hematological AEs, in particular neutropenia, regardless of the treatment arm. We were unable to assess any relation between DPYD c.1679T>G and 5-FU-related AEs, because none of our patients harbored a G allele, consistently with its low minor allele frequency (around 0.1% among Caucasians) [DPYD and UGT1A1 genotypes allows predicting with high specificity grade ≥ 3 hematological AEs, including febrile neutropenia, and stomatitis. Furthermore, in DPYD and UGT1A1 variant allele carriers we observed that grade ≥ 3 AEs occurred more frequently early during the treatment, within the first four cycles of therapy. These events were often clinically relevant, requiring dose modifications and/or delays in therapy administration as well as preventing treatment continuation in more serious cases.Through the analysis of 443 patients from TRIBE trial, we identified statistically significant associations between DPYD or UGT1A1*28 variants, some reports recommended to adopt a reduced starting dose of 5-FU and irinotecan, respectively, to be then increased based on reported toxicities, while not affecting treatment efficacy [i.e., FOLFOXIRI, FOLFIRI, XELIRI), supposing that the personalization of treatment starting doses according to DPYD and UGT1A1 genotypes might contribute to decrease the incidence of some preventable AEs. The lack of planned DPYD and UGT1A1 genotype-guided dose modifications in the TRIBE study clearly prevents us from deriving definitive conclusions about the effectiveness of the dose titration approach in reducing the incidence of AEs and improving treatment adherence, while not compromising its efficacy.In order to prevent the occurrence of treatment-related AEs in patients known to be carriers of efficacy , 28–32. DPYD variants, including c.2194G>A [A potential contribution to fluoropyrimidines toxicity has been recently hypothesized for other .2194G>A , 22. How.2194G>A –35, so tDPYD c.1905+1G>A and c.2846A>T and UGT1A1*28 variants for the assessment of the risk of AEs in all mCRC patients candidate to first-line 5-FU- and irinotecan-containing regimens.In conclusion, based on present results and consistent with literature data, our work supports the upfront test of TRIBE (TRIplet plus BEvacizumab) was a multicenter, randomized, phase III trial conducted by the Italian Cooperative GONO (Gruppo Oncologico Nord Ovest) group (NCT00719797). Five-hundred and eight unresectable mCRC patients, untreated for the metastatic disease were randomized to receive either FOLFOXIRI plus bevacizumab or FOLFIRI plus bevacizumab as initial treatment. Patients received up to 12 cycles of induction treatment according to randomization, followed by maintenance therapy with 5-FU and bevacizumab until evidence of disease progression in both arms. Full details of the TRIBE study have been previously published . The emei.e., who had received at least one cycle of the assigned study treatment), providing their written informed consent to blood sampling and pharmacogenetic analyses were evaluated. The protocol was approved by local Ethics Committees at each participating site.Patients enrolled in the TRIBE trial, included in the safety population . The DPYD and UGT1A1 variants were analysed by pyrosequencing method. DNA was amplified using the “Fluoropyrimidines response” and “Irinotecan response” kit  on a RotorGene TM6000 , according to the manufacturer's instructions. The reaction products were run on a PyroMark Q69 ID system, and the results were analysed on the PyroMark Q24 1.0.9 software .One ml of whole blood were taken from all patients, stored in EDTA, and genomic DNA was extracted from 200 μl by the Biorobot EZ1  was used (Table DPYD variant was compared to the published frequencies in dbSNP [DPYD and UGT1A1 genotypes.The study endpoints were the development of grade ≥ 3 gastrointestinal AEs, including nausea, vomit, diarrhea and stomatitis, and/or hematological AEs, including neutropenia, febrile neutropenia, thrombocytopenia and anemia. Logistic regression modeling was used to test the hypothesis of associations between the genotypes of each in dbSNP  and testP values of 0.05 or less were considered statistically significant.All statistical tests were two-sided and


CELA3A (p.G241A) and CELA3B (p.A241G), genetic analysis can directly assess whether individual variability in complex formation might alter risk for chronic pancreatitis. Here we sequenced exon 7 of CELA3A and CELA3B in a cohort of 225 subjects with chronic pancreatitis  and 300 controls of Hungarian origin. Allele frequencies were 2.5% for CELA3A p.G241A and 1.5% for CELA3B p.A241G in controls, and no significant difference was observed in patients. Additionally, we identified six synonymous variants, two missense variants, a gene conversion event and ten variants in the flanking intronic regions. Variant c.643-7G>T in CELA3B showed an association with alcoholic chronic pancreatitis with a small protective effect . Functional analysis of missense variants revealed no major defects in secretion or activity. We conclude that variants affecting amino-acid position 241 in CELA3A and CELA3B are not associated with chronic pancreatitis, indicating that changes in complex formation between proelastases and procarboxypeptidases do not alter pancreatitis risk.Human chymotrypsin-like elastases 3A and 3B (CELA3A and CELA3B) are the products of gene duplication and share 92% identity in their primary structure. CELA3B forms stable complexes with procarboxypeptidases A1 and A2 whereas CELA3A binds poorly due to the evolutionary substitution of Ala241 with Gly in exon 7. Since position 241 is polymorphic both in  PRSS1 (cationic trypsinogen), SPINK1 (pancreatic secretory trypsin inhibitor), and CTRC (chymotrypsin C) stimulate activation of trypsinogen and result in elevated trypsin activity in the pancreas [CPA1 gene encoding carboxypeptidase A1 were shown to increase risk for early onset CP [CPA1 variants exhibited a secretion defect due to intracellular misfolding and retention. The mechanism of action of CPA1 variants was unrelated to trypsinogen activation or trypsin activity and seemed to involve endoplasmic reticulum stress as a consequence of misfolding [Chronic pancreatitis (CP) is a progressive, relapsing inflammatory disorder of the pancreas which often develops in the background of genetic susceptibility ,2,3. Mutpancreas ,6,7,8,9.onset CP . The majsfolding . Howeversfolding  and refeCELA1, CELA2A, CELA2B, CELA3A and CELA3B. Due to evolutionary mutations, CELA1 is not expressed in the human pancreas and CELA2B is an inactive protease [CELA3A and p.A241G in CELA3B between subjects with CP and controls without pancreatic disease.The human chymotrypsin-like elastase (CELA) family is encoded by five genes: protease ,13,14. Cprotease ,15,16,17protease . We founprotease . NotablyCELA3A and c.722C>G (p.A241G) in CELA3B in subjects with CP and controls without pancreatic disease. We sequenced exon 7 and flanking intronic regions of CELA3A and CELA3B in 225 patients and 300 controls from the registry of the Hungarian Pancreatic Study Group. This CP cohort consisted of 120 alcoholic chronic pancreatitis (ACP) and 105 idiopathic chronic pancreatitis (ICP) patients. Sequence analysis of CELA3A and CELA3B was successfully completed for all patient samples and for 295 and 293 of the 300 control samples, respectively. In CELA3A we found 8 variants which included 4 intronic variants, 3 synonymous variants and 1 non-synonymous (missense) variant  variants and a gene-conversion event resulting in five nucleotide alterations that changed three amino-acids at the protein level  in  variant . Synonymin level . SynonymCELA3B variant c.643-7G>T in intron 6, which occurred with an allele frequency of 16% in patients and 21.3% in controls  while it was not significant in ICP patients  variant was found in one ACP patient and one control, whereas the heterozygous c.740G>C (p.R247P) variant was found in one control subject only. Both subjects with the p.V232L variant also carried a heterozygous c.643-7G>T CELA3B variant, while the subject with the p.R247P variant carried a heterozygous c.643-26C>T CELA3B variant. An unexpected observation in CELA3B was a heterozygous gene conversion event detected in a patient with ICP. A minimum of seven nucleotides in CELA3B between c.736 and c.742 was converted with the corresponding CELA3A sequence. This resulted in five nucleotide changes  and three amino-acid changes  in CELA3B , whereas the same association could not be verified in the ICP group (p = 0.52)  . Using a = 0.52) .CELA3B variant, we found no other association of CELA3A and CELA3B variants with CP or disease subgroups ACP and ICP. Importantly, the lack of association between variants at amino-acid position 241 and CP indicates that changes in complex formation between proelastases and procarboxypeptidases have no appreciable effect on the risk of CP.In summary, with the exception of the apparently protective c.643-7G>T CELA3A and CELA3B and the gene conversion in CELA3B alter elastase secretion and activity, we transfected human embryonic kidney (HEK) 293T cells with expression plasmids and measured proelastase levels in the conditioned medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and elastase activity by enzymatic assays. When elastase secretion into the medium was determined at 48 h after transfection, all variants were secreted normally, as judged by Coomassie Blue staining and densitometry of stained gels (CELA3B variant p.V232L exhibited a slight (~20%) but reproducible increase in secretion. Similarly, elastase activity of the conditioned medium after activation with trypsin showed no major differences among the variants and wild-type enzymes when measured with the Suc-Ala-Ala-Pro-Ala-p-nitroanilide chromogenic peptide substrate  in an independent cohort. None of the other variants showed a significant difference in frequency between CP patients and controls. Finally, we performed functional analysis on all missense variants detected in this study and found no major defects in proelastase secretion or elastase activity.In addition to the position 241 variants discussed above, we identified six synonymous variants, two missense variants, a gene conversion event and ten variants in the flanking intronic regions. Intriguingly, CELA3A and CELA3B in an individual with ICP. Gene conversion is a non-reciprocal exchange of genetic information between homologous DNA sequences, most likely as a result of mismatch repair following a heteroduplex formation between the donor and acceptor genes [PRSS1 and PRSS2 or between PRSS1 and the pseudogene PRSS3P2 were shown to generate pathogenic alleles that cause hereditary pancreatitis [CEL) and its pseudogene CELP was described as a novel genetic risk factor for CP [SBDS gene and its paralogous duplicated pseudogene SBDSP cause Shwachman-Bodian-Diamond syndrome, an autosomal recessive disorder with pancreatic exocrine insufficiency, hematologic dysfunction, and skeletal abnormalities [CELA3A and CELA3B was detected in a CP patient, however, this is likely an accidental finding as we found no functional defect with respect to secretion or activity of the converted CELA3B that might suggest a pathogenic role in CP.We discovered a rare gene conversion event between exons 7 of or genes . DNA excor genes  includinreatitis ,20,21. Mr for CP . Finallymalities . The newCELA3A and CELA3B are not associated with CP, indicating that changes in complex formation between human proelastases and procarboxypeptidases do not influence the risk for CP. The observation that intronic variant c.643-7G>T in CELA3B was significantly underrepresented in ACP patients suggests this might be a protective variant.In summary, our study demonstrated that variants affecting amino-acid position 241 in human CELA3A and CELA3B reference sequences . Note that the reference sequence for CELA3A contains the minor alleles at positions c.722 (p.241) and c.795+21. Similarly, in CELA3B positions c.780 (p.260) and c.795+21 represent minor alleles. To better contrast position 241 between the two genes, we used the major c.722G allele (p.G241) as a reference for CELA3A and designated the variant at this position as c.722G>C (p.G241A). Amino-acid variants are numbered starting with the initiator methionine of the primary translation products of CELA3A and CELA3B.Nucleotide numbering reflects coding DNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the CELA3A and CELA3B was successfully completed for 295 and 293 of the 300 control samples, respectively. Patient characteristics are summarized in SPINK1 variants; the p.N34S variant was found in three ICP patients and in one control subject while the c.194+2T>C variant was detected in two ACP and one ICP patients but not in controls [De-identified genomic DNA samples were obtained from the registry of the Hungarian Pancreatic Study Group . Individuals in the registry were recruited from 11 Hungarian centers between 2012 and 2016 and all gave informed consent according to the ethical guidelines of the Declaration of Helsinki. The study was approved by the Institutional Review Board at Boston University . A total of 225 unrelated patients with CP, including 120 with alcoholic CP (ACP) and 105 with idiopathic CP (ICP) and 300 control subjects with no pancreatic disease were studied. Sequence analysis of controls .CELA3A and CELA3B. Primer sequences and amplicon sizes are given in CELA3A were also sequenced with the forward PCR primer. New variants were confirmed by a second independent PCR amplification and also by sequencing the other DNA strand.Primers were designed against intronic sequences flanking exon 7 in CELA3A and CELA3B carrying C-terminal His tags in the pcDNA3.1(−) vector was described previously [Construction of expression plasmids for human eviously . Mutatio6 cells per well in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum, 4 mM glutamine, and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Transfections were performed using 4 μg expression plasmid and 10 μL Lipofectamine 2000  in 2 mL DMEM. After overnight incubation at 37 °C, cells were rinsed and overlaid with 2 mL Opti-MEM reduced serum medium (Gibco). Media and cells were collected 48 h after this medium change.Human embryonic kidney (HEK) 293T cells were cultured in 6-well tissue culture plates at a density of 102 solution. Proelastases were activated with 100 nM human cationic trypsin  for 30 min at 37 °C. Aliquots (20 μL) of activated elastases were then mixed with 175 μL assay buffer (0.1 M Tris-HCl (pH 8.0), 1 mM CaCl2, 0.05% Tween 20) and elastase activity was measured by adding 5 μL of 6 mM Suc-Ala-Ala-Pro-Ala-p-nitroanilide substrate. The release of the yellow p-nitroaniline was followed at 405 nm in a SpectraMax Plus384 microplate reader  for 5 min. Rates of substrate cleavage were calculated from fits to the initial linear portions of the curves and expressed in milliOD/min units.Aliquots (100 μL) of conditioned media were supplemented with 0.1 M Tris-HCl (pH 8.0) by adding 10 μL from a 1 M stock solution and 1 mM calcium by adding 1 μL of a 0.1 M CaClConditioned media (180 μL) were precipitated with 10% trichloroacetic acid ; the precipitate was collected by centrifugation and resuspended in 20 μL Laemmli sample buffer containing 100 mM dithiothreitol, heat-denatured at 95 °C for 5 min and run on 15% SDS-polyacrylamide gels. The gels were stained with Coomassie Blue R-250. Densitometric quantitation of bands was carried out with the Gel Doc XR+ gel documentation system and Image Lab software .


All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 3 Hemophilia A is an X-linked recessive bleeding disorder caused by a quantitative or qualitative deficiency of coagulation factor VIII (FVIII). FVIII is a cofactor for factor IXa (FIXa) that accelerates the activation of factor X, playing an essential role in the intrinsic pathway of the coagulation cascade3567Most proteins that pass through the eukaryotic secretory pathway are glycosylated on asparagine (N) residues of the consensus sequence N-X-S/T (X≠P) as they enter the ER1315161718Two major chaperone systems in the ER, BiP (GRP78) and calnexin/calreticulin (CNX/CRT), facilitate proper folding of proteins including FVIII1112www.factorviii-db.org). Some missense mutations were reported to decrease secretion or stability of the mutant protein2627293031The most common mutations in hemophilia A patients are missense mutations, with over 1,400 such mutations reported to date , the A2 domain (N582), the A3 domain (N1810) and the C1 domain (N2118). To investigate which consensus sites are actually glycosylated in cells, we expressed individual domains of FVIII  in both COS-1 and HEK293T cells and treated cell lysates with PNGase F to cleave N-linked glycans. After PNGase F treatment, proteins that contain N-linked glycans exhibit increased mobility in SDS-PAGE compared to untreated controls. The A1, A3, and C domains all showed increased mobility after digestion with PNGase F, but the A2 domain remained unchanged . Next, w333435Three reported missense mutations at N582 cause moderate to severe hemophilia A . These mutations are termed glycosylation defective mutations in the FVIII variant database . We intr10-helical turn or short helix composed of residues D580 to S584  is also associated with multiple mutations. All 3 amino acid mutations  were introduced into the A2 expression construct and analyzed for their glycosylation status in COS-1 and HEK293T cells  to block protein translation and carried out a CHX chase to compare the intracellular decay rates of four mutants, D580H, D580V, N582D and S584T in HEK293T cells. The WT A2 domain had a half-life of ~5.6 h with relatively slower degradation kinetics during the first 8 h after cycloheximide treatment . At 24 hAs part of ERAD, proteins that fail ER quality-control checkpoints are dislocated into the cytoplasm and degraded by the 26S proteasome. We used the proteasome inhibitor MG132 to explore the degradation mechanism of A2 missense mutations. MG132 was added to cell culture media 24 hours after transfection. After 12 h of treatment with 10 μM MG132, the intracellular level of the D580V mutant was increased approximately 4-fold . AlthougWe investigated the interactions between ER chaperones and five representative A2 mutants, D580H, D580V, E581K, N582D and S584T. Thirty-six hours after transfection with plasmids of WT A2 and A2 mutants, we collected cell extracts to analyze protein levels of BiP, CRT and CNX. Expression of all mutants other than D580V increased the protein levels of BiP to 1.5- to 2-fold of the WT A2 . However10-helix described above into a full-length FVIII expression plasmid (pMT2-FVIII). Forty-eight hours after transfection, cell extracts and conditioned media of COS-1 cells were harvested to detect the antigen level or activity level of FVIII. The protein level of FVIII D580V inside cells was lower than that of WT FVIII , consistent with our observation. Previous studies have compared N-glycosylation of recombinant FVIII expressed in different cell types, as well as plasma-derived FVIII . Full-length FVIII is N-glycosylated in 21 of 25 potential sites. Among the four N-glycans outside the B domain, N41 and N1810 carry complex structure, while N239 and N2118 are of mostly high-mannose type34333440Our results confirm that all of the consensus sequences outside the B domain are glycosylated in cells, with the notable exception of the A2 domain consensus site residue N582. Expression of isolated FVIII domains allowed detection of partially glycosylated species, such as the minor non-glycosylated A3 domain. A recent quantitative proteomic study found that N1810 at the A3 domain is ~80% occupied by an N-linked glycan  may nevertheless reduce the stability of the local structure and perturb overall A2 domain conformation, as the N582D mutant is not secreted  FVIII were cloned into the pED (A1 (1–379) and A2 (380–711)) or pZ (A3 1689–2019) and C 2020–2332)) expression vectors and C (22) expresMonoclonal antibodies against Flag, CRT and CNX were purchased from Sigma  and the monoclonal anti-BiP was from Santa Cruz Biotechnology . Monoclonal anti-human FVIII antibody, a gift from D. Pittman (Bayer), was conjugated to the AminoLink coupling gel agarose beads ThermoFisher . Cycloheximide (CHX) and MG132 were obtained from Sigma and the Peptide-N-Glycosidase F (PNGase F) enzyme was purchased from the New England BioLabs . Protein A/G Plus-agarose beads were purchased from Santa Cruz Biotechnology .2. Cells were transfected using FuGENE 6  according to the manufacturer’s instruction. Cells and conditioned media were harvested at various times after transfection for analysis. To control for variation in transfection efficiencies, experiments were independently performed 2–3 times. For stability assays, cells were treated with 200 μM CHX or 10 μM MG132 at 24 h post transfection. For CHX chase assays, cells were chased for 0, 2, 4, 8, 16, 24 hours, harvested, and lysed. After 12 h treatment with MG132, cell extracts were harvested for Western blot analysis.HEK293T and COS-1 cells were grown in DMEM supplemented with 10% FBS, 100 IU/ml penicillin and 100 IU/ml streptomycin at 37 °C and in 5% CO2) 36 h after transfection with WT A2, A2-N582D, A2-D580H or A2-D580V plasmids. After removal of debris by centrifugation , 30 μg proteins were digested on ice for 15 min by varying concentrations of trypsin (0–100 μg/ml) in 15 μl reaction volume.Cells were lysed in NP-40 lysis buffer . Aliquots of cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and Western blot analysis was performed using the appropriate antibodies. Images of chemiluminescent blot signals from horseradish peroxide-conjugated secondary antibodies were scanned and analyzed using ImageJ softwareCells were lysed in NP-40 lysis buffer supplemented with EDTA-free protease inhibitor cocktail (Roche Applied Science) on ice for 10 minutes. Lysates were cleared by centrifugation  for 1 hour at 4 °C. Beads were collected by centrifugation at 7,000 rpm for 1 min, washed three times with the NP-40 buffer and incubated with 40 μl SDS sample buffer at 100 °C 5 minutes. The supernatant was harvested and separated in a 10% SDS-PAGE gel. Western blot analysis was performed using appropriate primary antibodies.2 and 5% glycerol (buffer A). Immunoprecipitated FVIII in buffer A were divided into two aliquots for incubation in the absence or presence of 5 U/ml thrombin (Sigma) at 37 °C for 30 minutes. The resulting samples were separated in a 12% SDS-PAGE gel and visualized by exposing to an X-ray film using a Kodak Biomax Transcreen LE.Twenty hours after transfection, COS-1 cells transfected with PMT2-FVIII plasmids were metabolically labeled with [35S]-methionine/cysteine (250 μCi/mL in methionine/cysteine-free DMEM)  for 45 minutes, followed by a 30-minute incubation in complete medium. Cell extracts were prepared by lysis in the NP-40 lysis buffer and immunoprecipitated with anti-FVIII antibody coupled to CL-4B sepharose. Immunoprecipitated proteins were washed with NP-40 lysis buffer and PBS, and resuspended in 50 mM Tris-Hcl pH 7.5, 150 mM NaCl, 2.5 mM CaClForty-eight hours after transfection, conditioned media were collected for FVIII activity and antigen assays. The Coatest SP4 Factor VIII kit  was used to measure FVIII activity in conditioned media. WT mouse plasma was used as standards for activity assays. The VisuLize FVIII antigen kit  was used to analyze the antigen levels of FVIII in cell lysates and conditioned media. Activities and antigen levels of WT and mutant FVIII were subtracted from mock-controls.All data are presented as mean ± SEM. Statistical significance was calculated using two-tailed Student’s t test. p-values < 0.05 were considered significant for all assays.How to cite this article: Wei, W. et al. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII. Sci. Rep.7, 45033; doi: 10.1038/srep45033 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Fabry disease is a rare X-linked disorder characterized by deficiency ofα-galactosidase A, leading to progressive accumulation of glycosphingolipid in various organs, including the heart. Several studies have pointed out the unique pre-contrast T1 value character of classic Fabry cardiomyopathy, which is lower than the normal myocardium. In Taiwan, several recent studies pointed out a surprisingly high incidence of a later onset Fabry mutation (IVS4+919G>A). There is a lack of information about the T1 value of this Fabry variant cardiomyopathy in the literature. We aim to present the T1 value of this subtype of Fabry disease on cardiac MR.As referred from pediatric cardiologist, we collected cases with Fabry mutation (IVS4+919G>A), proved via gene analysis from blood sampling and endomyocardial biopsy. They underwent MR study from March 2015 to July 2015. We recorded the location of delayed myocardial enhancement according to AHA 16-segment model and measured the thickest segment of left ventricular (LV) in short axis view at end diastole. We used conventional look-locker sequence (pre- and post contrast) and the Circle CVi42 software to calculate T1 value of target myocardium. Another 16 normal volunteers (with age and gender matched) were collected and they underwent non-contrast cardiac MR with T1 map evaluation. The myocardial pre-contrast T1 values were compared by t test as follows: Fabry cases vs. control group, Fabry cases with left ventricular hypertrophy  vs. Fabry cases without LVH. The pre-contrast T1 values are compared by paired t test as follows: whole LV myocardium vs. interventricular setum vs. LV inferio-lateral wall, enhanced myocardium vs. non-enhanced myocardium.2. The T1 value of LV myocardium was significantly lower in Fabry variant cases than the normal control . No significant difference of T1 value of LV myocardium was noted between Fabry variant cases with and without LVH . The T1 value of whole LV myocardium and inferiolateral wall of fabry cases were both higher than that of interventricular septum of fabry cases . In fabry cases with delayed enhancement of LV, The enhancing myocardium demonstrated higher T1 value than non-enhancing myocardium .A total of 16 patients  were enrolled in this study and the basic characters of LV were as follows: ejection fraction of LV=64.0 ± 10.5%, LV stroke volum=103.2 ± 28.3 ml, LV mass=167.4 ± 68.3 gm, LV mass index=94.5 ± 37.8 gm/mIn our study, Fabry variant cases demonstrated lower pre-contrast T1 value of LV myocardium comparing with normal control cases. No significant difference of pre-contrast T1 value was noted between fabry variant cases with and without LVH.


Salsola soda allows a transition from C3 to C4 photosynthesis. A developmental transcriptome series revealed novel genes showing expression patterns similar to those encoding C4 proteins.The genome of  3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.Some species of Salsoleae (Chenopodiaceae) convert from C C4 is a carbon concentration mechanism that is thought to have evolved from C3 photosynthesis (C3) in response to conditions favouring photorespiration, for example hot and/or dry environments (4 plants takes place between mesophyll (M) and bundle sheath (BS) tissues of leaves  has become feasible and allowed extensive comparisons of gene expression between closely related C3 and C4 species. The understanding of C4 evolution has been significantly promoted by differential gene expression (DGE) analyses, for example in eudicots between C3 and C4 species of the genus Flaveria or from the Cleomaceae  Bunge were compared. The differences detected are not caused by variation between species, but can be associated with differences between C3 and C4, or to organ-related variation. However, due to its tree-like growth form, H. ammodendron is, in practical terms, not ideal as a model species. On the other hand, it belongs to the tribe Salsoleae and within this tribe, several species are known that conduct C3 in cotyledons and C4 in assimilating shoots/leaves . Finally, S. soda belongs to a tribe  that is diverse in C3, C3-C4 intermediate and C4 species .In the current study, we introduce 3 to a C4 metabolism by conducting deep sequencing of transcriptomes (RNA-seq) of five different developmental stages, from young seedling to adult plant. We reveal detailed insights into gene expression patterns based on the different organs and/or stages. All the transcripts predicted for a NADP-malic enzyme (NADP-ME) type species were highly abundant in leaves while being mostly absent in cotyledons. Consequently, the results of this study present the opportunity to perform detailed targeted investigations of genes encoding potentially novel C4 proteins.We investigated the transition from a CSalsola soda seed sample used in this study was collected on a stony beach of the Adriatic coast south-west of Trieste, near Strunjan, in the Naravni nature reserve  in Slovenia by M. Kaligaric s.n. (25 October 2012). A voucher of this collection is deposited at the herbarium of the University of Mainz (MJG no. 014562).The Salsola soda plants were grown in potting soil  in a glasshouse at the University of Mainz with supplementary light under a light intensity of ~300 µmol m−2 s−1. Samples were harvested during April and May 2014, between 10:30 and 13:00, and dried in silica, or immediately frozen in liquid nitrogen and stored at −80 °C for measurement of carbon isotope values and RNA extraction, respectively.Fresh plant material was fixed in AFE , dehydrated using an ethanol series, and embedded in Technovit 7100 (Heraeus Kulzer). Transverse sections, 5–10 µm thick, of the middle regions of fully developed cotyledons and leaves were made using a rotary microtome (Leitz) and stained in a 6:6:5:6 mixture of Azur II, Eosin Y, methylene blue and distilled water. Eukitt (O. Kindler) was used as the mounting medium and images of the sections were taken using a Leitz Diaplan light microscope combined with Leica Application Suite 2.8.1. For a more detailed description of the sectioning method, see S. soda were harvested (see above) and dried for several days in silica gel. Dry leaf samples were pulverized using the mixer mill MM 301 (Retsch). Approximately 200mg of each sample were used to determine stable carbon isotope ratios (i.e. 13C/12C) relative to the Pee Dee belemnite standard : stage 1 (yS), whole imbibed seed 2 d after germination; stage 2 (Cot), fully expanded, mature cotyledon, where the tip (±10mm) of the first leaf pair was visible (~6 d after germination); stage 3 (1L), mature first leaf pair and at this stage the tip of the second leaf pair was visible (~20 d after germination); stage 4 (2L), mature second leaf pair and again at this stage the tip of the third leaf was visible (~40 d after germination); and stage 5 (oL), mature leaf pair of an adult plant (~4 months after germinating).Total RNA was isolated from 18–120mg ground plant tissue with the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol (January 2011), including DNase digestion with the RNase-Free DNase Set (Qiagen). RNA quality and quantity were determined with the 2100 Bioanalyzer (Agilent Technologies), NanoDrop (Thermo Fisher Scientific), and Qubit (Life Technologies). For each sample, 500ng of high-quality, total RNA (RNA Integrity Number between 8.8 and 9.6) were used for cDNA library preparation with the TruSeq RNA Sample Preparation Kit (Illumina Inc.), following the Low Sample Protocol . Sequence determination of 100bp single reads was performed on an Illumina HiSeq2000 platform. Five different developmental stages of individual http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and filtered and trimmed using FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Raw reads were mapped onto coding sequences of the ‘minimal’ TAIR 9 transcriptome  of ≥1 was applied to call differentially expressed genes. To identify and visualize expression patterns of interest, hierarchical clustering using Euclidean distance (http://www.tm4.org/mev.html). To investigate which biological functions were active at each stage, transcriptional investment  was analysed using MapMan  in ground glass homogenizers following the method of S. soda are slightly flattened to biconvex and moderately succulent. Transverse sections of cotyledons show an isobilateral C3 anatomy, with one plane of vascular bundles (VB), including one distinct, main VB in the centre and up to six smaller VB surrounded by weakly developed water storage (WS) tissue  smaller VB at the periphery, most in close contact with Kranz cells  tissue . Towards) tissue . In contells KC; . ChloropS tissue . These w4 metabolism, since C4 plants discriminate less than C3 plants against 13C (13C) of the ten developmental stages from dry seed to the mature leaf of an adult plant confirm the anatomical observations: cotyledons of S. soda appear to perform C3 whereas leaves perform C4 (JXB online). Starting with C4-like δ13C values in imbibed seeds and young cotyledons (−15.83 and −17.34), the δ13C value clearly decreases and falls into a C3-like range during the mature cotyledon and young first leaf pair stages (−22.37 and −22.39). After this C3 phase of the development, the δ13C value increases again, and C4 values are exhibited from the mature first leaf pair stage onwards  imbibed seed (yS), (ii) mature cotyledons (Cot), (iii) mature primary leaves (1L), (iv) mature secondary leaves (2L), (v) leaves of a 4-month-old plant (oL).We looked at DGE by means of mRNA sequencing in five developmental stages of http://www.ncbi.nlm.nih.gov/sra/  and thus mapping was comparable between all five stages  terms that are significantly  more abundant at a particular stage compared with the other stages correlate with results from the MapMan bin analysis. Enriched GO terms within the 3311 transcripts showing increased abundance in yS compared to Cot encode proteins involved in processes related to transport, especially ‘nuclear transport’, ‘protein transport’ and ‘RNA transport’ . The 363When the 1L stage is compared to Cot, mRNA metabolism, including modification and editing of mRNA, anatomical structure organization and development, stomata and epidermis morphogenesis, carbohydrate metabolism, and secondary metabolite biosynthesis are enriched processes among the 1046 transcripts . While lComparing stage oL to stages 1L and 2L, we found the 786 transcripts showing increased abundance were enriched in GO terms that reflect regulatory functions in response and/or defence to stress and biotic stimuli, for example ‘regulation of defence response’, ‘regulation of response to stress’, ‘ion transport’ and ‘response to stimulus’ .enolpyruvate carboxykinase (PEP-CK) to form CO2 and PEP, with the latter entering gluconeogenesis. In Cucurbita pepo, PEP-CK activity is low in the first 2 d of seed germination and dramatically increases between days 2 and 4, followed by no detectable activity in young leaves , and 1L and 2L (representing C4).Since a major focus of this study was the developmental transition from C4 were significantly  more abundant in leaves compared to Cot (enolpyruvate carboxylase (PEPC) 1 and PEPC2, NADP-dependent malate dehydrogenase (NADP-MDH), aspartate aminotransferase 5 (Asp-AT5), NADP-dependent malic enzyme (NADP-ME) 1, NADP-ME2 and NADP-ME4, alanine aminotransferase 1 (Ala-AT1) and pyruvate orthophosphate dikinase (PPDK).Transcripts encoding most of the known proteins of the NADP-ME subtype of Cd to Cot . These i4 pathway, and transcript abundance does not always correlate with protein levels  1, DiT2, bile acid:sodium symporter family protein (BASS) 2, BASS4, sodium:hydrogen antiporter 2 (NHD2), phosphoenolpyruvate/phosphate translocator (PPT) 1  and PPT2 , pyrophosphatase 6 (PPase6), phosphoenolpyruvate carboxylase kinase 1 (PEPC-K1), PEPC-related protein (PEPC-RP) and PPDK regulatory protein 2 (PPdK-RP2).Increased abundance of transcripts encoding Cand PPT2 . Increas4, however, were not increased in one, two or all three leaf stages. These exceptions include: beta carbonic anhydrase 3 (β-CA3), which shows higher transcript abundance in 1L and 2L, than in oL; triose phosphate translocator (TPT) mRNA levels, which are increased in 2L, but not in 1L or oL; transcripts encoding PPDK-RP1 are higher in 2L and oL, but not in 1L; PEPC-K1 mRNA abundance is higher in all leaf stages than in earlier developmental stages, but still very low in 1L; transcripts coding for PEPC-K2 are higher in 2L and oL, than in 1L; PPase6 transcript abundance is increased in 1L and 2L, but not in oL  or PEP-CK C4 subtype behave as predicted if C3 and C4 conducting tissues are compared .Taken together, these results indicate that most of the genes coding for known proteins of NADP-ME C3 and C4 species found the components of photorespiration to be significantly higher in C3 than in C4 species  2-phosphoglycolate phosphatase (PGLP), (ii) glycolate oxidase (GOX), (iii) glutamate:glyoxylate aminotransferase (GGT), (iv) the multienzyme glycine cleavage system (GDC) comprising three enzymes P-, T- and L-protein and the substrate H-protein, (v) serine hydroxymethyltransferase (SHMT), (vi) serine:glyoxylate aminotransferase (SGT), (vii) hydroxypyruvate reductase (HPR1) and (viii) glycerate 3-kinase (GLYK)  or higher levels in stages 1L and 2L compared to Cot   . Enriche3 and 7) . Cluster3 and 7) . Cluster3 and 7) .4 cycle enzymes such as PEPC1 and PEPC2, NADP-MDH, Asp-AT5, NADP-ME isoforms 1, 2 and 4, Ala-AT1, PPdK, DiT2, BASS2 and BASS4, PPT1/CUE1 and PPT2, AMK2, and PPdK-RP1 and PPdK-RP2. Consequently, we carefully investigated the other genes included in cluster 3 and found two promising genes that code for transporters/transport-related proteins that might be involved in C4. The transcript encoding triose phosphate/phosphate translocator-related protein  is significantly more abundant (log2FC between 2.4 and 2.8) in all leaf stages compared to Cot. Transcripts coding for two other proteins with transport activity are also significantly increased (log2FC between 1.6 and 2.6) in leaves compared to Cot: phosphate transporter4.1  and PHT4.4 (AT4G00370), also known as anion transporter (ANTR) 1 and ANTR2, respectively.Cluster 3 includes many genes encoding C2FC between 1.9 and 2.2) in all leaf stages compared to Cot. PDH kinase is thought to control metabolite exit from the C4 pathway via pyruvate decarboxylation for entry into citric acid cycle, and transcripts encoding the protein were increased in Megathyrsus maximus, a NAD-ME subtype C4 species  are also significantly increased in all leaf stages compared to Cot (log2FC between 3.7 and 5.7).Increased abundance of transcripts encoding pyruvate dehydrogenase kinase  is also significant  behind the transition from C, and 18 . In tota, and 18 .S. soda, we expected to find a salsoloid type C4 anatomy in both organs, since this was previously reported in the literature .When we initially looked at cross sections of cotyledons and leaves of 4-like δ13C even though no C4 pathway is functional. This is because they develop using carbon stored in the seed; carbon fixed by an adult plant via the C4 pathway. The dry seed probably has a more negative δ13C value, compared to the imbibed seeds, because of its large lipid fraction, which was shown to have lower δ13C than other fractions/parts of the plant , suggesting that cotyledons do not contribute to carbon assimilation and energy gain in S. soda at all, and the occurrence of C3 in cotyledons is an evolutionary relict. However, carbon isotope data refute this suggestion in that cotyledons assimilate carbon via C3 to such an extent that a C3-like δ13C signature is detected in the young first leaf  of the C3 to C4 transition demonstrated by S. soda.Our rationale for Cral Asia , and seetabolism . Hence SS. soda conducts C3 in cotyledons before it switches to C4 in leaves. Salsola soda has ideal features to be an additional model plant for studying the genetic background and the biochemical assembly of setting up the C4 pathway: an annual life cycle, easy and fast to grow in the greenhouse, production of many seeds per plant each year, seeds with a high germination rate (in the first year), and the switch from C3 to C4 occurs within a single species and individual .In summary, we were able to provide evidence that JXB online.Supplementary data are available at Figure S1. K-means clustering (K=20 clusters) of all transcripts in all five stages. Table S1. List of carbon isotope values of ten stages including technical triplicates see .Table S2. Data availability of raw short read data of all samples uploaded to National Center for Biotechnology Information Sequence Read Archive.Table S3. Mapping of reads, transcript abundance and statistical tests of differential gene expression.Table S4. Enriched GO (gene ontology) terms highly represented in one stage (first column) compared to a certain other stage (second column).4 photosynthesis in the five developmental stages.Table S5. Expression pattern of genes involved in glyoxylate cycle, photosynthesis, photorespiration and CTable S6. Enriched GO (gene ontology) terms in yS-related clusters 8 and 11.Table S7. Enriched GO (gene ontology) terms in oL-related cluster 19.Table S8. Enriched GO (gene ontology) terms in Cot-related clusters 1 and 18 see .4-related clusters 3 and 7 (see Table S9. Enriched GO (gene ontology) terms in Cnd 7 see .Table S10. Transcriptions factors (TF) included in k-means clusters 1, 2, 3, 7, and 18 that show decreased (clusters 1 and 18) or increased transcript levels  in 1L and 2L compared to Cot . Supplementary_Figure_S1Click here for additional data file.Supplementary_Table_S1Click here for additional data file.Supplementary_Table_S2Click here for additional data file.Supplementary_Table_S3Click here for additional data file.Supplementary_Table_S4Click here for additional data file.Supplementary_Table_S5Click here for additional data file.Supplementary_Table_S6Click here for additional data file.Supplementary_Table_S7Click here for additional data file.Supplementary_Table_S8Click here for additional data file.Supplementary_Table_S9Click here for additional data file.Supplementary_Table_S10Click here for additional data file.