To examine concentration of Paraoxonase 1 enzymes across both Sudanese patients suffering from Type-I and Type-II diabetes.This was a cross-sectional study done in Khartoum/Sudan during the period from June 24th 2018 to August 23, 2018. One hundred seven diabetic patients (40 T1DM and 67 T2DM) compared with 45 healthy people from both genders. Biochemical parameters include PON1, FBG, HbA1C, and lipids were done and compared between study groups.PON1 was reduced in patients than controls (P < 0.01), in addition PON1 was lower in T1DM than T2DM (P < 0.01), moreover, FBG, HbA1c and lipids was higher in diabetes than controls (P < 0.05). PON1 inversely correlated with LDL and apo B in T1DM (P < 0.01) and T2DM (P < 0.05), in addition PON1 correlated with HDL and apo A1 in T1DM (P < 0.01), inversely correlated with LDL in T2DM (P < 0.05) and with apo A1 in T2DM (P < 0.01). Moreover, PON1 inversely correlated with diabetes duration in T1DM (P < 0.01) and T2DM (P < 0.05).Sudanese T1DM and T2DM have a lower PON1 concentration than healthy subjects, T1DM have lower level of PON1 than T2DM. PON1 was inversely correlated with bad lipids and duration of diabetes, but it has positive correlation with good lipids. PON1 decreases lipid peroxide accumulation in LDL due to its antioxidant ability against hydroperoxides thus reduce LDL and phospholipid oxidation, Thus, PON1 may be involved against atherosclerosis protection.3Paraoxonase 1 (PON1) is a 44 kD CaIn recent meta-analysis study that involved 35 studies worldwide, PON1 has been shown to decrease in DM and to be associated with the risk of diabetic macroangiopathy and microangiopathy.5The prevalence of diabetes in Sudan was high and increased from 3.4% in 2001In this cross-sectional study, 107 diabetic patients (mean age = 45±4.8) (40 T1DM and 67 T2DM)  were enrolled in study from Jabir Abu Aliz Diabetes Centre  during the period June 24th 2018 to August 23, 2018. In addition, 45 healthy subjects  as a control group were included for comparison. Inclusion criteria for diabetes group: HbA1c more than 6.5%, random blood glucose (FBS) ≥ 7.7 mmol/l. Exclusion criteria included kidney failure, chronic illness like cancerous patients, liver diseases, anemias and thyroid disease. The permission of study was taken by Research Ethics Committee - Applied Medical Sciences - Taibah University, Madinah, Saudi Arabia. In addition, ethical approval was taken from Khartoum State - Ministry of Health - Research Ethics Committee – Khartoum – Sudan. Both committees followed the ethical standards of the 1964 Declaration of Helsinki and its amendments.2) according to Quetelet equation.2), overweight was (25–29.9 kg/m2) and obese was (≥30.0 kg/m2).Body mass index (BMI) was measured as (kg/mBlood samples (3 ml) were collected from all participants in plain tubes after 12-hour overnight fasting and serum was obtained using standard centrifugation method. FBG, total cholesterol, triglyceride, HDL, LDL, apolipoprotein A1 (apo A1) and apolipoprotein B (apo B) were measured by using an auto-analyzer (Hitachi 704 Roche Diagnostics Switzerland). HbA1c measured by D-10™ Hemoglobin Analyzer Bio-Rad (Nyocard), PON1 concentration was measured with Elabscience’s ELISA kit (Sandwich-ELISA principle) using fresh serum. According to protocol, 100 µl of samples and standard were added to ELISA wells (pre-coated microplate PON1) and incubated for 90 minutes at room temperature (RT), then biotinylated  was added and incubated for one hour in RT. After wash (3 cycles), avidin-horseradish peroxidase (HRP) was added as secondary antibody and incubated for 30 minutes at RT. The plates were washed 5 times and the substrate reagent was then added to wells and incubated for 15 minutes at RT. The reaction was terminated with 50 µl of Sulphuric acid solution and plates were read at wavelength 450 nm using ELISA reader. The concentration of PON1 was calculated by plotting optical density on the standard curve.The data were analyzed using GraphPad Prism version five statistical program . Results represent as mean±SD. ANOVA (Tukey post hoc test for comparing 3 groups), t-test (for comparing 2 groups), chi-square (for non-parametric), and Pearson correlation were applied when needed. The scatter diagram designed with the GraphPad. P value ≤ 0.05 considered significant.Demographics  and biochemical data are shown in The most important finding in the current study was the significant decrease of the concentration of PON1 enzyme in both T1DM and T2DM compared to healthy people. This is in agreement with a previous study from Czech in both T1DM and T2DM,21Prolong hyperglycemia in patients with diabetes due to impairment of insulin pathway leads to oxidative stress (atherogenesis) and the subsequent induction of antioxidant mechanisms as a protective measure. PON1 plays a vital role as antioxidant enzyme, and has important role against oxidative damage. In DM, hyperglycemia causes glycosylation of proteins including enzymes and this process may lead to decrease their activity.With respect to other biochemical parameters, glucose, HbA1c, total cholesterol, triglyceride, LDL and apo B they were significantly higher in T1DM & T2DM than controls (P < 0.001). This is in agreement with Indian T2DM,12The results showed that HDL and apo A1 were decreased in both diabetic groups than control subjects (P < 0.001). This is in agreement with the Slovakian T2DM studyIt is well known that PON1 enzyme decreases accumulation of oxidized lipids from LDL because of its ability to decrease hydroperoxides as well as HDL that reduce the accumulation of lipid peroxides in LDL,23The decrease in PON1 enzyme activity reduces the ability of the body to normalize lipid-peroxidation, which thereby accelerates the oxidative stress. In diabetes there is overproduction of reactive oxygen species due to hyperglycemia or increased in free fatty acids and/or dyslipidemia.There were significant association between PON1 and period of diabetes in T1DM and T2DM. This is supported by previous findings that document a strong association between duration of diabetes and elevated the risk of developing of CHD, as an independent risk factor.Sudanese T1DM and T2DM have a lower PON1 concentration than healthy subjects, T1DM patients have lower level of PON1 than T2DM. PON1 was inversely correlated with bad lipids and duration of diabetes, but it has positive correlation with good lipids.


Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis. Human papillomaviruses cause various diseases associated with cellular hyperproliferation, including cervical cancer. Structural, biochemical, and cellular analyses reveal the molecular basis and significance of the intermolecular interaction between the E7 protein of human papillomavirus 18 and the human tumor suppressor protein PTPN14. Human papillomaviruses (HPVs) are small, double-stranded DNA viruses belonging to the papillomavirus family, which infect cutaneous or mucosal epithelia and induce cellular hyperproliferation –3. AmongPTPN14—also called PTPD2, PTP36, or PTP-Pez—is a member of the protein tyrosine phosphatase (PTP) protein family. In coordination with protein tyrosine kinases, PTP proteins control protein tyrosine phosphorylation, one of the key posttranslational modifications intimately associated with the regulation of various cellular signaling and biological processes ,30. PTPNIn this study, we confirmed the direct and potent interaction between the two domains and determined the crystal structure of the PTP domain of human PTPN14 in a complex with the C-terminal domain of HPV18 E7. It is the first crystal structure of HPV E7’s C-terminal domain bound to a host protein, and to the best of our knowledge, it is also the first PTP domain structure bound to a viral protein. The complex structure provided a basis for in-depth analysis of the E7-binding specificity of PTP proteins, which led to not only the identification of the nonconserved PTPN14 residues critical for complex formation but also verification of PTPN21 as a direct interacting partner of HPV E7. Through structure-based mutagenesis abrogating the HPV18 E7–PTPN14 binding, we demonstrate that the intermolecular interaction is significantly associated with HPV18-mediated proteasomal degradation of PTPN14, up-regulation of Hippo signaling downstream effectors, and enhanced cell proliferation, migration, and invasion, which was verified at a cellular level using nontumorigenic keratinocytes and HPV-positive or negative cervical cancer cells.6–maltose binding protein (MBP)-tagged C-terminal domain (residues 54–105) of HPV18 E7 were separately expressed in Escherichia coli, after which their cell lysates were mixed for copurification. After Ni–nitrilotriacetic acid (NTA) affinity chromatography, the eluted protein sample was treated with tobacco etch virus (TEV) protease and further purified using a second round of Ni-NTA affinity chromatography to remove the His6–MBP tag. Finally, the protein sample was subjected to size-exclusion chromatography (SEC), and the resulting sample was assayed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). As shown in KD) of the interaction was determined to be 18.2 nM   . The int8PWC101) . In deta8PWC101) . Additio8PWC101) . Notably8PWC101) .Based on the complex structure, we selected Arg84 and Leu91 of HPV18 E7 and Phe1044, Gly1055, and Glu1095 of PTPN14 as key residues in the binding interaction between the two proteins. ITC measurements confirmed that alanine substitution (R84A∙L91A) of the HPV18 E7 key residues or introduction of PTP1B-mimicking mutations (F1044S∙G1055Q∙E1095A) into PTPN14 completely abrogate the binding between the two proteins .Despite the fact that the asymmetric unit of our crystal contains one PTPN14 molecule bound to a single HPV18 E7 molecule, previous cellular –43 and sClassical PTP proteins, including receptor and nonreceptor types, share considerable sequence and structural homology among their catalytic PTP domain . This raThis hypothesis was validated by using ITC to measure the binding affinities between HPV18 E7 and PTP proteins including PTPN21, PTP1B, PTPN3, PTPε, and PTPσ. Indeed, it was confirmed that the C-terminal domain of HPV18 E7 interacts with the PTP domain of PTPN21 very potently, with a dissociation constant of 5.81 nM, but not with the other PTP domains . SEC-MAL6–MBP tag for further purification, we noticed that only the E7 C-terminal domain from HPV18, but not those from other genotypes we tested, was able to be purified to homogeneity  and PTPN14  were identified . We atteHaving known that human keratinocytes are the targets of HPV infection causing oncogenesis, the necessity of HPV18 E7 binding to PTPN14 in nontumorigenic keratinocytes was our next issue to be addressed. For this purpose, we transduced HaCaT cells with retroviruses encoding wild-type or the PTPN14 binding-defective AA mutant HPV18 E7 and with empty-vector control. The protein level of endogenous PTPN14 was confirmed to be significantly reduced in keratinocyte cell lines stably expressing wild-type HPV18 E7 but not in those expressing the AA mutant HPV18 E7 , furtherIn high-risk HPV-infected cervical cancer cells, the protein level of endogenous PTPN14 was reported to be very low because of E7-mediated proteasomal degradation ,24, consGiven that Hippo signaling is directly associated with cell proliferation, migration, and invasion, we suspect that degradation of PTPN14 might be one of the key processes implicated in cellular transformation and tumorigenesis. To assess this hypothesis, we established stable HeLa cell lines constitutively expressing empty vector (mock), wild-type PTPN14, or the SQA mutant PTPN14 and investigated the effects of PTPN14 expression on cervical cancer cells. E6 and E7 proteins are two defined oncoproteins encoded by HPV that are critically responsible for immortalization and transformation of HPV-infected human cells. Therefore, they have been under intensive investigation in order to understand their functions and molecular mechanisms. In this study, we verified the direct and potent molecular interaction of the C-terminal domain of HPV18 E7 and the PTP domain of human PTPN14. This was demonstrated by measuring the molecular weight of the complex by means of SEC-MALS , calculaIdentification of key residues of PTPN14  and HPV18 E7 (Arg84 and Leu91) for the complex formation enabled us to design mutant E7 and PTPN14 proteins defective in binding each other. It was confirmed by ITC using recombinant proteins  and coimOur structure-based sequence alignment led to the identification of PTPN21 as another direct binding partner of HPV18 E7 . The potIn this study, we provided the structural and molecular basis for how HPV recognizes human PTPN14 to be degraded via its E7 protein. They were subsequently proven to be relevant in various aspects of the HPV18-mediated cervical cancer cell physiology. Our structural analysis also identified human PTPN21 as a direct binding partner of HPV18 E7, which was biochemically verified. Our work therefore expands our understanding of the pRB-independent tumorigenesis pathway mediated by the HPV-encoded oncoprotein E7. We also expect that our observations would be rational grounds for therapeutic approaches toward HPV-implicated malignant diseases.6-linked MBP. Recombinant proteins were separately produced in the E. coli BL21(DE3) RIL strain (Novagen) cultured in 2 L (for PTPN14) or 4 L (for HPV18 E7) Luria-Bertani media at 25 °C for 16 hours. After centrifugation at 1,800g for 30 minutes at 4 °C, each of the cells expressing PTPN14 or HPV18 E7 were resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, and 5 mM βME and then mixed for copurification as the complex form. After sonication on ice, cell debris was removed by centrifugation of the cell lysate at 19,000g for 50 minutes at 4 °C. The supernatant was loaded onto a Ni-NTA column (QIAGEN) for initial purification. About 250 mg of the PTPN14–HPV18 E7 protein complex was eluted with 50 mL buffer containing 50 mM Tris (pH 7.5), 200 mM NaCl, 3 mM βME, and 300 mM imidazole. After the removal of the N-terminal tag from HPV18 E7 through 3 mg TEV protease treatment at 4 °C for 16 hours, proteins were subjected to a second Ni-NTA column and equilibrated with 50 mL buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 3 mM βME, and 50 mM imidazole. Eluted samples were then subjected to a HiTrap Q anion exchange column  with a gradient from 0 to 300 mM NaCl in a buffer solution containing 50 mM Tris (pH 7.5) and 3 mM βME. Recombinant proteins were further purified using a HiLoad 26/600 Superdex 75 pg gel filtration column  and equilibrated with a buffer solution containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 2 mM dithiothreitol. Crystals were obtained by the sitting-drop vapor-diffusion method at 18 °C by mixing and equilibrating 1 μL protein solution (10 mg/mL) and 1 μL precipitant solution containing 0.1 M Tris-HCl (pH 8.0), 25% (w/v) polyethylene glycol 3350, 0.25 M lithium sulfate monohydrate, and 2% (v/v) tert-butanol. Before the data collection process, crystals were immersed briefly in a cryoprotectant solution, which was a reservoir solution containing 10% glycerol. Diffraction data were collected on the beamline 5C at the Pohang Accelerator Laboratory, Korea, and processed using the program HKL2000 [The DNA fragment coding for the PTP domain of human PTPN14 (residues 886–1187) was cloned into the pET21a plasmid (Novagen). The DNA fragment coding for the C-terminal domain of HPV18 E7 (residues 54–105) was cloned into the pET28a plasmid (Novagen) that was modified to tag the protein with N-terminal (His) HKL2000 . The com HKL2000 , using t HKL2000  were use10-linked green fluorescent protein. The PTPN21 PTP domain was purified using a Ni-NTA column and subjected to TEV protease treatment and subsequent secondary Ni-NTA column purification. Each of the C-terminal domains of E7 from HPV1a (residues 39–93), HPV4 (residues 44–100), HPV11 (residues 45–98), HPV16 (residues 45–98), and HPV38 (residues 45–100) were cloned into the MBP-coding gene-inserted modified pET28a plasmid and purified similarly with that of E7 from HPV18.The PTP domain of PTPN14 containing the F1044S∙G1055Q∙E1095A mutation and the C-terminal domain of HPV18 E7 containing R84A∙L91A mutation were prepared and purified in a manner similar to that for the wild-type proteins. Each of the PTP domains of PTP1B (residues 1–321), PTPN3 (residues 628–913), and PTPσ (residues 1367–1948) were cloned into the pET28a plasmid, and that of PTPε (residues 107–398) was cloned into the pET21a plasmid (Novagen). These four PTP proteins were purified using a Ni-NTA column and dialyzed against a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 10 mM β-mercaptoethanol. The PTP domain of PTPN21 (residues 873–1174) was cloned into the pPRoEX HTa plasmid (Invitrogen) that was modified to tag the protein with N-terminal (His)Protein samples were diluted to a concentration of 6 mg/mL for the HPV E7 C-terminal domain or 3 mg/mL for the PTP domain and for the complex in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 2 mM dithiothreitol. SEC-MALS was carried out using a Superdex 75 10/300 GL column . The differential refractive index spectra were obtained using Optilab T-rEX (Wyatt Technology), which was combined with high-performance liquid chromatography (Shimadzu) and DAWN HELEOS-II (Wyatt Technology). The weight-average molar mass was calculated using ASTRA 6 software (Wyatt Technology).All measurements were performed at 25 °C on a VP-ITC microcalorimetry system . Protein samples were dialyzed against the solution containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 10 mM β-mercaptoethanol. In all experiments, PTP was titrated into HPV E7, in which a monomeric PTPN14 molecule interacts with a single HPV18 E7 molecule in a one-to-one binding mode. Prior to the measurements, the samples were centrifuged to remove any residuals. Dilution enthalpies were measured in separate experiments (titrant into buffer) and subtracted from the enthalpies of the binding between the protein and the titrant. Data were analyzed using the Origin software (OriginLab) as the one-to-one interaction mode. All experiments were performed in triplicate.Plasmids p6641 MSCV-P C-FlagHA 18E7-Kozak   and pcDN2. C33a cells were purchased from ATCC and cultured in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37 °C, 5% CO2. Transient transfection of plasmids into 293T, HeLa, and C33a cells was carried out using polyethylenimine (Sigma-Aldrich) and Lipofectamine 3000 (Invitrogen), respectively, according to the manufacturer’s instructions. For establishment of stable cell lines, retroviral supernatants were generated by transfection of 293T cells with target plasmids and viral packaging plasmids VSV-G and GAG-pol (Addgene) using Lipofectamine 3000 for 48 hours. Supernatants were filtered using a 0.45-μm filter and transferred into HeLa and HaCaT cells in the presence of 8 μg/mL polybrene. Infected cells were selected with 1–2 μg/mL puromycin for 7 days.HeLa, 293T, and HaCaT cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotic-antimycotic (GIBCO) at 37 °C, 5% COFor western blot analyses, cells were resuspended in phosphate-buffered saline (PBS). Samples were mixed with the 2X SDS sample buffer in a 1:1 ratio and boiled at 4 °C for 10 minutes. For immunoprecipitation analysis, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM ethylenediaminetetraacetic acid, and a protease inhibitor cocktail (Roche). The cell lysates were subjected to sequential immunoprecipitation using anti-Flag M2 affinity gel  overnight at 4 °C and Protein G-Sepharose  for 2 hours at 4 °C. The immunoprecipitates were then mixed with 2X SDS sample buffer and boiled for 5 minutes. Proteins were separated by SDS-PAGE and transferred onto a 0.2-μm nitrocellulose membrane . The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 for 1 hour and probed with the appropriate primary antibodies for overnight at 4 °C. The membranes were then washed with Tris-buffered saline with Tween 20 three times and probed with appropriate HRP-conjugated secondary antibodies . Proteins were detected using ECL reagents (Amersham) according to the manufacturer’s instructions. Antibody-bound proteins on the membrane were detected with ChemiDoc (Vilber Lourmat). The following antibodies were used for immunoblotting: anti-PTPN14 , anti-HSP90 , anti-Flag , anti-HA , anti-HPV18 E7 , and anti-GAPDH . MG132 and CHX were purchased from AG Scientific (M-1157) and Sigma-Aldrich (C4859), respectively.HeLa, 293T, and C33a cells were seeded in 24-well culture plates a day before transfection and transfected with TEAD-binding element reporter (8xGTIIC-luc), the control Renilla luciferase plasmid (pRL-SV40), and the plasmids expressing PTPN14 for 24 hours (HeLa and 293T cells) or 48 hours (C33a cells). After removing the growth medium, plates were washed with PBS to remove any detached cells and residual growth medium. The cells were lysed with 1X passive lysis buffer by rocking at room temperature, and the lysates were transferred to 96-well luminescence plates. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.Immunohistochemistry was performed on HeLa cells using an antibody against YAP/TAZ , PTPN14 , Alexa Fluor 594 , and Alexa Fluor 488 , which were used as fluorescent secondary antibodies compatible with the primary antibody. Cells were cultured on the confocal dishes and transfected with appropriate genes using Lipofectamine 3000 for 24 hours. The cells were then washed with PBS containing 0.1% bovine serum albumin and incubated with 4% paraformaldehyde for 20 minutes to fix them onto the dishes. To allow antibodies to pass through the cell membrane and to minimize nonspecific interactions, the cells were incubated with PBS-supplemented 10% goat serum and 0.3% Triton X-100 for 30 minutes. Then, the cells were incubated overnight at 4 °C with the primary antibodies against YAP and PTPN14 in 10% goat serum. The plates were washed three times for 5 minutes with PBS containing 0.1% bovine serum albumin prior to incubation with fluorescent secondary antibodies. The next day, the cells were washed and incubated with fluorescent dye–conjugated secondary antibodies for 1 hour at room temperature. ProLong Gold antifade reagent with DAPI  was used to mount immunostained cells and to stain cell nuclei. Then, the plates were covered with 0.8-mm coverslips and viewed using a fluorescence microscope IX2-UCB (Olympus). The numbers of total and YAP/TAZ-nuclear excluded cells were counted manually based on immunofluorescence images for calculating the percentage of cellular localization of HeLa cells in cytoplasm and nucleus. Data are expressed as the means ± SD of four immunofluorescence images from two independent experiments.For cell growth and survival assays, stable HeLa and HaCaT cells were seeded in a 48-well plate at 2,000 cells per well and were cultured for 0–4 days in 200 μL DMEM media containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution without medium change. C33a cells were transfected with appropriate genes for 24 hours using Lipofectamine 3000 prior to seeding cells in a 48-well plate at 2,000 cells per well for the short-term proliferation assay. Cell proliferation was analyzed using CellTiter-Glo luminescent cell viability assay kit (Promega). After 10 minutes of incubation, luminescence was measured with a luminometer Wallac Victor X3 (Perkin Elmer).4 cells per well in triplicate and then grown for 6 days. Stable HeLa cells were seeded in a 6-well plate at 1,000 cells per well in triplicate and then cultured for 15 days. Medium was changed every 5 days. For crystal violet staining, PBS-washed plates were incubated in 4% paraformaldehyde in PBS for 20 minutes and then stained with 0.5% crystal violet solution in filtered distilled water for 20 minutes. Plates were washed subsequently with filtered distilled water and dried, and images were captured using a camera. Stained area of cells or colonies was quantified using ImageJ Plugin. The number of stained cells or colonies was photographed and counted.Stable HaCaT cells were seeded in a 6-well plate at 4 × 104 cells/mL, 3 × 105 cells/mL, and 1 × 105 cells/mL for migration, respectively, in 100 μL of serum-free DMEM medium. Stable HeLa cells were also collected and suspended at a density of 1.5 × 106 cells/mL for the invasion assay in 100 μL of the same medium. The media containing 10% FBS was subsequently added to the wells of the plates. For the invasion assay, a thin layer of Matrigel  was added to the membrane matrix prior to seeding. On top of the inserts and Matrigel-coated inserts, 100 μL of prepared cells was added. The chamber plate was incubated for 24 hours in a cell incubator at a temperature of 37 °C and in an atmosphere of 5% CO2. The cells that invaded into the membrane were fixed with 4% paraformaldehyde (Biosesang) in PBS for 30 minutes and stained with 0.5% crystal violet  for 20 minutes. Migrated and invaded cells were observed with a microscope Eclipse Ts2 (Nikon), and images were obtained to count the total number of migrated and invaded cells.The migration and invasion assay was performed using Costar Transwell chambers containing an 8.0-μm polycarbonate membrane . HeLa cells stably expressing wild-type and the SQA mutant PTPN14, HaCaT cells stably expressing wild-type and the AA mutant HPV18 E7, and C33a cells transiently expressing wild-type and the SQA mutant PTPN14 were collected and suspended at a density of 3 × 10RNA isolation was performed with the easy-spin total RNA extraction kit (iNtRON) according to manufactures’ instructions. Extracted RNA was utilized for cDNA synthesis using MMLV reverse transcriptase (Promega) as described in the manufactures’ protocol. cDNAs were amplified by CFX Connect RT-PCR detection system (Bio-Rad) using specific primers and the EvaGreen RT-PCR kit (SolGent) to determine mRNA levels. Experiments were performed in triplicate, and each value was normalized to β-Actin. Primer sequences are listed in S1 FigCopurified protein samples were subjected to a HiLoad 26/600 Superdex 75 pg gel filtration column for final purification, and the resulting fractions were loaded onto SDS-PAGE and visualized by Coomassie blue staining together with size marker. (A) PTPN14 and HPV18 E7; (B) PTPN21 and HPV18 E7; (C) PTPN14 and E7 proteins from five different HPV genotypes. HPV, human papilloma virus; PTPN14, nonreceptor-type protein tyrosine phosphatase 14; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography.(TIF)Click here for additional data file.S2 Figα traces of two forms of the PTPN14 PTP domain are superimposed and shown in a stereo view. (B) Conformation of the catalytic pocket residues are structurally aligned and shown in a stereo view. Residues constituting the phosphate-binding loop and the WPD loop are present in sticks and labeled. HPV, human papilloma virus; PTP, protein tyrosine phosphatase; PTPN14, nonreceptor-type PTP 14; WPD, tryptophan-proline-aspartate.(A) C(TIF)Click here for additional data file.S3 FigDirect or water (represented as red spheres)-mediated hydrogen bonds between PTPN14 (violet) and HPV E7 (green) shown in sticks are indicated by dashed lines. Labeled are residues involved in hydrogen bonds. HPV, human papilloma virus; PTPN14, nonreceptor-type protein tyrosine phosphatase 14.(TIF)Click here for additional data file.S4 Figα traces represent proteins, and red circles indicate coordinated zinc ions. HA, hemagglutinin; HPV, human papilloma virus; PTPN14, nonreceptor-type protein tyrosine phosphatase 14.(A) Intermolecular interaction between Flag-tagged and HA-tagged C-terminal domain of HPV18 E7. The 293T cells were transfected with the indicated plasmids for 24 hours, and the interaction was examined by coimmunoprecipitation and immunoblotting. (B) Stereo view of packing of PTPN14 and HPV18 E7 molecules in crystals. Dimerization between symmetry-related HPV18 E7 molecules is indicated by a dashed rectangle. (C) Structural comparison between the E7 C-terminal domain from three different genotypes of HPV. C(TIF)Click here for additional data file.S5 FigThe sequence of the HPV18 E7–binding interface of PTPN14 is aligned with those of the corresponding regions of the PTP D1 domain (A) or PTP D2 domain (B) of receptor-type PTP proteins, as in (TIF)Click here for additional data file.S6 FigThe PTP domain of PTPN14 bound to HPV18 E7 is superimposed onto those of four nonreceptor-type PTP proteins  and two receptor-type PTP proteins (PTPσ and PTPε), as in (TIF)Click here for additional data file.S7 Fig(A) Sequences of the C-terminal domain of E7 from six HPV genotypes are aligned. The secondary structures of HPV18 E7 are shown at the top. Conserved residues are shaded in orange. Six residues in HPV18 E7 that play a key role in the intermolecular interaction with PTPN14 are marked by red arrows. (B) SEC-MALS analysis confirms the complex formation between the PTPN14 PTP domain and the E7 C-terminal domain from the indicated HPV genotypes. Solid lines, molar masses in kg/mol; dotted lines, refractive indexes. Constructs are listed in (TIF)Click here for additional data file.S8 Fig6–MBP-tagged E7 C-terminal domain protein samples from six different HPV genotypes were treated with TEV protease for 16 hours at 4 °C and then analyzed using a HiLoad 26/600 Superdex 75 pg gel filtration column. The peak fractions in each analysis were analyzed and visualized by SDS-PAGE and Coomassie staining. Red arrows indicate the fractions containing HPV18 E7(54–105) separated from His6–MBP. HPV, human papillomavirus; I, input; MBP, maltose binding protein; S, size marker; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TEV, tobacco etch virus.His(TIF)Click here for additional data file.S9 FigTwo PTPN14 samples were diluted to 0.6 mg/mL and prepared in a 20 μL reaction buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2 mM dithiothreitol, and 8X diluted SYPRO orange fluorescent dye (Applied Biosystems). Samples were pipetted into 8-well PCR tubes and sealed with optical flat strips (Bio-Rad). All experiments were performed using a Bio-Rad CFX96 real-time system, and the ROX reporter was chosen to collect fluorescent emission signals. Temperature was held for 5 sec at 0.5-degree intervals from 25 °C to 99 °C. The numerical data are included in (TIF)Click here for additional data file.S10 FigP < 0.001 in the Student t test. The numerical data are included in (A) Protein levels of transiently expressed wild-type and the SQA mutant PTPN14 were not affected by 20 μM MG132 treatment for 8 hours in 293T cells. (B) Interactions of wild-type and the SQA mutant PTPN14 and Hippo components YAP and LATS1 transiently expressed in 293T cells were examined by coimmunoprecipitation and immunoblotting. (C) Transcriptional activity of YAP in 293T cells with or without wild-type or the SQA mutant PTPN14 was measured by quantifying luciferase activity. The YAP activity was suppressed significantly by transient expression of both the PTPN14 constructs. ***(TIF)Click here for additional data file.S11 FigP < 0.05; **P < 0.01; ***P < 0.001 in the Student t test. The numerical data are included in The indicated PTPN14 constructs were transiently expressed in C33a cells. *(TIF)Click here for additional data file.S1 TableResidues that are derived from tags or translation-initiating methionine are marked in red. SEC-MALS, size-exclusion chromatography–multiangle light scattering.(DOCX)Click here for additional data file.S2 TableResidues that are derived from tags or translation-initiating methionine are marked in red. Residues removed during purification are underlined.(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableqRT-PCR, quantitative real-time polymerase chain reaction.(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Lastly, we demonstrated the presence of CX3CR1 on BM CD14+CD16+ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138 Tumor-promoting inflammation and angiogenesis are hallmarks of cancer . MultiplC-X3-C motif chemokine ligand 1 (CX3CL1), also known as fractalkine, is a protein of 373 amino acids with three main domains: Chemokine, mucin-like stalk, and transmembrane . In partThe pathophysiological role of the CX3CL1/CX3CR1 axis in the interaction between tumor cells and the microenvironment has been demonstrated in several solid tumors, such as breast and prostate and in different types of B cell lymphomas and chronic lymphocytic leukemia . A possiP < 0.0001, Kruskal–Wallis test). Specifically, CX3CL1 levels were significantly increased in the BM plasma of MM patients compared to HD, MGUS, and SMM patients   (median range BM CX3CL1 level in ISS I = 0.770 (0.46–1.530) ng/ml; II = 0.71 (0.394–1.460) ng/ml; and III = 1.38 (0.53–2.23) ng/ml). Moreover, BM soluble CX3CL1 levels were positively correlated with the percentage (%) of BM PCs checked by flow cytometry in the BM aspirates   in MM patients , 25 with smoldering myeloma (SMM), and 70 with active MM, and 10 healthy donors (HD) as controls. BM CX3CL1 levels significantly increased across the groups of subjects analyzed (median range BM CX3CL1 level in HD = 0.560 (0.420–0.730) ng/ml; MGUS = 0.665 (0.435–0.902) ng/ml; SMM = 0.720 (0.398–2.090) ng/ml; MM = 1.008 (0.394–0.230) ng/ml) (ey test) A. Based elation) C. On thepatients .+CD16+ shares both pro-osteoclastogenic and angiogenic properties and increased in MM patients compared to patients with asymptomatic disease [+CD16+ cells. Interestingly, we found that BM CX3CL1 levels positively correlated with the percentage of BM CD14+CD16+ monocytes  in the sub-cohort of the patients analyzed . A multivariate analysis confirmed that CX3CL1 BM levels significantly correlated with the percentage of both PCs (P = 0.003) and BM CD14+CD16+ monocytes (P = 0.0001).Moreover, since the monocytic subset CD14 disease ,25, we aonocytes D  . Multivariate analysis showed that BM microvessel density (MVD) significantly correlated with the number of PCs (P = 0.0001) and with the BM CX3CL1 levels even though statistical significance was not reached (P = 0.08).On the basis of the well-known involvement of CX3CL1 in angiogenesis , we sougelation) A. Represelation) C (P = 0.CX3CL1 mRNA expression by primary CD138+ cells and human myeloma cell lines (HMCLs) in public datasets (GSE16122) (GSE6205). Primary CD138+ cells and HMCLs expressed CX3CL1 (+ cells from four newly diagnosed MM (MM-ND) and four relapsed MM (MM-R) patients  and P = 0.0007; HBMESC + CM OPM2 vs. Cnt P = 0.0002; HBMESC + CM MM1S vs. Cnt P = 0.0006, Student’s t-test). In the same experimental setting, the treatment with anti-TNFα neutralizing antibody (2.7 µg/ml) for 24 h significantly inhibited the production of CX3CL1 by ECs induced by CM HMCLs.    A,B.stimuli from MM cells, we explored the CX3CR1 expression by MM cells and microenvironment cells. We found that HUVECs expressed CX3CR1 at protein levels  evaluated as median fluorescence intensity (MFI) by flow cytometry . The treatment with blocking anti-CX3CL1 monoclonal antibody (mAb) significantly reduced this pro-angiogenic activity   assay. As expected, BM plasma from four MM patients induced a significant increase of neo-vessel numbers compared to the negative control MM-11: BM plasma vs. negative control  t-test) A. FigureP < 0.0001; rhCX3CL1 2.25 ng/ml 418.25 ± 233.08 vs. anti-CX3CL1 mAb 55.5 ± 22.8, P = 0.027; rhCX3CL1 0.45 ng/ml 288.75 ± 99.72 vs. anti-CX3CL1 mAb 33 ± 9.8P = 0.021, Student’s t-test)  CX3CL1. For this aim, rhCX3CL1 was used at 20 ng/ml, which is known to be the maximum dose able to induce tube formation in HUVEC cells , and at  t-test) B. All thThe expression of the CX3CL1/CX3CR1 axis in MM cells and their microenvironment was defined in this study. We showed that higher levels of soluble CX3CL1 were present in the BM plasma of MM patients as compared with indolent monoclonal gammopathies. Moreover, we found that the CX3CL1/CX3CR1 axis was mainly involved in the increased BM vascularization and, consequently, in MM-induced angiogenesis.CX3CL1, also known as fractalkine, is a chemokine found either as a membrane-bound protein or in a soluble form after cleavage by the metalloproteases ADAM10  and ADAM+ PCs did not express CX3CR1 mRNA. Consistently, gene and protein expression analysis revealed the absence of CX3CL1 expression by PCs. Our data also indicated that ECs may contribute to the higher BM CX3CL1 levels in MM patients. The levels of BM CX3CL1 positively correlated with both the number of CD34+ vessels and with the percentage of BM PCs in bone biopsies. In agreement with what was reported by others [stimuli produced high levels of soluble CX3CL1. In the same way, ECs showed high CX3CL1 production after treatment with CM of several HMCLs supporting the notion that the cross talk between MM and the microenvironment cells stimulated the production of inflammatory cytokines. Indeed, it is well reported that MM cells secreted TNFα [Multiple Myeloma is typically characterized by a pro-inflammatory and pro-angiogenic microenvironment that supports MM cell growth and the progression of the disease . Howevery others ,40, ECs ted TNFα . Our in ted TNFα . Our stuAmong the different microenvironment cell types, we then confirmed that ECs expressed CX3CR1. Previous data demonstrated that the angiogenic property of CX3CL1 was mediated by the phosphorylation of extracellular signal-regulated kinases (ERK), Protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production . CX3CL1 +CD16+ also exists. In a multivariate analysis, we demonstrated that BM CX3CL1 plasma levels correlated with the number of BM CD14+CD16+. We and others previously reported higher levels of this subpopulation of monocytes in MM patients as compared to MGUS patients that are able to generate osteoclasts in vitro [+CD16+ non-classical monocytes either in physiological conditions or in the tumor microenvironment [+CD16+ monocytes [Our data in MM patients indicated that, other than the relationship between BM plasma CX3CL1 levels and BM MVD, a statistical correlation between CX3CL1 and the number of BM CD14in vitro ,42,43. Tironment . Moreoveonocytes ,45 suppoonocytes ,21.Thus, based on the relationship between CX3CL1 soluble levels with the angiogenic properties of MM patients, we next explored the contribution of the CX3CL1/CX3CR1 axis in MM-induced angiogenesis in pre-clinical models. The pro-angiogenic role of CX3CL1 has been investigated in two different models both in vivo and in vitro. In CAM assay, as expected, BM plasma from MM patients increased the neo-angiogenesis effect that was inhibited by the treatment with blocking anti-CX3CL1 mAb. Similarly, rhCX3CL1 stimulated tube formation at concentrations comparable to those found in MM plasma whereas the addition of blocking anti-CX3CL1 mAb inhibited this effect. Overall, these data demonstrated the role of this system in the pro-angiogenic properties of MM cells through the interaction with ECs and the activation of the CX3CL1/CX3CR1 axis.Our data support the possible role of this system as a potential therapeutic target in MM. Currently, there are no data reporting the use of modulators of the CX3CL1/CX3CR1 axis as anti-angiogenic drugs. However, the anti-inflammatory effect of blocking CX3CL1/CX3CR1 antagonists has been previously reported . A humanIn addition, the high-affinity small-molecule inhibitor of CX3CR1 (AZD8797) has been investigated in a rat model of multiple sclerosis where it blocked the infiltration of CX3CR1-expressing cells into the central nervous system . More reA total cohort of 111 patients  with PC disorders were included in the study until September 2018: 16 patients with MGUS , 25 with SMM , and 70 with active MM  including 49 MM-ND and 21 MM-R.Ten HD were used as controls. All patients were diagnosed according to the International Myeloma Working Group (IMWG) revised criteria . PatientBone Marrow aspirates  and bone biopsies were obtained from the iliac crest of each patient. BM plasma was collected after centrifugation, and stored at −20 °C until the analysis. Patient samples were obtained after informed consent, according to the Declaration of Helsinki. The study was included in a project approved by the Institutional Ethical Review Board of Parma Hospital (0006639-6.2.2 of 22 February 2017).® Cell Separation Media, CEDARLANE, ON, Canada). CD138+ PCs were isolated from BM MNCs by an immunomagnetic method with anti-CD138 antibody conjugated with microbeads  from MM patients, as previously described [Bone marrow mononuclear cells (MNCs) were obtained after gradient centrifugation with Ficoll solution  and analyzed with the OLYMPUS Stream software, adjusting tone and contrast to ensure the best image quality.Bone biopsy sections were incubated with mouse anti-human CD138 clone B-A38  and the reaction was revealed with polymeric ultraView Universal DAB detection  or with rabbit anti-human CD34 . After washing, sections were incubated with a secondary antibody (rat anti-Immunoglobulin G horseradish peroxidase (HRP); Millipore, Burlington, MA, USA; 1:250) and the reaction was revealed with a solution of 3,3′-diaminobenzidine tetrahydrochloride . MVD was evaluated as the number of CD34After MNC isolation, cells were collected and analyzed by flow cytometry using the following mAbs : Anti-CD138 R-phycoerythrin (PE) (552026), anti-CD14 Allophycocyanin (APC) (cod. 555399), anti-CD16 fluorescein isothiocyanate (FITC) (cod. 555406), and anti-CX3CR1 PE (cod. 565796). The analyses were performed on a two-laser FACSCalibur instrument (BD Biosciences) using CellQuest software v.4 (BD Biosciences).The gene expression profiles of CX3CL1 were evaluated in PCs of 4 HD, 11 MGUS, 133 MM, 9 PCL patients (GSE16122), and 23 HMCLs (GSE6205). The data were extracted from CEL files using a robust multi-array average (RMA) normalization procedure and custom chip definition file (CDF) annotation package , as previously described .The HMCLs JJN3, OPM2, U266, MM1S, NCI-H929, RPMI-8226, and the acute monocytic leukemia (THP-1) cell lines were purchased from Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH . The HMCLs XG1 and INA6 were obtained by the Laboratory of INSERM from Dr. Martine Amiot .The human bone marrow stromal cell line, HS-5, was purchased from ATCC . The human telomerase reverse transcriptase transduced mesenchymal stromal cell line (hTERT-MSCs) was kindly gifted from Dr. Giuseppe Gaipa . Immortalized human osteoblast-like (HOBIT) cells were kindly provided from Dr. B. L. Riggs . The human pre-osteocytic cells (HOB-01) were established from human bone and kindly provided by Julia Billars . HUVEC were obtained by ATCC. The human BM derived endothelial stem cells (HBMESC) were purchased from Celprogen  and both were maintained in culture with HBMESC complete growth media with serum (Celprogen) on a flask pre-coated with collagen type 1 . All cell lines were authenticated and tested for mycoplasma contamination.CM from HMCLs were collected after 48 h of culture and stored at −20 °C until use. In collagen-coated six-well plates, HUVEC and HBMESC were incubated in the presence or absence of diluted CM (1:3) of OPM2, JJN3, and MM1S for 24 h and with or without recombinant TNFα (50 ng/ml)  and/or neutralizing anti-TNFα mAb   for 24 h. HUVEC and HBMESC treated with TNFα (50 ng/ml) and IFNγ (10 ng/ml)  for 24 h were used as a positive control. At the end of the experiment, the culture media were collected and analyzed by enzyme-linked immunosorbent assay (ELISA).Soluble CX3CL1 was measured by ELISA  in BM plasma samples and in HMCLs, HUVEC, HBMESC, and co-cultured CM, according to the instructions of the manufacturer.+ cells purified from four MM-ND and four MM-R patients, as previously described [Western blotting was performed on the HMCLs  and on primary CD138escribed . Blots wovo using a stereomicroscope equipped with a camera and image analysis system . Blood vessels entering the sponges within the focal plane of the CAM were counted by two observers in a double-blind fashion at a magnification of 50×.Fertilized White Leghorn chicken eggs were incubated at 37 °C in constant humidity. On day 3 of incubation, a square window was cut in the shell of each egg, and 2–3 ml of albumen was removed to allow detachment of the developing CAM. The window was sealed with a glass coverslip, and the eggs were returned to the incubator. On day 8 of incubation, the coverslips were removed and the growing CAMs (10 eggs per group) were treated with 1 lL PBS (negative control); 1 µl PBS with 250 ng vascular endothelial growth factor (VEGF) ; 1 µl MM BM plasma treated or not with a neutralizing antibody to CX3CL1 0.3 µg/ml (R&D Systems). The coverslips were replaced after these treatments, and the CAMs were examined daily until day 12 of incubation, when they were photographed in In vitro angiogenesis was assessed by a V2a angiogenesis assay kit obtained from Cellworks . Endothelial cells were stimulated with VEGF  or suramin  or with recombinant CX3CL1  or with a neutralizing antibody to CX3CL1 (0.6 µg/ml) (R&D Systems). At day 14, cells were fixed and stained using an anti-CD31 Ab provided with the V2a angiogenesis assay kit following the manual instructions. Angioquant software was used to quantify the number and length of the formed tubules and the number of junctions.P value <0.05 was considered significant. GraphPad Prism 6 software was used for all the statistical analyses.Comparisons among the three groups were made with the Kruskal–Wallis test and pairwise comparisons with the Mann–Whitney test or with parametric unpaired t-test. The Spearman test was used for correlations. Relationships adjusted for potential covariates were examined by multiple linear regression. A In conclusion, our data indicate that BM CX3CL1 plasma levels are increased across the progression of monoclonal gammopathies from MGUS to MM and that the high CX3CL1 BM levels in MM correlated with the presence of a high grade of vascularization. The major source of CX3CL1 seems to be ECs that release CX3CL1 in the presence of MM cells with the involvement of the pro-inflammatory cytokine TNFα. By both in vitro and ex vivo angiogenic models we finally demonstrated the role of the CX3CL1/CX3R1 axis in the pro-angiogenic switch induced by MM cells. Our results underline the importance of the BM CX3CL1 levels as a possible new biomarker for the progression of MM related to the increase of the BM vascularization. The role of the CX3CL1/CX3CR1 axis as a new possible anti-angiogenic therapeutic target in MM is also suggested by our results.


A dual intramolecular electrochemiluminescence resonance energy transfer process is proposed with triple-component Pdots to enhance ECL emission, which greatly improves the ECL efficiency and can be used for sensitive and specific visual quantification of different targets. vs. 1 mM Ru(bpy)32+). Using metal–organic frameworks to carry the triple-component Pdots, a highly luminescent probe is proposed. By integrating the probe with target-mediated enzymatic circulation amplification and DNA arrays, a highly sensitive ECL imaging method is designed for simultaneous visual analysis of two kinds of proteins, mucin 1 and human epidermal growth factor receptor 2, on living cells, which exhibited linear ranges of 1 pg mL–1 to 5 ng mL–1 and 5 pg mL–1 to 10 ng mL–1 with limits of detection of 1 pg mL–1 and 5 pg mL–1, respectively. The proposed strategy showed promising application in bioanalysis.Polymer dots (Pdots) have become a type of attractive illuminant for electrochemiluminescence (ECL). However, the low ECL efficiency severely limits their practicability. Here, we design a dual intramolecular resonance energy transfer (RET) mechanism with newly synthesized triple-component Pdots to achieve great ECL enhancement. This mechanism efficiently shortens the path of energy transmission, thus greatly promoting the ECL amplification by 380 and 31 times compared to systems with no and single RET, and results in a relative ECL efficiency of 23.1% ( Owing to their good biocompatibility, low cellular toxicity, easy functionalization, high photostability, and size/surface-trap controlled luminescence, polymer dots (Pdots) have been widely applied as promising fluorescent bioprobes in single particle tracing, biosensing and cellular labeling.To improve the ECL performance of Pdots, some efficient approaches have been developed, such as introducing donor–acceptor (D–A) type32+ into Pdots, the RET from the excited Pdots to encapsulated Ru(bpy)32+ can enhance the ECL emission by 15.7 fold.2(mcpbpy)2+ (acceptor) into one molecule has showed enhanced ECL emission owing to the short path of energy transmission and less energy loss.P1 dots) are composited with carbazole, tetraphenylethene (TPE), and 2,2-difluoro-3-(4-methoxyphenyl)-4,6-diphenyl-2H-1,3l4,2l4-oxazaborinine (DMDO)  and human epidermal growth factor receptor 2 (HER2), on living cells was developed with specific recognition and DNA coding technology. The excellent performance of the proposed method indicates the promising application of the dual RET mechanism along with the triple-component Pdots in bioanalysis.As well known, ECL–RET from the donor to acceptor, similar to Förster RET (FRET), has demonstrated its ability to extend ECL applications to biosensinge (DMDO) . The care (DMDO) , the P1 P2–P4) were designed  (PSMA) as a functional reagent.In the dual intramolecular RET mechanism, AIE-active TPE plays key roles (A1/D2). As the proof-of-concept, three model polymers , indicating the intramolecular charge transfer process from the electron-rich carbazole donor to the electron-deficient DMDO acceptor via the TPE bridge linker.P1 dots with the emission of P2 dots in H2O demonstrated the feasibility of the second FRET process from the TPE moiety (A1/D2) to DMDO (A2) (ectively . The peaMDO (A2) .P1 in THF showed a weak emission situated at 412 nm with a shoulder peak at 433 nm  reached 70%. Meanwhile, the peak at 496 nm due to the emission of the TPE moiety gradually increased from fw = 0 to 30%, and then disappeared at fw = 40%, indicating the first intramolecular FRET process from the carbazole moiety to the TPE moiety.fw, a new emission peak assigned to the DMDO chromophore appeared at 527 nm, which gradually redshifted to 548 nm at a fw of 90% with an abrupt increase of the peak intensity at fw in the range of 80% to 90%, demonstrating the presence of the second intramolecular FRET process in P1 dots at high water fractions.The fluorescence spectrum of t 433 nm , which cP1 dots showed a fluorescence lifetime τ of 8.00 ns (CHISQ = 1.03), which was much longer than 1.58 ns of P2 dots (CHISQ = 1.00) (P1 dots. The fluorescence lifetime of 2.68 ns (CHISQ = 1.09) for P3 dots was also slightly longer than that of P2 dots. Therefore, the fluorescence spectra of P1 and P3 dots exhibited different emission peak wavelengths from that of P2 dots  , demonst P2 dots . When thn-propylamine (TPrA) as a co-reactant, a P1 dot modified glassy carbon electrode (GCE) showed the maximum ECL intensity, compared with the responses in the presence of other coreactants such as K2C2O4, Na2SO3 and TEA  and P4 dots (+0.93 V) but much lower than that of P3 dots (+1.13 V) (P1 dots (+ (D1˙+–A1/D2–A2), which produced the excited Pdots* (D1*–A1/D2–A2), and the energy was transferred to TPE and then to DMDO to generate excited D1–A1/D2–A2* for ECL emission. As a result, the ECL intensity for P1 was about 31- and 380-fold higher than those of single (P2) and no RET (P3) systems, respectively  . These p(P1 dots : TPrA waectively .–1P1 dots vs. 1 mM Ru(bpy)32+ was calculated to be 23.1% , a sensitive ECL imaging platform was developed. The detailed DNA sequences are listed in Table S3.The designed y Fig. S8, providiThe specific recognition of Apt1 toward targets was further demonstrated with confocal laser scanning microscopy (CLSM) imaging on the living cell surface . CompareP1 dots and metal–organic framework (MOF) nanoparticles (PCN-224) showed the spherical and monodisperse feature with an average diameter of around 11 nm  images of nd 11 nm  and 83 nnd 11 nm , respectsorption , which c PCN-224 . Moreover Fig. S9, which pr Fig. S9, and posr Fig. S9 could eadots–DNA . After ld 358 nm , attribut 348 nm , respectRct) successively increased. After incubating with S-DNA, the Rct further increased, indicating the successful capture of the released S-DNA in the target recycling mixture.The assembly process of DNA sequences on Au/ITO for ECL imaging was examined with electrochemical impedance spectroscopy Fig. S10. Upon th–1 to 5 ng mL–1 for MUC1 and 5 pg mL–1 to 10 ng mL–1 for HER2 with limits of detection (LODs) of 1 pg mL–1 and 5 pg mL–1 at a signal-to-noise ratio of 3, respectively  . The aveectively . These rectively , indicatP1 dots to greatly enhance the ECL emission of Pdots. The P1 dots contain two pairs of energy donors and energy acceptors, which reduce the path of energy transmission and energy loss, and thus show a high ECL efficiency at a relatively low ECL potential. Through loading DNA functionalized P1 dots on MOFs as a carrier, a sensitive ECL probe has been structured for highly sensitive bioanalysis by the introduction of target-mediated enzymatic circulation amplification as the third amplification process. The proposed ECL imaging platform shows excellent performance for sensitive and specific quantification of cell surface MUC1 and HER2 with an LOD in the pg mL–1 level, and can be used for screening of breast cancer cells. By using different aptamers to specifically code the targets, the designed method can be conveniently extended for simultaneous visual analysis of different targets. This dual intramolecular RET strategy provides a new concept for ECL enhancement, and broadens the application of Pdots in ECL imaging and bioanalysis.A dual intramolecular RET mechanism is proposed with newly designed triple-component The authors declare no competing financial interests.Supplementary informationClick here for additional data file.


Human papillomavirus type-6 (HPV6) is the major etiological agent of anogenital warts both men and women. The present study aimed to characterize the genetic diversity among HPV6 in Southwest China, and to investigate the origin of, selective pressure experienced by, and impact of the resultantly identified genetic variants on the HPV6 secondary structure.E6/E7 genes were estimated by Phylogenetic Analyses by Maximum Likelihood version 4.8 software.Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by Molecular Evolutionary Genetics Analysis version 6.0. The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6 and E7 gene sequences of HPV6 isolated from patients were sequenced and compared to GenBank HPV6 reference sequence X00203. The results of these analyses revealed that both the HPV6 E6 and E7 were highly conserved within the analyzed patient samples, and comprised only 3 types of variant sequence, respectively. Furthermore, the analysis of HPV6 E6 and E7 sequences revealed seven/five single-nucleotide mutations, two/four and five/one of which were non-synonymous and synonymous, respectively. The phylogenetic analyses of the E6 and E7 sequences indicated that they belonged to sub-lineage A1 and sub-lineage B1, whereas the selective pressure analyses showed that only the E7 mutation sites 4R, 34E, and 52F were positive selection.HPV6 was the most prevalent low risk HPV type in southwest China. In total, 143 E6 and E7 sequences were highly conserved within the analyzed patient samples in southwest China, indicating that the low risk HPV6 can adapt to the environment well without much evolution.HPV6 (detection rate = 13.10%) was very prevalent in southwest China, both the HPV6  Papillomaviridae, genus Alphapapillomavirus, species 10) [Human papillomaviruses (HPVs) are associated with a variety of epithelial lesions, including benign genital warts and cervical intraepithelial neoplasia . To datecies 10) , which bcies 10) . Generalcies 10) . Howevercies 10) –9.To date, extensive research has been conducted to investigate sequence variation among carcinogenic HPV types; nevertheless, only limited data is available regarding HPV6 variants, despite their significant impact on human health. Structurally, the HPV is a double-stranded, circular DNA virus that encodes E1, E2, E4, E5, E6, E7, L1 and L2 proteins . HPVs inE6 and E7 sequence variability among HPV6 isolated from cervical papilloma samples collected from patients in Southwest China. Phylogenetic analyses were conducted to compare the identified nucleotide sequences with those previously described in other ethnic populations. In addition, the secondary structure of the identified sequences were predicted to assess the probably impact of the low risk variants on overall viral function. The results of the study could provide important data for the research on HPV6 prevention, diagnostic, therapeutic and even the design of therapeutic vaccines based on proteins E6 and E7 in Southwest China.Genetic variability analyses have proven essential to facilitate an improved understanding of the evolution of the papillomavirus. A number of carcinogenic variants have been identified in HPV variants isolated from populations in Southwest China; however, only limited research has been conducted to identify low risk HPV variants. Thus, the present study aimed to analyze From May 8, 2013 to June 1, 2016, cervical swabs were obtained from patients  at the Affiliated Hospital of Zunyi Medical University, Angel Women’s and Children’s Hospital, Sichuan Reproductive Health Research Centre Affiliated Hospital, and the Chengdu Western Hospital Maternity Unit. Women over 18 years old with visible cervical lesions and/or HPV-related diseases  were eligible for inclusion. Specimens were stored at − 20 °C until DNA extraction and HPV typing. Specimens DNA were extracted and tested using the Human Papillomavirus Genotyping Kit For 23 Types  according to the manufacturer’s instructions . This kit enabled the classification of the 23 HPV types .E6 and E7 gene primers . The reaction mixture comprised 2.5 mM dNTPs, 2 U Taq DNA polymerase, 90 pmol of each primer, and sufficient 10× Mg2+ PCR buffer to reach a final volume of 25 μL. Amplification of the sample sequences was achieved using thermal cycling conditions comprising an initial denaturation step (5 min at 95 °C), followed by 39 amplification cycles . All amplified HPV6 E6 and E7 DNA products were detected via electrophoresis using an agarose gel that was impregnated with ethidium bromide, and sequenced using Sanger sequencing , and all the data were confirmed by repeating the PCR amplification and sequence analysis at least twice.In total, 216 samples were subjected to PCR amplification with http://bioinf.cs.ucl.ac.uk/psipred/), using default parameters. This software uses a very stringent cross-validation technique to achieve an average Q3 score of 81.6% [https://blast.ncbi.nlm.nih.gov/Blast.cgi), Primer Premier 5 (Premier Biosoft), and DNAMAN version 5.2.2 . HPV6 nucleotide positions were numbered in accordance with the GenBank HPV6 reference sequence (X00203).The secondary structures of the identified sequences were predicted with PSIPRED online software v.3.2 (of 81.6% . Any mutE6 and E7 variants using MEGA v.6 software (http://www.megasoftware.net/home) to apply Kimura’s two-parameter model. Tree topology was evaluated by bootstrap resampling 1000 times [“Maximum-likelihood” phylogenetic trees were constructed for the identified 00 times . The refE6 and E7 HPV6 gene sequences that were likely to be subject to positive selection were predicted by using PAML 4.8 software  to perform likelihood ratio tests (LRTs) to infer nonsynonymous/synonymous nucleotide divergence, according to the method described by Nei and Gojobor [ftp://ftp.ebi.ac.uk/pub/software/clustalw2/) [Sites within the  Gojobor –18. The stalw2/) .https://www.ncbi.nlm.nih.gov/nuccore/). Seven reference sequences were obtained .The sequence results were compared to reference sequences in NCBI  were collected. The overall positive rate of detectable 23 HPV types was 33.33% (3116/9349). 22.01% (2058/9349) specimens were high risk HPV types and 11.32% (1058/9349) were low risk HPV types. In all HPV-positive women involved in the study, the most common detected HPV types were HPV16 (detection rate = 13.12%) and HPV6 (detection rate = 13.10%)  identified seven polymorphic sites, two of which comprised non-synonymous , and five of which comprised synonymous mutations. One of the identified non-synonymous mutations, D14Y, occurred within a region that encodes the alpha helix, whereas three of the synonymous mutations occurred in regions encoding the α-helix or β-strand. Notably, the mutation A120T and G372A were discovered in all specimens  E6 sequences with reference sequences belonged to sub-lineage A1 (70.63%) and sub-lineage B1 (29.37%) , of which four substitutions were non-synonymous, and one substitutions were synonymous. The most common non-synonymous mutation, T155A, occurred with a frequency of 34.27%, resulted in the amino acid (AA) change of F52Y (Phe to Tyr). The C294A was the most common synonymous mutation, and occurred with a frequency of 34.26%. One non-synonymous mutation (F52Y) occurred in the E7 sequence encoding β-strand (Table E7 variants. The (297 bp) E7 variants mainly belonged to sublineages A1 (65.73%) and sublineages B1 (31.47%) , five single-nucleotide changes were identified within the 297 bp 03, five None of the identified variant sequences were predicted to introduce changes to the reference HPV6 E6 and E7 secondary structures , E34K (glutamic acid to lysine), and F52Y , were predicted to be subjected to positive selection.The non-synonymous/synonymous rate ratio (dN/dS), can measures selective pressure. When one non-synonymous mutation has advantage in fitness, it will rise to a higher rate than synonymous mutation, resulting in dN/dS > 1. The variable dN/dS ratios were tested among various lineages using the PAML 4.8 software. This program uses the Nei and Gojobori method of estimating synonymous substitutions, which is an unweighted pathway method. The first step in the procedure requires enumeration of the number of synonymous and non-synonymous sites present at each codon, where each site may be both partially synonymous and non-synonymous. The next stage is to determine the number of synonymous and non-synonymous changes between each pair of aligned sequences, codon-by-codon. The results of the conducted selective pressure  analysis of the identified HPV6 E6, the mutations A120T, C150G, A264T, and C291T were discovered in all sequence listed. The mutation G40 T was only found in HPV6vc . AA exchanges identified at nucleotide positions 40 (D14Y) were additional characteristic features. The mutation at nucleotide positions 55 (T19S) was not found in this study. For E7, A10G, G11A, and G100A were proved as new mutations in this study. The mutations C156T and A262G were not discovered in the research. All sequence listed contain the mutation C294A. Point mutations altered the E7 AA sequence of non-prototypic isolates, which contained specific mutations at nucleotide positions 155 (F52K). AA exchanges identified at nucleotide positions 10, 11 (R4E) and 100(E34K) were additional characteristic features. The mutation at nucleotide positions 262 (D88N) was not found in this study.Comparation between the results of our sequence with the reference sequence has been listed in the Table p53 can cause abnormal replication cell to lose control. The HPV genome can only be replicated along with the replication of the host genome. Therefore, E6 and E7 are especially important in HPV lifecycle. The present study sequenced the E6 and E7 ORFs in 143 HPV6 isolates from patients in Southwest China, to assess the HPV6 genetic diversity and evolution characteristics within this population, and help further identify specific HPV6 variants.E6 and E7 are essential HPV E-gene products, they target P53 and retinoblastoma (Rb) tumuor-suppressor proteins, respectively. The degradation of PRb can initiate abnormal cell replication; the inhibition of HPV6 was the second prevalent HPV type and the most prevalent low risk HPV type in southwest China, indicating that HPV6 is highly adaptable to the environment in southwest China compare with most HPV types that are not so prevalent.E6 sequence variants were identified, two of which induced corresponding E6 amino acid changes. One of these, H50Q (His to Gln), was identified in 30.08% of the analyzed HPV6 sequences, the amino acid at position 50 of prototype HPV6 E6 may be located centrally between the two internal Zn-binding motifs, which is important for the E6 protein stability [E6 sequence was shown to be highly conserved within the analyzed patient samples, which exhibited only two main genotypes, HPV6E601 and HPV6E603, that represented 67.13% (96/143) and 29.37% (42/143) of the samples, respectively. Likewise, five E7 variants were identified, four of which exhibited a corresponding E7 amino-acid change. The most common E7 nucleotide mutations were T155A and C294A. Notably, T155A mutation affected 34.27% (49/143) patients, and caused a F52Y (Phe to Tyr) amino acid conversion that was predicted impact the protein’s secondary structure. Furthermore, the E7 sequence also was proved to be highly conserved within the patient specimens, comprising only two main genotypes HPV6E702 and HPV6E703 that were exhibited by 31.47% (45/143) and 65.73% (94/143) of the analyzed patients, respectively, and the sequence of HPV6E703 is in conformity with the reference (X00203). The fact that both sequences were so highly conserved supports the hypothesis that they play vital roles in HPV6 structure and function [E6 and E7 were much more conserved than main high risk HPV types , may indicating low risk HPV has lower evolutionary pressure in gene level [Seven tability . These vfunction , 24, confunction . Thus, trence X003. The fane level , 26.Structurally, some of the detected mutations affected amino acids at critical positions related to known biological functions. In E6, one (I97) and three  mutations were found to affect the β-strand and α-helix-encoding regions, respectively, that are critical for structural stability. In E7, only one mutation (F52Y) was detected may affect the β-strand-encoding region. The nucleotides as positions 58, 61, 91, and 94 in the E7 protein sequence have been previously shown to act as zinc binding sites , but no E6 and E7 sequences are subject to positive selection in Southwest China. The main characteristic of positive selection is that it causes an unusually rapid rise in allele frequency, thereby enabling a species to adapt rapidly to environmental changes [E7, they may have evolutionary significance in making HPV6 adaptive to their environments.Notably, the present study is first to conduct an analysis to assess whether the  changes . The selE6 and E7 sequences within the patient specimens belonged to sub-lineage A1  and sub-lineage B1  [E7, a new sub-lineage was found, however, only three samples were detected, and therefore, it requires further study.Previous studies revealed the existence of two variant lineages  and five variant sub-lineages   among HP, LP243) . Lineage, LP243) , 31, and, LP243) , and forCertain strengths of this study were that all clinical samples were collected from the Southwest region of China which had a strong regional representation and several new mutations were discovered, which will provide real and valid data for development of therapeutic vaccines for affected people in the Southwest China. Thus, the results of the present study significantly expand the current knowledge of HPV6 genetic diversity in Southwest China, and also provide a valuable resource for future studies of HPV6 epidemiology, evolution, function, pathogenesis, and use as a therapeutic target. However, the cellular level research is required, and a series of cellular experiments about the mutations should be designed for future studies.E6 and E7 sequences were highly conserved within the analysed patient samples in southwest China, may indicating that the low risk HPV6 can adapt to the environment well without much evolution.HPV6 was very prevalent in southwest China, both the HPV6 Additional file 1: Table S1. HPV6 E6 and E7 primers. (DOC 32 kb)Additional file 2: HPV6 E6 reference sequence predicted secondary structure. Secondary structure within the reference sequence of HPV6 E6 protein. (PDF 14 kb)Additional file 3: HPV6 E6 variation sequence predicted secondary structure. Secondary structure within the variation sequence of HPV6 E6 protein. Variations of HPV6E601, HPV6E602 and HPV6E603 were integrated into one sequence to predict. (PDF 15 kb)Additional file 4: HPV6 E7 reference sequence predicted secondary structure. Secondary structure within the reference sequence of HPV6 E7 protein. (PDF 11 kb)Additional file 5: HPV6 E7 variation sequence predicted secondary structure. Secondary structure within the variation sequence of HPV6 E7 protein. Variations of HPV6E701, HPV6E702 and HPV6E703 were integrated into one sequence to predict. (PDF 11 kb)


Exhibiting high consistence in sequence and structure, S100 family members are interchangeable in function and they show a wide spectrum of biological processes, including proliferation, apoptosis, migration, inflammation and differentiation and the like. While the prognostic value of each individual S100 in ovarian cancer is still elusive. In current study, we investigated the prognostic value of S100 family members in the ovarian cancer.We used the Kaplan Meier plotter (KM plotter) database, in which updated gene expression data and survival information are from 1657 ovarian cancer patients, to assess the relevance of individual S100 family mRNA expression to overall survival in various ovarian cancer subtypes and different clinicopathological features.P = 0.012), S100A7A ,S100A10 ,and S100A16  were significantly correlated with worse OS in all ovarian cancer patients, while the expression of S100A1 , S100A3 , S100A5 , S100A6 , S100A13  and S100G  were associated with better prognosis. Furthermore, we assessed the prognostic value of S100 expression in different subtypes and the clinicopathological features, including pathological grades, clinical stages and TP53 mutation status, of ovarian cancer patients.It was found that high expression of S100A2  contains supplementary material, which is available to authorized users. As the leading cause of gynecologic cancer-related deaths worldwide, almost 75% ovarian cancer patients are diagnosed in late stage, losing the best time for operation. Thus, it is urgent to find new prognosis or therapeutic targets for ovarian cancer.2+ binding proteins, which are highly conserved and functioned as both intracellular Ca2+ sensors and extracellular factors [In humans, S100 family is consisted of 21 acidic-CaDysregulated expression of multiple S100 proteins have been found to be involved in the progression of numerous tumors. In addition, the protein expression of S100 family members has been used to facilitate diagnosis, predict prognosis, determine treatment options and monitor therapeutic efficacy , 3.The expression and function of S100 protein vary across different malignant tumors or tumor subtypes. For instance, in oral carcinoma, S100A2 acts as a tumor-suppressor, while tumor-promoter in lung carcinoma , 5. In owww.ncbi.nlm.nih.gov/geo/) and TCGA , which integrates the gene expression with clinical information. In order to examine the prognosis effect of an individual gene, the patients were divided into two groups in accordance with the expression level of this gene.We used an online database to assess the relevance of individual S100 mRNA expression to OS. The database was developed by use of gene-chip or RNA sequencing data with clinical information of the 1647 EOC  patients from GEO , 95 %CI  and log rank http://gepia.cancer-pku.cn/index.html), which is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx (Genotype-Tissue Expression) projects [Differential expression data between normal and ovarian cancer tissues was from the GEPIA  database , S100A7A , S100A10  and S100A16  mRNA high expression was significantly correlated with worse OS in all ovarian cancer patients . We also observed that the high expression of S100A1 , S100A3 , S100A5 , S100A6 , S100A13 , S100G  were associated with longer survival time , S100A5 , S100A6 , S100A8  and S100A13 , high mRNA expression predicted favorable OS in serous ovarian cancers respectively. However, S100A10  predicted worse survival. The rest members of S100 family were not related with prognosis effect in serous ovarian cancer , S100A13  and S100G  were associated with better OS, while S100A2  and S100A10, P = 0.022) predicted worse survival , S100A6 , S100A16  and S100B  was found to be associated with significantly worse OS in grade II ovarian cancer patients respectively. While, the S100A12 , S100A13  and S100Z  expression predicted better prognosis in grade II ovarian cancer patients (Table P = 0.0051), S100A10 , S100A16  and S100P  expression were found to be correlated with shorter overall survival time. While, the S100A3 , S100A5 , S100A6 , S100A8  and S100B  expression were related to better prognosis respectively.Furthermore, we examined the relationship between S100 expression and the clinicopathological features in the ovarian cancer patients, including pathologic grades, clinical stages and TP53 mutation status  and S100P  were associated with worse survival, while, S100A7 , S100A13  and S100G  were associated with better prognosis in stage I + II patients. In stage III + IV ovarian cancer patients, the expression of S100A10 , S100A16  and S100P  were correlated with worse OS and S100A11  with modestly worse OS; while, for S100A1 , S100A3 , S100A5 , S100A8 , S100A12  and S100B , the mRNA expression levels of them predicted favorable overall survival. S100G  was only slightly correlated with favorable OS but with no statistic difference. Figure P = 0.031), S100A11  and S100B  were related with worse survival in wild-TP53-type ovarian cancer patients, while, S100A2 , S100A3  and S100A5  were related with better OS in wild-p53-type EOC patients. We also found that the level of S100A7A , S100A12 , S100A14 , S100P  and S100G  predicted worse OS in mutant-p53-type EOC patients; however, S100A1 , S100A13  and S100A16  were associated with better prognosis in ovarian cancer patients , S100A7 , S100A9 , S100A12  and S100G  were associated with better OS for patients with surgery of optimal debulk.Compared with suboptimal debulk, the high expression of S100A3 , S100B  and S100G  high expression were associated with better OS. As for chemotherapy containing taxol. S100G  mRNA predicted better OS. As for chemotherapy containing platin and taxol, S100A12  and S100G  were associated with better OS for EOC patients. With regard to chemotherapy containing docetaxel, S100A3 , S100A4 , S100A7 , S100A8 , S100A12  and S100G  were associated with better OS.As for chemotherapy strategies containing platin, S100A5 . Interestingly, we also found that, compared to the other normal organs across the whole body, each S100 family member has extremely low expression in the normal ovarian tissue. However, in the tumor tissues, most members have a relative higher expression level in the ovarian cancer versus other organ tumor tissues 2 heterotetramer (AIIt), which results in the translocation of S100A10 to the plasma membrane [S100A10 is known to form (annexinA2)membrane . Extracemembrane .Furthermore, these processes mentioned above were thought to be associated with tumor invasion, metastasis and angiogenesis , 54. In S100A13 is a novel member of S100 family that characterized by its specificity in various form of cancer. Previously, S100A13 has been indicated to be a powerfully angiogenic biomarker for melanoma and astrocytic gliomas –59. ReceS100A16 was a recent addition to the S100 family that isolated from astrocytoma . The expS100P was first discovered in placental tissue. Accumulating evidences have suggested a link between S100P expression and progression of various types of cancers –72. In pAs intracellular Ca2+ sensors and as extracellular factors, this family of proteins modulates a wide spectrum of other cellular functions in the normal or non-cancerous conditions. We had reviewed some recently published literatures, and found that S100A12 has pro-inflammatory properties that are likely to be stable in an oxidative environment. Conversely, S100A8 and S100A9 may have important protective mechanisms in inflammation . In endoP value were fully analyzed, and the multivariate analysis by Logistical or COX regression could not be achieved for the reason of data limitation. Thus, the survival according to expression level of S100 family members might be cross- influenced by variable factors such as grade, stage, surgery and so on. Secondly, as for grade, secondary classification system about ovarian serous cancer, namely the low-grade serous carcinoma (LGSC) and high-grade serous carcinoma (HGSC), were recommended by 2014 WHO for the superiority that can be simple and easy to use, high repeatability, better able to guide the clinical treatment. While, the Kaplan-Meier database still used 2003 edition of the WHO classification method, namely, Silverberg grading system, which were divided into tumor Grade 1 (high differentiation), Grade 2 (moderate differentiation), Grade 3 (poorly differentiation). Thirdly, we can only obtain the differential expression information of each S100 family member between normal and ovarian cancer tissues, which provides clues to enhance the points indirectly. Additional file 2:Table S7. Patient numbers of low or high mRNA expression of S100 family members in different clinical and pathological features. Abbreviations: exp. -expression, n-number. (XLSX 41 kb)Additional file 3:Figure S2. Differential expression of S100 family members in normal and tumor tissues across 31 tumor types from the TCGA and GTEx data. Abbreviation: N-normal tissues; T-tumor tissues; ACC-Adrenocortical carcinoma; BLCA-Bladder Urothelial Carcinoma; BRCA-Breast invasive carcinoma; CESC-Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL-Cholangio carcinoma; COAD-Colon adenocarcinoma; DLBC-Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA-Esophageal carcinoma; GBM-Glioblastoma multiforme; HNSC-Head and Neck squamous cell carcinoma; KICH-Kidney Chromophobe; KIRC-Kidney renal clear cell carcinoma; KIRP-Kidney renal papillary cell carcinoma; LAML-Acute Myeloid Leukemia; LGG-Brain Lower Grade Glioma; LIHC-Liver hepatocellular carcinoma; LUAD-Lung adenocarcinoma; LUSC-Lung squamous cell carcinoma; MESO-Mesothelioma; OV-Ovarian serous cystadenocarcinoma; PAAD-Pancreatic adenocarcinoma; PCPG-Pheochromocytoma and Paraganglioma; PRAD-Prostate adenocarcinoma; READ-Rectum adenocarcinoma; SARC-Sarcoma; SKCM-Skin Cutaneous Melanoma; STAD-Stomach adenocarcinoma; TGCT-Testicular Germ Cell Tumors; THCA-Thyroid carcinoma; THYM-Thymoma; UCEC-Uterine Corpus Endometrial Carcinoma; UCS-Uterine Carcinosarcoma; UVM-Uveal Melanoma. (TIF 849 kb)Additional file 4:Table S8. Differential expression of S100 family members in normal and tumor tissues across 31 tumor types from the TCGA and GTEx data. Abbreviations: exp. -expression, n-number. (XLSX 16 kb)Additional file 5:Figure S1. Progressive free survival of S100 members in different ovarian cancer subtypes. Notes: (A-C) Survival curves of S100A1  are plotted for all/endometrioid/serous ovarian cancer patients. Abbreviation: HR: hazard ratio (TIF 131 kb)


Dear Editor,Among the wide array of human neoplasms, bone and soft tissue tumors originating from the skull bone are extremely rare, making up < 2% of all the musculoskeletal tumors . Skull bA total of 426 skull bone tumors including 27 subtypes diagnosed over a period of 10 years (from March 2005 to December 2016) were retrieved from the Sanbo Brain Hospital of the Capital Medical University . The mean age of the investigated patients was 33.0 years . Among all cases, 60 (14.1%) were diagnosed as benign tumors, 299 (70.2%) were malignant tumors and 67 (15.7%) were UNNTs , followed by parasellar  and frontal  regions  and pathological phenotype of tumor tissues. As shown in Additional file Compared with the benign population of this study, it was worthy to note that the calcification as one benign index was found to be predominated in the chondrosarcoma because most of the chondrosarcoma in this study  were at low grades with well differentiation. In addition, some specific neuroimaging signs such as the osteolytic lesion of solitary plasmacytoma of bone (SPB) and Ewing sarcoma  were the most common lesions in this study. They have many similarities in clinicopathology and neuroradiology , 10. CliThis study reported the largest series of skull bone tumors and provided a comprehensive assessment of their epidemiology, clinicopathology and neuroradiology to improve their differential diagnosis. The malignant tumors present the main subgroup across the whole neoplasms arising from skull bone and the operation combined with radiation remains the chief therapeutic strategy for the malignancies. It is noticeable that neuroradiography plays a critical role in approaching the differential diagnosis of skull bone tumors, thus providing an objective protocol for patient management.Detailed materials and methods are available in the Additional file Additional file 1: Table S1. Summary of general information, clinicopathologic features and follow-up results for 426 patients with skull bone tumors.Additional file 2: Table S2. Statistical analysis of CT and MRI characteristics of 426 cases with skull bone tumors.Additional file 3: Figure S1. Representative CT and MRI features in the current study (A) Sagittal bone-window CT presenting the actinomorphous-shape destruction of haemangioma. (B) Axial CT showing the irregular structural calcification in chondrosarcoma. (C) Axial enhanced T1WI displaying the extensive erosion in intraosseous meningioma. (D) Axial T2WI showing the cystic space with FFLs in haemangioma. (E) Axial contrasted T1WI showing the large cystic areas in myxoma. (F) DWI scan showing the obvious hyperintensity in myxoma. (G) Axial Flair image showing the expansive involvement in EWS. (H) Coronary T1WI with enhancement showing the invasive involvement in haemingioperithelioma. T1WI, T1 weighted image; T2WI, T2 weight image; DWI, diffuse weight image; FFLs, fluid-fluid levels.Additional file 4: Figure S2. Neuroimaging and histopathological findings in representative case of GCT. A 22-year-old man presented with mild frontal headache for about 3 months and the progressive blurred version for 1 month. Preoperative sagittal (A) and axial bone window (B) CT scans showing a large tumor in the sellar region with the hyperintensity and eggshell-like calcification. Preoperative axial T2WI (C) and DWI (D) displaying a solid-cystic lesion with hyperintensity on T2WI and hypointensity on DWI. Preoperative sagittal gadolinium-enhanced MRI (E) showing the lesion with heterogeneous enhancement. GCT consisted of mounts of osteoclast-like giant cells containing numerous round or spindle-shaped nuclei (F) and it was positive for CD68 (G) and CK14 (H). GCT, giant cell tumor; T2WI, T2 weight image; DWI, diffuse weight image. Scale bar, 10 μm.Additional file 5: Figure S3. Neuroimaging and histopathological findings in representative case of EWS. A 48-year-old woman suffering from the relapsed EWS presented with a right parietal headache and left upper limb dyskinesia for approximately 3 months. Axial bone window CT scan (A) showing the primary EWS eroding the right parietal skull bone. Sagittal enhanced T1WI (B) noting the secondary tumor involving the normal cerebral tissues with obvious peritumoral edema. Intraoperative photograph (C) presenting the EWS metastasizing to the brain with expensive involvement. EWS constituted the uniform small round cells with round nuclei and clear eosinophilic cytoplasm (D) and it was immunopositive for CD99 (E) and FLI-1 (F). EWS, Ewing sarcoma; T1WI, T1 weighted image. Scale bar, 5 μm.Additional file 6: Figure S4. Representative neuroradiological and histopathological images of FD A 25-year-old young woman suffered from frontal protrusion and facial appearance deforming for about 8 years. The preoperative coronary CT scan (A) showing the bone-forming calcification in the frontal bone. Preoperative T2WI (B) and contrasted T1WI (C) displaying the hypointensity and obvious enhancement with the ambiguous boundary in FD. The H.E. staining (D) showing the distributed bland fibroblastic cells and irregular trabeculae of woven bone. T1WI, T1 weighted image; T2WI, T2 weight image. Scale bar, 5 μm.Additional file 7: Table S3. Summary of general information, clinicopathologic features and follow-up results for 232 patients with chordomas and chondrosarcomas.Additional file 8: Table S4. Statistical analysis of immunostaining status of 232 patients with chordomas or chondrosarcomas.Additional file 9: Figure S5. Representative immunostaining features of chordoma and chondrosarcoma (A–C) Micrographs showing that chordoma was immunopositive for EMA and CK and negative for D2-40. (D–F) Images showing that chondrosarcoma was immunonegative for EMA and CK and positive for D2-40. Scale bar, 10 μm.Additional file 10: Figure S6. Survival analysis by Kaplan–Meier estimate among the patients with chordoma and chondrosarcoma (A) The survival of patients with dedifferentiated chordoma was poorer than the other groups (P = 0.0005). (B) There existed no significant difference of survival time among the three subgroups of patients with chondrosarcoma (P = 0.069).Additional file 11. Additional methods and materials.