The calcium-binding protein S100A12 correlates with measures of disease activity in patients with rheumatoid arthritis (RA). The protein reflects neutrophil activation and the present objective was to explore in a pilot study the associations between S100A12 and other inflammatory markers, clinical assessments as well as degree of synovitis detected by a comprehensive ultrasonography (US) examination in RA patients during biologic treatment.Twenty patients with RA were examined clinically and by use of US as well as laboratory markers S100A12, calprotectin, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) before starting adalimumab, with follow-up after 1, 3, 6 and 12 months. Ultrasonographic B-mode (BM) and power Doppler (PD) assessments of 78 joints, 36 tendons/tendon groups and 2 bursas were performed, and sum US scores calculated. Wilcoxon signed rank test assessed treatment response and Spearman rank correlation test was used to calculate correlations.The concentrations of S100A12 decreased after 3 months (p < 0.01) and significant correlations were found between S100A12 and the other laboratory markers during follow-up . Of the clinical assessments, S100A12 had highest correlations with the assessor’s global VAS . Compared with CRP and ESR, S100A12 showed higher correlations with the sum US scores (both BM and PD), with median (range) correlation coefficients of 0.55 (0.35-0.78 (NS-p < 0.001)) for sum BM scores and 0.45 (0.27-0.75 (NS-p < 0.001)) for sum PD scores.The S100A12 protein was significantly associated with other inflammatory markers, clinical assessments as well as sum US scores, indicating that S100A12 is a potential marker of inflammation in RA patients.The online version of this article (doi:10.1186/1471-2474-15-335) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is characterized by synovitis that untreated may lead to joint damage and functional disability. Even in clinical remission, a majority of patients with RA had ongoing synovial inflammation as detected by magnetic resonance imaging (MRI) or ultrasonography (US) . Use of S100A12 is a calcium-binding protein, expressed predominantly in neutrophils, but also in monocytes . It is rUS is a sensitive imaging modality for detecting synovitis in RA patients. The amount of synovitis is detected by B-mode (BM). The degree of vascularization, reflecting the inflammatory activity, is evaluated by use of power Doppler (PD). A comprehensive US examination of joints and tendons of RA patients may thus be able to reflect the total amount of ongoing inflammation in these structures.The major leukocyte S100 protein, calprotectin (hetero-complex of S100A8/A9), was investigated earlier in plasma samples of this cohort and correlated with the US sum scores . The preTwenty patients (15 women and 5 men) with RA according to the American Rheumatism Association 1987 revised criteria  with medBlood samples were drawn at inclusion and after 1, 3, 6 and 12 months of treatment with adalimumab. Conventional inflammatory markers included ESR and CRP, analysed by use of in-house methodology, with upper normal levels of 20 mm/h for ESR and 4 mg/L for CRP. Calprotectin in plasma was examined by use of enzyme-linked immunosorbent assay (ELISA) kits from CALPRO AS, Norway. Sera from all five examinations were frozen at −70°C. S100A12 concentrations in serum were determined by ELISA as follows: 96-well high binding Costar 3590 micro plates from Costar Corporation, USA, were coated with mouse monoclonal anti-S100A12 5 μg/mL in 0.1 M sodium citrate pH 6, 150 μL per well, for 18 h to 4 weeks at +5°C. Samples were diluted 1:5 using the sample dilution buffer. After washing the wells three times, 50 μL of calibrators or samples were added in triplicate wells and incubated at room temperature with shaking (500 rpm) for 40 min. A second monoclonal was conjugated with alkaline phosphatase. After washing again, all wells were added 50 μL of the ALP conjugate diluted 1:1000 and incubated again for 40 min. After a final wash, all wells were given 100 μL ALP substrate. Reading at 405 nm was performed when the optical density (OD) of the higher standard (512 ng/mL) was between 2.0 and 3.0. For samples with concentrations of 512 and 16 ng/mL the intra-assay coefficients of variation were 6.5% and 10.6% and inter-assay coefficients of variation were 2.3% and 10.9% respectively. The lower limit of quantification of the ELISA was 4 ng/mL. The reactivity and specificity of the antibodies were previously tested by ELISA using micro wells coated with S100A12 or calprotectin, which has considerable amino acid sequence homology to S100A12 [Forty joints  were assessed by one of two experienced nurses for tenderness and swelling, blinded for the results of the US examination. They also scored the global disease activity on a visual analogue scale . The disease activity score DAS28  was calcUS examinations were performed by one experienced sonographer  at the day of inclusion and after 1, 3, 6 and 12 months as previously described . A totalWilcoxon signed rank test was used to analyse the differences between the levels of S100A12, ESR, CRP and US scores before starting adalimumab treatment and during follow-up. Associations between laboratory markers, clinical evaluations and US scores were analysed by use of Spearman rank order correlations. IBM SPSS Statistics version 20 was used for the statistical analyses and p < 0.05 was considered statistically significant. All tests for significance were two-sided.The patients had median (range) S100A12 levels of 49 (18 to 475) ng/mL at baseline, 34 (18 to 687) ng/mL after 1 month, 23 (16 to 173) ng/mL after 3 months, 33 (19 to 394) ng/mL after 6 months and 40 (12 to 620) ng/mL after 12 months. The level of S100A12, ESR and CRP changed significantly from baseline to 3 months follow-up and ESR already after 1 month, while the sum US scores decreased significantly during follow-up (Figure There were significant correlations between S100A12 levels and sum US scores of all joints, tendons and bursae for BM and PD at baseline, after one month and 6 months. During the study, S100A12 showed higher correlation coefficients with the US scores than the conventional inflammatory parameters CRP and ESR (Table The present one-year follow-up study of RA patients starting treatment with adalimumab is the first to analyse the associations between serum levels of S100A12 and a comprehensive US joint examination, and significant correlations were currently found.All the inflammatory laboratory markers decreased during the study. However, they tended to increase at 6 months onward Figure , which cThe S100A12 serum levels were lower in this study than those reported for RA patients with active arthritis . The decS100A12 correlated better and more consistently with the comprehensive US sum BM and PD scores than the conventional inflammatory parameters (Table Although both S100A12 and calprotectin are released by neutrophil activation, their correlations during follow-up were presently found to be variable. S100A12 was not superior to calprotectin as a biomarker for RA in this cohort, but the associations between these two S100 proteins and clinical assessments and US scores should be explored in further studies. Also S100A12’s ability to predict destruction should be assessed in larger cohorts of RA patients.In a large cohort of patients with juvenile idiopathic arthritis it was found that increased levels of S100A12 and calprotectin, in addition to high-sensitivity C-reactive protein, indicated subclinical inflammation and thereby could identify patients with increased risk of relapse when in clinical remission . WhetherThe present study has several strengths. Primarily, this is the first study exploring the associations between S100A12 serum levels and a comprehensive US examination. In addition, it is a longitudinal 12 months follow-up study, giving the possibility to study changes in S100A12 during treatment with biologic medication. The weakness of this pilot study is the limited number of patients included, which reduces the strength of the present findings.In this pilot study of 20 RA patients followed for 12 months after initiation of adalimumab treatment, serum levels of S100A12 were associated with clinical and laboratory assessments as well as with a comprehensive ultrasonographic examination. This indicates that S100A12 could be a biomarker of synovitis in RA, and these promising findings should be explored further in larger cohorts of RA patients to evaluate S100A12 as a marker of inflammation.


Cervical cancer (CC) is a leading cause of mortality in females, especially in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (hrHPV), and hrHPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. STK31 gene of which the expression has been proven to be regulated by the methylation status of its promoter, is one of the novel cancer/testis (CT) genes and plays important roles in human cancers. Reasearches have indicated that viral infection is correlated to the methylation statuses of some genes. Herein, we detected methylation status of the STK31 gene in cervical tumors and explored its interaction with HPV16 or/and HPV18 (HPV16/18) infection.Bisulfite genomic sequencing PCR (BGS) combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation statuses of the STK31 gene promoter/exon 1 region in HPV16/18-positive, HPV-negative CC cell lines; ectopically expressed HPV16 E6, -E7, and -E6/E7 CC cells; normal cervical tissues and cervical tumor tissues of different stages. The mRNA and protein expressions of STK31 were detected by RT-PCR and western blotting.The STK31 gene promoter/exon 1 was hypomethylated in the HPV16/18-positive cell lines HeLa, SiHa and CaSki, and the mRNA and protein expression were detected. In contrast, the STK31 gene exhibited hypermethylation and silenced expression in the HPV-negative CC cells C33A and HT-3. Compared with the primary HPV-negative CC cell lines, the STK31 methylation was downregulated, and STK31 expression was induced in the HPV16E7/E67 transfected cells. The methylation statuses and expressions of STK31 were verified in the cervical tumor samples at different stages. Additionally, chemotherapy treatment may influence STK31 expression by regulating its methylation status.STK31 may be a novel cellular target gene for the HPV16 oncogeneE7. The HPV16 oncogene E7 may affect STK31 expression through a methylation-mediated mechanism. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor of human cervical carcinogenesis and a potential DNA aberrant methylation biomarker of conditions ranging from precancerous disease to invasive cancer.The online version of this article (doi:10.1186/s12985-016-0515-5) contains supplementary material, which is available to authorized users. CC is a leading cause of mortality in females, especially in developing countries . The twoResearch has indicated that epigenetic abnormalities, particularly aberrant methylation changes, play causative roles in tumorigenesis , 7. ThouIn this study, we investigated the influences of HPV16 E6 and E7 on the expression and methylation status of the STK31 gene in CC cell lines. Additionally, we tested the methylation levels and expression changes in STK31 in HPV16/18-positive and HPV-negative cervical lesions in different stages. We also explored the correlations between chemotherapy and STK31 gene methylation changes in CC cases.The transfection efficiency was tested by western blotting, which revealed that the transfected cells successfully expressed the E6, -E7, or -E6/E7 proteins Fig. . The RT-To investigate whether STK31 expression could be regulated by its promoter/exon 1 region, we tested the methylation status of STK31 promoter/exon 1 region. The analyzed STK31 gene sequence contains 37 CpG sites in its promoter/exon 1 region. The BGS statistics  were studied with qPCR. The expression of STK31 was higher in the HPV16/18-positive cervical tissues among CC tissues and CIN3 specimens than those in the HPV-negative ones  and 22.3 % of the CC cases (33/148). Examples MSP analyses are presented in Fig. p < 0.05), and no expression was detected in the HPV16/18-positive normal cervical tissue, HPV16/18-positive CIN1 or HPV16/18-positive CIN2 tissues. The qPCR results .Regarding the clinical samples, the statistical tests of the qPCR data Fig.  demonstrlts Fig.  revealedTo further validate whether STK31 expression is regulated by its methylation status, HeLa, SiHa, CaSki, C33A and HT-3 cell lines were treated with 5 μM and 10 μM 5-aza-dC for 4 days. BGS analysis  and was not associated with age, the type of cancer, lymph node metastasis, cancer grade, the FIGO stage, tumor size, HPV genotyping or Squamous cell carcinoma antigen (SCC-Ag) level .The relations between the methylation status of STK31 and the clinicopathological characteristics in the patients with CC were analyzed. The hypomethylation status of STK31 was significantly associated with HPV16/18 infection, chemotherapy treatment and chemotherapy resistance (p < 0.05); thus, we assessed the correlation between chemotherapy and STK31 methylation status in the CC patients. The methylation status of the STK31 promoter/exon 1 was evaluated via the BGS and MSP methods in 10 HPV16 cervical patients before and after neoadjuvant chemotherapy. The BGS data revealed that 8 of the 10 CC patients exhibited nearly complete hypermethylation STK31 prior to neoadjuvant chemotherapy and exhibited no methylation changes after chemotherapy (T1-T4 are shown as examples of the cancer patients exhibited nearly complete hypermethylation STK31 prior to neoadjuvant chemotherapy and exhibited no methylation changes after chemotherapy), and 2 cases (T5-T6) of the 10 CC samples had statuses of hypomethylation prior to neoadjuvant chemotherapy and exhibited hypermethylation after chemotherapy , revealing the aberrant methylation of STK31in the patients with CIN3 may indicate a higher risk for the development of CC. Because the aberrant methylation of STK31 in the CIN3 biopsy cases indicated an increased possibility of developing into CCs, individual therapy may be needed and of great importance for such CIN3. Interestingly, CIN2 and CIN3 are classified together as high-grade intraepithelial lesions (HSIL), but our study found that the STK31 methylation in CIN2 is different from that in CIN3. Although CIN2 and CIN3 are HSILs, studies have shown some different indexes between CIN2 and CIN3 [A small proportion of women with HPV infections that are left untreated develop invasive cervical carcinomas; therefore, the positive predictive value (PPV) of HPV testing for CC screening is very low. Additionally, there is no well-established national CC screening program in China , 17. Theand CIN3 , 23, it’The development of chemotherapy resistance requires changes in the expressions of a number of genes. It has been hypothesized that epigenetic therapy holds the potential to reverse chemotherapy resistance . A varieBased on the reversible nature of epigenetic aberrations, a number of DNA methylation and histone deacetylase (HDAC) inhibitors are being preclinically tested in combination for cancer therapy. These epigenetic therapeutic drugs have potential therapeutic effects due to abilities to exert synergistic effects on gene expression , 29 and In conclusion, though the processes that govern the degree of gene promoter methylation in HPV-induced carcinogenesis is quite complex, the present study primarily revealed that STK31 may be a novel cellular target gene for the HPV16 oncogene E7. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor for human cervical carcinogenesis and thus a potential DNA aberrant methylation biomarker of the progression of precancerous disease to invasive cancer. Overall, the STK31 gene represents a potential tool for the diagnoses, treatments and prognoses of CCs.The HPV16/18-positive CC cell lines HeLa, CaSki, SiHa and the HPV-negative CC cells C33A and HT-3, all of which were obtained from Shanghai institute for biological sciences, Chinese academy of Sciences Institute of Cell Resource Center . In the present study, we established the ectopically expressed HPV16 E6, -E7, and -E6/E7 cell models of C33AE6, C33AE7, C33AE6/E7, HT-3E6, HT-3E7, and HT-3E6/E7 by transfecting HPV16 E6, -E7, and -E6/E7 oncogenes with lentivirus vectors into the HPV-negative CC cell line C33A and HT-3 cells in our lab. The C33A-vector (C33A-V) and HT-3 vector (HT-3 V) cells were established by transfecting C33A and HT-3 cells with lentivirus vectors that did not code for the HPV16 E6, -E7, or -E6/E7 proteins as controls. Stable transfectants were selected with 10 ug/ml puromycin for 3 weeks. The transfection efficiency was tested by western blotting, which revealed that the transfected cells successfully expressed the E6, -E7, or -E6/E7 proteins Fig. .4 cells/25 cm2 flask and treated with various concentrations (5 μM and 10 μM) of 5-aza-dC for 96 h. We also cultured the five cells in a 10 μM 5-aza-dC medium for 0 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h.For the demethylation experiments, demethylation was induced with 5-aza-dC treatment at a concentration that was able to induce the demethylation of the DNA without killing the cells. HeLa, SiHa, CaSki, HT-3 and C33A cells were plated at a density of 8 × 10This study was approved by the Institutional Review Board (IRB) of the Affiliated Hospital of Qingdao University. All human cervical samples, including 148 HPV16/18-positive and 18 HPV-negative CC samples that were without preoperative adjuvant chemotherapy (PACT), 55cases of HPV16/18-positive CC samples with PACT, 42 HPV16/18-positive normal cervical samples, 20 cases of HPV16/18-positive CIN1, 20 cases of HPV16/18-positive CIN2, 48 cases of HPV16/18-positive CIN3 were used in the study and were obtained with written informed consent from the donor who underwent primary surgical treatment for cervical tumors or other benigh uterine tumors from the Affiliated Hospital of Qingdao University between 2003 and 2015. All cases were reviewed by at least two pathologists to confirm the diagnoses. Paraffin blocks of the HPV16/18-positive CC biopsy samples before preoperative adjuvant chemotherapy (PACT), CIN1, CIN2, and CIN3 were taken from the Pathology Department of our hospital and processed under strict conditions to avoid contamination.The detection of HPV DNA was performed by using HPV MY-PCR primers (MY09/MY11), GP5+/6+ and SPF1/2 methods. Both MY09/11 and GP5+/6+ were performed in a final reaction volume of 20 uL, containing 3 uL of the isolated DNA, 2 uL of the 10 × Buffer, 0.8 uL of each deoxynucleoside triphosphate, 0.4 uL of forward and reverse primers, and 1.0 U of Taq DNA polymerase . PCR conditions of MY09/11 were as follows: preheating for 5 min at 94 °C was followed by 40 cycles of 1 30 s at 94 °C, 1 min at 45 °C, and 1 min at 72 °C and a final extension of 7 min at 72 °C; PCR conditions of GP5+/6+ were as follows: preheating for 10 min at 94 °C was followed by 40 cycles of 1 min at 94 °C, 2 min at 40 °C, and 1.5 min at 72 °C and a final extension of 7 min at 72 °C. Each PCR experiment was performed with positive and several negative PCR controls. The final PCR products were separated by electrophoresis in 1.8 % agarose gel. SPF was performed in a final reaction volume of 40 uL, containing 4 uL of the isolated DNA, 4 uL of the 10 × Buffer, 3.2 uL of each deoxynucleoside triphosphate, 0.5 uL of forward and reverse primers, and 2.5 U of Taq DNA polymerase . PCR conditions were as follows: preheating for 1 min at 94 °C was followed by 40 cycles of 1 min at 94 °C, 1 min at 45 °C, and 1 min at 72 °C and a final extension of 5 min at 72 °C. Each PCR experiment was performed with positive and several negative PCR controls. The products of PCR were then fractionated on 12 % polyacrylamide gel electrophoresis, PAGE gels. As for all methods of HPV-DNA detection and genotyping, see Table Total RNA was isolated from the CC cells and cervical tissues using the Trizol reagent . A RNeasy FFPE Kit  was used to extract the RNA from paraffin-embedded cervical tissues. One microgram of RNA was used to obtain cDNA using the Transcriptor First Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. The cDNAs were then used for reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR).Conventional RT-PCR and qPCR were performed to examine the expression of the STK31 gene. A PCR assay with the specific primers for GAPDH cDNA was performed for each sample to ensure the integrity of the RNA. The final PCR products were separated by electrophoresis in 2 % agarose gel. See Table The DNA was extracted by using SDS-proteinase K and purified with phenol:chloroform:isoamyl alcohol. A QIAampDNAFFPE tissue kit  was used of the DNA extraction from the paraffin-embedded tumor tissues. The bisulfite conversion of the DNA was performed using an EpiTect Fast DNA Bisulfite kit  according to the instruction of the manufacturer. The methylation statuses of the promoter/exon 1 region of STK31 in the human CC cells and cervical specimens were analyzed by BGS. The primer sequences and conditions for the BGS are presented in Table MSP was performed to test the methylation status of the STK31 gene promoter/exon 1 region. The specific primer pairs and PCR conditions are listed in Table The harvested cells were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail. Each protein lysate (30 ug) was mixed with 5× SDS-PAGE sample loading buffer, and the mixtures were boiled for 5 min. The boiled mixtures (50 μg) were then fractionated on 12 % SDS-PAGE gels and subsequently transferred onto PVDF membranes . The primary antibodies used for the western blot analyses were anti-STK31 and anti-β-actin. β-actin was used as a housekeeping protein to normalize the protein loads.t-test. The correlations between the clinicopathological characteristics and the numbers of methylated specimens were tested using the χ2-test. Differences were considered significant at p < 0.05, and all statistical tests were performed with SPSS version 17.0 .The comparison of the STK31 expressions between the groups were performed with a Student’s


In particular, we show that when the plasmonic resonance of silver nanolplatelets is tuned to 656 nm, matching the emission wavelength, an upconversion enhancement factor ~5 is obtained. However, when the plasmonic resonance is tuned to 980 nm, matching the nanocrystal absorption wavelength, we achieve an enhancement factor of ~22 folds. The precise geometric arrangement between fluorescent nanoparticles and silver nanoplatelets allows us to make, for the first time, a comparative analysis between experimental results and numerical simulations, yielding a quantitative agreement at the single particle level. Such a comparison lays the foundations for a rational design of hybrid metal-fluorescent nanocrystals to harness the upconversion enhancement for biosensing and light harvesting applications.By using silver nanoplatelets with a widely tunable localized surface plasmon resonance (LSPR), and their corresponding local field enhancement, here we show large manipulation of plasmonic enhanced upconversion in NaYF4:Yb They are often co-doped with Yb3+/Er3+ to obtain better emission efficienciesThe upconversion photoluminescence (PL), a process resulting in the emission of photons with higher energy than the excitation source135794 nanoparticles doped with lanthanide47Manipulating light-matter interactions at the nanoscale using metallic nanostructure has been shown to be a very promising route for enhancing light emitters via plasmonic resonances181920212223242526272829303132333435363738394041424344252627282930313334353637383940424345464748495051525354555657584:Yb3+/Er3+) to plasmonic nanostructures (silver nanoplatelets) at the single particle level. Experimentally, we track single fluorescent particles before and after coupling to triangularly shaped silver nanoplatelets (AgNPs), whose plasmon resonance is widely tunable by adjusting the lateral length. Significantly, we determine the actual structural position of single emitters and plasmonic enhancers, allowing a quantitative comparison between experimental results and numerical modeling ( http://www.CST.com) at the single particle levelIn this communication, these critical questions are addressed here by manipulating the coupling of upconversion fluorescent nanoparticle  was spun on top of the emitter layer to control the space between the emitter layer and the AgNP layer (see more details in Supporting Information). We measured the fluorescence of the emitters before depositing AgNPs as plasmonic enhancers and then tracked them again after the deposition of the AgNPs as the third layer using spin coating.In order to track individual fluorescent nanoparticles before and after plasmonic enhancement, we created a substrate with registration markers serving as a constellation coordinate for single nanoparticles . The enhancement factor is wavelength-dependence, as studied in the Ref. Fig. S3. In contrast, when “emission matching” AgNPs are used, only an enhancement factor of 5 is achieved  (30 mmol/ml) under magnetic stirring. Then, 9 ml of polyvinyl pyrrolidone (PVP) , 0.36 ml of hydrogen peroxide (H2O2) (30%), 3 ml of silver nitrate (AgNO3) (0.85 mg/ml), and 1.5 ml of sodium borohydride (NaBH4) (3.78 mg/ml) were added into the solution in sequence. The second step starts by placing 10 ml of as-prepared silver nanoplatelet seeds into a flask. Then 10 ml of aqueous solution containing L-ascorbic acid (AA) (1.2 mmol/ml) and Na3CA (0.4 mmol/ml) were insert into the seeds under magnetic stirring. It was then followed by the injection of AgNO3 solution (0.6 mmol/ml) using a syringe pump at a rate of 10 mL/h. The silver nanoplatelets with different plasmonic resonance wavelength were pulled out at different time and collected by centrifugation at 11000 rpm for 12 minutes with water tow times under 4 degrees.Seed-mediated growth method was applied to synthesize the silver nanoplatelets. Two steps were involved. In the first step, the suspension of seeds were prepared as follows: 147 ml DI water was mixed with 9 ml of sodium citrate  at a speed of 4000 r/min. The marked substrates were made by photolithography method.AgNPs were synthesized using the method of Jie Zeng with a little modification. After synthesis, they were centrifuged and re-dispersed into ethanol at a speed rate of 12000 r/min for 12 minutes under 4 The samples used for transmission electron microscope (TEM) characterization were dropped on copper grids and dried at room temperature. TEM images were taken on JEOL 2010 FET transmission electron microscope (operated at 200 kV). The AFM images were taken on Multimode scanning probe microscope (MM-SPM).p-polarized laser for the measurements of PL was generated by a pulsed Ti:Sapphire laser with a pulse width ~3 ps and a repetition rate 76 MHz. The excitation wavelength was tuned to 980 nm. The PL from the sample was collected by anx100 objective. The PL spectra were recorded by using a spectrometer  coupled with a liquid nitrogen cooled CCD.The upconversion photoluminescence (PL) were collected by the reflection measurement. An The samples of silver nanoplates and upconversion nanocrystals were prepared by Y.L.W., H.Y.L.; the experimental measurements and data collection were carried out by Y.L.W., A.J., and L.X.X. with assistance of C.K.S. and A.A.; data analysis and theoretical modeling were performed by Y.L.W. N.M.E., A.A. and C.K.S. The manuscript was written by A.A., C.K.S. and Q.Q.W. with assistance of Y.L.W. and Z.Z.; the project was supervised by Z.Z., Q.Q.W., A.A. and C.K.S.How to cite this article: Wang, Y.-L. et al. Tailoring Plasmonic Enhanced Upconversion in Single NaYF4:Yb3+/Er3+ Nanocrystals. Sci. Rep.5, 10196; doi: 10.1038/srep10196 (2015).


We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of novel anti-inflammatory therapeutics.Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca Inflammation is a coordinated host response to local injurious stimuli . It trigNetrin-1’s classical role is in the development of the central nervous system (CNS). It is secreted at the ventral midline of the embryonic neural tube and creates a gradient along which neurons differentially migrate . Axonal et al that netrin-1, secreted by macrophages within atherosclerotic lesions, acts to promote atherosclerosis by inhibiting macrophage emigration from plaques in an UNC5b dependent manner [in vitro towards the chemokines CCL2 and CCL19, which are respectively implicated in monocyte recruitment to, and macrophage egress from atherosclerotic plaques [Several published studies have shown the wide-ranging actions of netrin-1 across the immune system. These include the observations that netrin-1 dampens inflammatory peritonitis in vivo  and thatt manner . In part plaques –14. IndeAlthough CC chemokines play a key role throughout an inflammatory response, other chemoattractants are equally important for monocyte recruitment and macrophage activation. However, the action of netrin-1 on non-CC chemokine mediated monocyte and macrophage chemotaxis is relatively poorly studied. In contrast to CC chemokine receptors, we observed that RAW264.7 and bone marrow derived macrophages (BMDMs) expressed high surface levels of C5a receptor 1 (C5aR1). C5a is a complement peptide that has an important role in innate immunity with potent chemotactic and anaphylatoxic properties that upregulate endothelial adhesion molecules, increase vascular permeability, and localise leukocytes and inflammatory molecules at sites of infection . As such2+ release, cell spreading, or altered PI3K/MAPK signalling. Although the mechanism remains unresolved, a better understanding of netrin-1’s impact on macrophage chemotaxis could identify further therapeutic avenues to explore in the development of novel anti-inflammatory drugs.Using two different real-time chemotaxis platforms, we show for the first time that netrin-1 inhibits human monocyte and murine macrophage migration towards C5a. We explored the possible mechanism behind the reduction both at C5aR1 proximal events and at signalling events further downstream of receptor ligation. We conclude that netrin-1 inhibition of macrophage chemotaxis towards C5a is not mediated through C5a receptor internalisation or changes in macrophage CaMurine CCL2, C5a, netrin-1 and the rat UNC5b blocking antibody were obtained from R & D systems . CIM-16 plates were purchased from Cambridge Bioscience . complete, EDTA free protease inhibitor cocktail tablets were purchased from Roche . Rabbit anti-phospho-ERK1/2 (D13.14.4E), rabbit anti-phospho-Akt (D9E), rabbit anti-phospho-p38 (D3F9), rabbit total ERK1/2 (137F5) and rabbit total p38 (D13E1) were purchased from Cell Signalling Technologies . HRP-conjugated Goat anti-rabbit secondary and Bio-gel polyacrylamide beads (P-100 fine 45–90 μm) were purchased from Biorad . Matrigel and all cell culture flasks and vessels were purchased from Corning . Fura-2 AM and Alexa Fluor® 488 phalloidin were purchased from Life Technologies. Phosphatase inhibitor cocktail 2, cell culture medium and all other laboratory chemicals were purchased from Sigma Aldrich .C57BL/6J mice were obtained from Harlan Laboratories . All animal studies were conducted with ethical approval from the Dunn School of Pathology Local Ethical Review Committee and in accordance with the UK Home Office regulations  Act 1986). Animals were humanely killed by exposure to carbon dioxide gas in a rising concentration, as set out in Schedule one of the Animals (Scientific Procedures) Act 1986, and all efforts were made to minimise distress and suffering of the animals used in this study.6 cells seeded into 10 cm non-tissue culture treated petri dishes in 10 ml of differentiation medium , 15% L929 cell conditioned supernatant, 2 mM l-glutamine and 100 U/ml penicillin/streptomycin). Three days later, an additional 5 ml of differentiation medium was added. BMDMs were then collected after 7 days by the addition of phosphate buffered saline (PBS) containing 2 mM EDTA and gentle agitation. BMDMs were then resuspended in chemotaxis buffer (RPMI 1640 supplemented with 0.5% BSA and 25 mM HEPES), counted and adjusted to the desired cell concentration.Bone marrow cells from the tibiae and femur of male C57BL/6J mice were collected, counted, and 4x106 cells/flask) and maintained at 37°C, 5% CO2. When approximately 80–90% confluent, the medium was removed and the cells lifted from the flask by the addition of PBS containing 10 mM EDTA followed by firm tapping. Cells were then resuspended in PBS and counted. For experimental usage, cells were then pelleted and resuspended in chemotaxis buffer to give the desired cell concentration. For propagation of the cell line, cells were resuspended in DMEM supplemented with 10% FCS and 100 U/ml penicillin/streptomycin and seeded into T75 flasks as detailed above. For all experiments, RAW264.7 macrophages were used at a passage number of 10 or lower.The RAW264.7 murine macrophage cell line , origina2. The dishes were then washed three times with 10 ml PBS to remove any non-adherent cells and the medium replaced with 10 ml chemotaxis buffer. After being left overnight at 37°C, 5% CO2, adherent macrophages were collected by the addition of PBS containing 10 mM EDTA and gentle agitation. Macrophages were then pelleted by centrifugation at 200 xg for 5 min at 4°C, resuspended in chemotaxis buffer, counted, and then adjusted to the desired cell concentration. These cells are subsequently referred to as bio-gel elicited macrophages and we have previously demonstrated that these are highly enriched for macrophages [Male C57BL/6J mice were injected intraperitoneally with 1 ml of sterile 2% bio-gel polyacrylamide beads  suspended in PBS. After four days, mice were sacrificed and the elicited cells collected by peritoneal lavage with 10 ml ice cold PBS containing 2 mM EDTA. Subsequently, peritoneal bio-gel elicited cells were pelleted by centrifugation at 200 xg for 5 min at 4°C and then re-suspended in 10 ml chemotaxis buffer before being transferred into 10 cm petri dishes  and left for 2 hours at 37°C, 5% CO+ monocytes were then isolated from the PBMCs by positive magnetic separation using human anti-CD14 coupled microbeads following the manufacturer’s instructions (Miltenyi Biotec). CD14+ monocytes were frozen for long term storage in FCS supplemented with 10% DMSO.Leukocyte cones were obtained from the NHS blood transfusion service or peripheral blood was taken from healthy volunteers, with informed consent and appropriate local ethics approval, and peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient centrifugation. Leukocyte cones were diluted 1 in 2 with PBS, whereas fresh blood was not, and 15 ml then layered on top of 5 ml Ficoll-Paque Plus  before centrifugation at 900 xg for 20 min at RT. The PBMC layer was carefully harvested and then washed repeatedly using 50 ml PBS and centrifugation at 300 xg for 5 min until the supernatant became clear. CD145 cells/well) were treated with either vehicle or 3 nM netrin-1 for 45 min at 37°C, 5% CO2. For experiments with bio-gel elicited macrophages 4x105 cells/well were used. Cells were then added to all upper wells (50 μl/well) and plate replaced into the RTCA-DP system. Cell Index (CI) measurements were then taken every 5 seconds over the 3–4 hour assay period. Change in Cell Index (Δ Cell Index) was calculated as maximum cell index minus minimum cell index and pooled data are displayed as a fold change relative to cells migrating towards vehicle alone. For the heat inactivation experiments, netrin-1 was incubated at 75°C for 25 min prior to BMDM treatment. For the UNC5b receptor blocking experiments, BMDMs were co-incubated with netrin-1 (3 nM) and an UNC5b blocking antibody (10 μg/ml) for 45 min at 37°C, 5% CO2, prior to being placed into the upper chamber of a CIM-16 chemotaxis plate.Real-time chemotaxis analysis was conducted as previously described . Briefly2 to adhere to the bottom gold electrode, with CI measurements made every 15 min. Cells were then treated with vehicle (chemotaxis buffer) or 3 nM netrin-1 for 45 min prior to being stimulated with vehicle or 10 nM C5a. CI measurements were taken every 5 s for 1 hour after agonist addition. Data are displayed as change in CI from the point of agonist addition (Δ Cell Index) and response was calculated as maximum CI–CI at point of agonist addition.Chemotaxis buffer (50 μl) was added to the wells of a 96 well E-plate and a background measurement taken. Afterwards, BMDMs were added to each well  and left for 2–3 hours at 37°C, 5% CO2 prior to being placed into the upper chamber of an IncuCyte® ClearView chemotaxis plate (60 μl—5000 cells/well), which had been coated with Matrigel (50 μg/ml for 30 min at 37°C and 30 min at RT). The plates were then left for 1 hour at RT to allow cell adhesion to the topside of the chemotaxis well. Vehicle or 100 nM C5a (200 μl) was added to the lower chamber of a ClearView chemotaxis plate and the top section carefully placed into the bottom. The plate was then placed into the IncuCyte® ZOOM microscope, which was housed inside a humidified incubator set to 37°C. The microscope was then set to take images of the top and underside of each well every 2 hours. Migration was then quantified by the IncuCyte® analysis software as the appearance of in focus cells, and the respective surface area they occupy , on the underside of the well. To validate equal cell loading, the surface area occupied by cells on the topside of the well at time point zero was calculated.BMDMs or RAW264.7 macrophages were incubated with either vehicle (RPMI + 10% FCS), 3 nM or 30 nM netrin-1 for 45 min at 37°C, 5% CO2. Cells were then treated with either vehicle (chemotaxis buffer) or 3 nM netrin-1 for 45 min at 37°C, 5% CO2 prior to being stimulated with either vehicle or 10 nM C5a for 5 or 30 min. Following stimulation, cells were fixed by the addition of 4% formaldehyde for 15 min at RT. Cells were washed twice with PBS and 0.5% triton X-100 was added for 5 min to permeabilise the cell membrane. The cells were then washed twice with PBS and 5 U/ml Alexa Fluor® 488 phalloidin was added to each well for 20 min at RT to visualise F-actin. Cells were then washed twice with PBS and the plate placed into the IncuCyte® ZOOM microscope and four images per well were then taken by the microscope. The IncuCyte® analysis software then determined individual cell areas by recognising unique green fluorescent objects in each image, and then calculated the average cell area for each well.BMDMs were seeded into 96 well black walled plates  in chemotaxis buffer and allowed to adhere for 2 hours at 37°C, 5% CO2. Cells were then treated with vehicle, 3 nM or 30 nM Netrin-1 for 45 min, 4 hours or 24 hours. Afterwards, cell viability was determined using the CellTiter-Glo® luminescent cell viability assay , which determines cell viability by quantification of ATP, following the manufacturer’s protocol. Briefly, an equal volume of CellTiter-Glo® reagent was added to each well and the plates were left for 15 min before luminescence was quantified using a PHERAstar plate reader .BMDMs were seeded into 96 well black walled plates  and allowed to adhere for 2 hours at 37°C, 5% CO5) were placed into polypropylene tubes and pelleted by centrifugation at 200 xg for 5 min at 4°C. These were then resuspended in FACS buffer  supplemented with mouse IgG and mouse SeroBlock FcR®  and left on ice for 30 min. Specific staining was conducted using the following antibodies and appropriate isotype controls: F4/80 , Ly-6B.2 , CCR2  and C5aR1  for 1 hour on ice. Cells were then pelleted by centrifugation at 200 xg for 5 min at 4°C and resuspended in 1% formaldehyde. For C5aR1 internalisation experiments, cells were treated with vehicle or 3 nM netrin-1 for 45 min at 37°C, 5% CO2 prior to being stimulated with 10 nM C5a for either 5 or 30 min. C5aR1 surface staining was then conducted as described above. For measurement of intracellular receptor levels, cells were first fixed in 1% formaldehyde for 15 min. Next, they were resuspended in 0.5% saponin for 15 min at RT to permeabilise the cell membrane. Cells were then incubated with fluorescently conjugated receptor antibodies in 0.5% saponin for 1 hour on ice, washed with FACS buffer and then resuspended in 1% formaldehyde. Analysis was conducted using a Dako Cyan ADP flow cytometer  and FlowJo software .Cells (5 x 102 for 24 hours. Cells were then treated with either vehicle (Cell assay reagent) or 3 nM netrin-1 for 45 min at 37°C, 5% CO2 before being stimulated with either vehicle or C5a at the indicated concentration for 30 min at 37°C, 5% CO2. Cell lysis and cAMP detection were then performed as per the manufacturer’s protocol. Luminescence measurements were taken using a PHERAstar microplate reader.Intracellular cAMP levels were measured using Discoverx cAMP Hunter™ eXpress kits  following the manufacturer’s protocol. Briefly, CHO-K1 cells stably expressing human C5aR1 receptor were plated into ½ area 96 well plates  and incubated at 37°C, 5% CO2 for 48 hours. Cells were then treated with either vehicle (Cell assay reagent) or 3 nM netrin-1 for 45 min at 37°C, 5% CO2 prior to stimulation with vehicle or C5a at the indicated concentration for 90 min at 37°C, 5% CO2. Cell lysis and detection of total recruited β-arrestin was determined following the manufacturers protocol. Luminescence measurements were taken using a PHERAstar microplate reader.Recruitment of β-Arrestin to C5aR1 was measured using the Discoverx PathHunter® eXpress β-Arrestin GPCR Assay following the manufacturer’s protocol. Briefly, CHO-K1 cells stably expressing murine C5aR1 were seeded into ½ area 96 well plates  and incubated at 37°C, 5% CO2 to adhere. The medium was then removed and replaced with RPMI containing 4 μM Fura 2-AM (Life Technologies) supplemented with either vehicle or 3 nM netrin-1. Plates were then left for 45 min at RT in the dark. Afterwards, cells were washed twice with PBS, the medium replaced with Fluorobrite™ DMEM (Life technologies) and the plate placed into a PHERAstar microplate reader set to 37°C. BMDMs were then stimulated with either vehicle or 10 nM C5a and changes in Fura-2 fluorescence were measured using excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. The change in the ratio of 340/380 nm stimulated emission at 510 nm was then calculated (referred to as Δ340/380 nm).BMDMs were seeded into black walled 96 well plates  and left for 2 hours at 37°C, 5% CO6 cells/well) in chemotaxis buffer and allowed to adhere for 2 hours at 37°C, 5% CO2. The cells were then treated with vehicle (chemotaxis buffer) or 3 nM netrin-1 for 45 min at 37°C, 5% CO2 prior to stimulation with vehicle or 10 nM C5a for 5 or 30 min. After stimulation, the medium was rapidly removed and the 6 well dishes placed at -80°C. Cell lysates were prepared by the addition of cell lysis buffer  supplemented with protease and phosphatase inhibitors, followed by manual disruption. Resultant supernatants were centrifuged at 16,000 xg for 10 min at 4°C and protein concentration of debris free supernatant was determined using a BCA protein assay kit  following the manufacturer’s protocol. Samples were then diluted 3:1 with 4x laemmli buffer , heated at 95°C for 5 min and either loaded directly onto an SDS-PAGE gel or placed at -80°C. SDS-PAGE was conducted using a 10% gel and 30 μg total protein per sample. Samples were then transferred onto Hybond ECL nitrocellulose  and membranes blocked using 5% BSA in TBS-T for 2 hours at RT or overnight at 4°C. After blocking, membranes were incubated with rabbit anti-phospho-ERK1/2, rabbit anti-phospho-Akt or rabbit anti-phospho-p38 , diluted in 5% BSA/TBS-T for 2 hours at RT or overnight at 4°C. Membranes were then incubated with a HRP-conjugated anti-rabbit secondary antibody  for 1 hour at RT. Protein bands were visualised by incubating the membranes for 5 min with Amersham™ ECL prime and subsequent exposure to x-ray film over a range of exposure times. To confirm equal protein loading between samples, bound antibodies were removed by incubating the nitrocellulose membranes in stripping buffer  for 30 min at 50°C. Membranes were then blocked with 5% BSA in TBS-T for 2 hours at RT and then incubated with rabbit anti-ERK1/2 or rabbit anti-p38 (both 1:1000) diluted in 5% BSA/TBS-T for 2 hours. Protein band detection was conducted as described above. Densitometric analysis was conducted using Image Studio Lite  and the phosphorylated protein band density was divided by total ERK1/2 band density. Data are displayed as a fold change relative to vehicle treated samples. Full, uncropped blots can be found in BMDMs were seeded into tissue culture treated 6 well dishes  following the manufacturer’s instructions. RNA concentration and quality was determined using a NanoDrop™ ND-1000 spectrophotometer. cDNA was subsequently produced using the QuantiTect® Reverse Transcription kit (Qiagen) following the manufacturer’s instructions. Expression of actin or CCL2 was determined using the following primers: (5’>3’): Actin Fwd CCAACAGCAGACTTCCAGGATT, Actin Rev CTGGCAAGAAGGAGTGGTAACTG, CCL2 Fwd CAGCACCTTTGAATGTGAAGTTG, CCL2 Rev TGCTTGAGGTGGTTGTGGAA and SYBR Select PCR master mix (Life Technologies). 500 ng of cDNA was used per reaction with the following thermal profile: 95°C for 5 min, 40 cycles of 95°C for 30 s, 60°C for 20 s, 72°C for 30 s and a final step of 72°C for 5 min using a StepOnePlus™ thermal cycler (Applied Biosystems). Cycle threshold values (Ct) were calculated using StepOne™ software version 2.3 and data are displayed as 2^-ΔCt (referred to as relative to actin) whereby ΔCt = Ct value of gene of interest minus Ct value of actin.All data were analysed using Graphpad Prism version 6. For comparisons between two groups, an unpaired two-tailed student’s t test was applied. For multiple comparisons with only one variable, a one-way ANOVA with Dunnett’s multiple comparisons correction was applied and for multiple comparisons with two variables, a two-way ANOVA with Sidak’s multiple comparisons correction was applied. The exact test applied to each data set is stated in the corresponding figure legends. Data are expressed as mean + or ± SEM, as stated in the figure legend. A P value of < 0.05 was taken to be statistically significant.We began our study by attempting to replicate the finding that netrin-1 is able to inhibit the chemotaxis of the RAW264.7 macrophage cell line towards the CC chemokine, CCL2 . SurprisAs the surface expression of CCR2 seemed low on BMDMs, we were interested to compare this with other primary macrophage populations. We selected bio-gel elicited macrophages, as we have previously shown that they migrate towards CCL2 in our real-time chemotaxis system [Given that our main objective with this study was to determine whether netrin-1 is capable of inhibiting macrophage chemotaxis to non-chemokine chemoattractants, and that all macrophage populations we tested had high surface levels of C5aR1, we next examined the effect of netrin-1 on C5a induced macrophage chemotaxis. Treatment of BMDMs with 3 nM netrin-1 for 45 minutes inhibited their migration towards 1.25, 2.5, 5 and 10 nM C5a , whereasIn order to corroborate our real-time chemotaxis results obtained using the ACEA xCELLigence system, we decided to employ a microscopy based assay that allows measurement of cell migration in real-time. The IncuCyte® ZOOM system uses custom Boyden-chamber style 96 well plates, where each well contains 96 8 μm diameter fixed location pores in the filters that separate the upper and lower halves. The microscope takes images of the topside and the underside of each filter and the analysis software then identifies the location of each pore and cells in focus on the top and the bottom of the filter in each well. As can be seen in the representative underside images in In preliminary experiments, we found that higher C5a concentrations than those used in our impedance based real-time chemotaxis system were needed to cause robust BMDM chemotaxis, and therefore 100 nM C5a was chosen as an optimal concentration . In compTo begin to understand how netrin-1 inhibits macrophage chemotaxis, we first tested whether netrin-1 needed to be in its native conformation to reduce cellular migration. To do this, we denatured netrin-1 by heating to 75°C for 25 minutes before BMDM treatment. In contrast to non-denatured netrin-1, which inhibited BMDM chemotaxis , howeveri/o-coupled GPCR activation. In CHO cells expressing C5aR1, C5a induced a concentration dependent decrease in forskolin-induced intracellular cAMP levels and a concentration dependent increase in β-arrestin recruitment to C5aR1, as would be expected. However, netrin-1 had no effect on either of these signalling pathways , demonstrating that chemotactic drive seemed to be correlated with surface receptor expression. Further support for this hypothesis comes from our cell spreading experiments. In contrast to van Gils preading , we founIn the search for the mechanism of action of netrin-1, we performed a proximal to distal analysis of the effect of netrin-1 on C5aR1 dynamics and signalling; a first in the field. A direct competitive effect of netrin-1 at C5aR1 was ruled out, as C5a induced cAMP signalling and β-arrestin recruitment remained unaffected by netrin-1 in C5aR1 expressing CHO cells. In BMDMs we also ruled out the hypothesis that netrin-1 effected receptor dynamics, as surface C5aR1 levels or its internalisation remained unchanged following netrin-1 treatment. Furthermore, netrin-1 had no effect on proximal receptor signalling following C5aR1 ligation, including the PI3K pathway that is central to cell migration . In lighRegardless of the precise mechanism, the finding that netrin-1 inhibits C5a induced monocyte and macrophage chemotaxis uncovers a novel area of clinical interest. Currently, the therapeutic potential of netrin-1 has largely centred on cardiovascular disease with debate surrounding whether netrin-1 contributes to, or reduces atherosclerotic plaque formation . This coin vivo.This context dependent regulation of netrin-1 has direct impact for our present study and the discussion of whether netrin-1 may have therapeutic value in modulating the host complement response. Complement driven pathology is characterised by excessive or inappropriate activation of complement and can manifest as a multitude of inflammatory diseases including sepsis . PeptidoS1 FigA-C) Representative histograms of surface receptor levels, for 3 independent biological samples, compared to isotype control. Quantification of GPCR surface levels by (D) median fluorescence intensity or (E) geometric mean of fluorescence intensity. Data are mean + SEM, n = 3 biological replicates. (F) Bio-gel elicited macrophages were placed into the upper chamber of a CIM-16 plate (2x105 cells/well) and allowed to migrate for 4 hours at 37°C, 5% CO2 towards vehicle or 5 nM CCL2. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction.* P < 0.05, ** P < 0.01, compared to isotype control.Bio-gel elicited macrophages were stained for surface expression of CCR2 and C5aR1. ((EPS)Click here for additional data file.S2 FigA) BMDMs were placed into the upper chamber of an IncuCyte® ClearView chemotaxis plate (5000 cells/well), which had been coated with Matrigel (50 μg/ml for 30 min at 37°C and 30 min at RT). Cells were then allowed to migrate towards vehicle, 100, 50 or 25 nM C5a and chemotaxis was measured by imaging the underside of the well every 2 hours and calculating total underside cell area. Data are mean of 3–4 technical replicates per condition. (B) BMDMs were incubated with either vehicle, 3 nM or 30 nM netrin-1 for 45 min at 37°C, 5% CO2 prior to being placed into the upper chamber of an IncuCyte® ClearView chemotaxis plate (5000 cells/well), which had been coated with Matrigel (50 μg/ml for 30 min at 37°C and 30 min at RT). To assess the uniformity of cell loading in the experiments conducted in ((EPS)Click here for additional data file.S3 Fig2. Cells were then treated with vehicle, 3 nM or 30 nM netrin-1 for 45 min, 4 hours or 24 hours. Cell viability was then determined using the CellTiter-Glo® luminescent cell viability assay. Data are mean SEM, n = 4 biological replicates with 2 technical replicates per condition. Statistical analysis was conducted by two-way ANOVA with Sidak’s multiple comparisons correction. ns P > 0.05.(A) BMDMs were seeded into black walled 96 well plates  and left to adhere for 2 hour at 37°C, 5% CO(EPS)Click here for additional data file.S4 Fig6 cells/dish) and then treated with either vehicle or 3 nM netrin-1 for 45 min at 37°C, 5% CO2. Cells were then stimulated with either vehicle or 10 nM C5a for 5 or 30 min at 37°C, 5% CO2. Cell lysates were prepared and western blotting conducted for phosphorylated p38 (phospho-p38) followed by stripping and re-staining for total p38 as a loading control. A blot, representative of n = 2 biological replicates, is shown.(A) BMDMs were seeded into 6 well dishes (2x10(EPS)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.


Background: We investigated the molecular basis of human IFN-α2A recognition by sifalimumab.Results: We determined the structure of the complex between the Fab of sifalimumab and IFN-α2A.Conclusion: The interferon-neutralizing properties of sifalimumab result from direct competition for IFN-α2A binding to IFN receptor subunit 1 and not IFN receptor subunit 2.Significance: These data provide the basis for the mechanism of action of sifalimumab. We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody . The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2). Interferons (IFNs) belong to the “4-helical cytokines” superfamily  and can 4–e.g. IFN-α2A, IFN-α2B, and IFN-β), unbound or bound to a single chain Fv (scFv), have already been determined using either x-ray crystallography or nuclear magnetic resonance (NMR)  –16. Howe6) was a generous gift from Sandrina Phipps (MedImmune). All antibody and antigen amino acid positions mentioned in the text were identified according to a consecutive numbering scheme. In these conditions, the Kabat-defined complementarity determining regions (CDR)All chemicals employed were of analytical grade. The histidine-tagged recombinant extracellular domain of the IFN-α receptor 1 , 1RH2 , and 1AU1 . None of these 3 potential models yielded a clear molecular replacement solution with PHASER or MolRep. However, the phases obtained through the solution of both sifalimumab Fab molecules yielded very clear electron density for the proximal region of IFN-α2A. Two rounds of Fab-only refinement and model adjustment using the “O” software (1RH2), which differed from IFN-α2A by only one amino acid (R23K). The tight non-crystallographic symmetry restraints were used throughout the refinement of the model with Refmac5  program running on its Web server (2+ ions (first and third). Another two Ni2+ ions (second and fourth) were coordinated by His-190 in the sifalimumab light chain. The fifth and sixth Ni2+ ions did not have histidine residues in the coordination sphere. Ni2+ ions were the only divalent metal ions present in the crystallization mixture, and could be identified by their fit to the corresponding electron density.Diffraction images were integrated and scaled using HKL 2000 . Moleculsoftware  further  Refmac5 . The subb server , 28. Furb server , the IFNm NaHCO3, pH 9.0, overnight at 4 °C according to the manufacturer's instructions. Coated beads were then separated from unreacted IFN-α2A using a gentle pulse spin and blocked for ∼15 min at 22 °C with 1 m Tris, pH 8.0, bovine serum albumin, 10 mg/ml. The slurry was then spun and the blocking solution removed. The blocking step was repeated for 2 h at 22 °C. Beads were then resuspended in 27 ml of run buffer , pH 7.4, 0.02% NaN3) and packed into a column. Typically, sifalimumab was prepared at concentrations of 40 and 200 pm. IFN-α2A was then titrated across these IgG solutions at concentrations of 313 fm to 16 nm and 980 fm to 50 nm, respectively, and incubated for 1–4 days at room temperature. The amount of free IgG in the samples was derived from the fluorescence signal obtained after the passing of Cy5-labeled goat anti-human IgG F(ab′)2  through the column. The dissociation constant (DK) was determined by fitting the individual equilibrium titration data to a 1:1 binding model using the KinExA Pro 1.0.3. software.The interaction of immobilized IFN-α2A with sifalimumab was monitored using a KinExA 3000 instrument . IFN-α2A was first coated onto UltraLink Biosupport beads  at concentrations of 5 and 10 μg/ml in 0.05 m in 10 mm sodium acetate buffer, pH 5.0) at a density of ∼4,800 resonance units according to the manufacturer's instructions. The extracellular domain of IFNAR2 (MedImmune) was also immobilized using standard amine coupling  at a density of ∼2,000 resonance units. IFN-α2A and sifalimumab were prepared in PBS, pH 7.4, containing 0.005% Tween 20. Sifalimumab competition was assessed by 2 consecutive injections of IFN-α2A and a mixture of IFN-α2A and sifalimumab over the IFNAR1 or IFNAR2 surfaces. For IFNAR1, IFN-α2A was first injected at 200 μg/ml (30 μl/min for 120 s), which was followed by a second injection (30 μl/min for 120 s) of the IFN-α2A (200 μg/ml)/sifalimumab (100 μg/ml) mixture. For IFNAR2, IFN-α2A was first injected at 10 μg/ml (30 μl/min for 120 s), which was followed by a second injection (30 μl/min for 120 s) of the IFN-α2A (10 μg/ml)/sifalimumab (100 μg/ml) mixture. The extent of competition was derived from the additional binding detected from the second injection. All sensorgram data were processed by ProteOn Manager 3.1 software (Bio-Rad), and the binding graphs were prepared with Prism (GraphPad).The ability of sifalimumab to inhibit the human IFNAR1/IFN-α2A and IFNAR2/IFN-α2A interactions was monitored using a ProteOn XPR36 instrument (Bio-Rad). The extracellular domain of IFNAR1 (MedImmune) was immobilized to the EDAC/Sulfo-NHS-activated surface of on a GLC biosensor chip (Bio-Rad) using standard amine coupling (200 n4YPG).We successfully determined the x-ray crystal structure of the complex between the Fab of an anti-human IFN-α therapeutic antibody  and IFN-α2A. The corresponding refinement statistics are given in A. A shape complementarity of ∼0.674 between sifalimumab Fab and IFN-α2A was calculated using the “sc” program from the CCP4 suite  , C (residues 78–101), and D (residues 112–133) helices as illustrated in Fig. 1D.The interface contributed by the sifalimumab Fab portion could best be described as a canyon, whereas IFN-α2A exhibited a remarkable shape complementarity to this groove as indicated in Fig. 1umber 4) . This re2 . The contact interface included ∼20 amino acids from each of the antibody polypeptides and ∼40 amino acids from the antigen. In particular, the heavy and light chains of sifalimumab Fab covered ∼600 and 700 Å2 of solvent-accessible area upon complex formation, respectively. The list of all hydrogen bonds between both chains of sifalimumab Fab and IFN-α2A can be found in Fig. 2A and Fig. 2B and Fig. 2C and Fig. 2D and Fig. 2E and DK shown in The buried surface area upon formation of the complex between sifalimumab Fab and IFN-α2A was estimated at more than 1,300 Ået al. . This comforts the notion that the active form of IFN-α molecules is monomeric, as determined by Klaus et al.  comprises 2 major transmembrane subunits, namely IFNAR1 and IFNAR2 , 31. IFNr IFNAR1 , 33. Ther IFNAR1 , 34. The3SE3) , and, thus, rule out a mechanism of action in which the antibody interferes with the corresponding receptor/ligand interaction.A number of studies have revealed critical residues on both IFNAR2 and IFN-α2 through mutagenesis, NMR, and x-ray crystallography –40. As c–3SE3) , both si . In fact, sifalimumab actually inhibited the binding of IFN-α2A to immobilized IFNAR1, as observed from the drop in signal upon injection of the IFN-α2A/sifalimumab mixture. Indeed, the interaction of sifalimumab/IFN-α2A (DK of 44 pm) is much stronger than that of IFNARI/IFN-α2A (DK of 1.5 μm (41–3SE3) . Thereff 1.5 μm , 41–43, .5 μm 41–. Moreove41–3SE3)  was supe41–3SE3) , it cleaIn summary, we conclude that sifalimumab acts as a direct competitive inhibitor of the IFN-α2/IFNAR1 interaction. A coherent model of the mechanism of action for sifalimumab thus emerges, and indicates the antibody does not interfere with the first step in the response to IFN-α2, namely the ligand·IFNAR2 complex formation. Rather, we suggest that sifalimumab sterically interferes with the recruitment of IFNAR1 and prevents formation of the IFN-α2·IFNAR1·IFNAR2 ternary complex.


The monodispersed MgO nanoparticles, 20 nm in size, were used to treat human cancer cell lines  at different concentrations  and incubation times . Both the genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized at 150 µg/mL nanoparticle treatment with 48 h incubation. However, the genotoxic effect of MgO nanoparticles was not significant compared with control experiments, which indicates its significant potential applications in nanomedicine as a diagnostic and therapeutic tool.Water-dispersible MgO nanoparticles were tested to investigate their cytotoxic effects on oxidative stress gene expression. In this  After evaporating the solvent, it was incubated at 75 °C in vacuum for 50 min. An addition of 4 mL water resulted in a clear and dark-brown suspension. After filtration and centrifugation, the resulting nanoparticles were then well dispersed in phosphate buffered saline . The cell lines were derived from the human hepatocellular carcinoma—HepG2 cells (ATCC HB-8065). The cell lines between 15 and 25 of the subculture passages were used as adherent cells. Cell lines were treated with 25, 75 and 150 µg/mL concentrations of MgO nanoparticles in serum-free culture medium and incubated for a period of 24, 48 and 72 h. The cell lines were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin —streptomycin . Cells were washed with PBS and incubated in a 3 mL trypsin-ethylenediaminetetraacetic acid  for 3–5 min at 37 °C in a CO2 incubator. Cell suspensions were then centrifuged for 3 min at 1300 rpm. Then the pellet was re-suspended with a fresh medium and the seed density was adjusted using a hemocytometer based cell counting by an inverted microscope. For the RT-PCR quantitation of ROS, cells were cultured in a 24 micro-well plate (1 × 105 cells/mL).Water-dispersible and biocompatible MgO nanoparticles were synthesized  ,31,32. Nm 58 °C, product size: 130 bp, gene bank number: NM_146421.2; TagMan probe sequence: 5'-CCCACGCCATCCGCCTGCTCCT-3', Tm 68.9 °C ) and catalase  genes were used in RT-PCR. The TaqMan probes were conjugated with the fluorophore, 5-carboxyfluorescein (FAM) at 5' end and black hole quencher (BHQ1) at 3' end. The 20 µL reaction mixture consisted of 1 µL of cDNA template, 0.25 µL of TaqMan probe (20 µM), 0.5 µL of forward and reverse primers (10 µM), 10 µL of Mastermix  and 7.75 µL of RNase free water. RT-PCR was performed in a qPCR thermocycler  with initiation at 90 °C for 15 min followed by 40 cycles of amplification at 90 °C for 15 s (denature) and 50 °C for 50 s . Samples were analyzed with the wavelength of excitation/emission at 488/530 nm with respect to the FAM fluorophore, and the relationship of the amplified cDNA copy number versus its cycle threshold (Ct) values was plotted in the graph.The MgO nanoparticle treated cells were used for the isolation of total RNA using a Qiagen RNeasy mini kit. The isolated total RNAs were collected in 20 µL RNase free water and quantified by nanodrop spectrophotometer. We conducted a RT-PCR using a two-step reaction which consisted of complementary DNA (cDNA) synthesis by reverse transcription reaction and then RT-PCR analysis for quantitative gene expression. The cDNA was synthesized by adding 1 µg total RNA to the reverse transcriptase premix tube  with a reaction volume made up to 20 µL with RNase free water. The tubes were placed in a thermocycler and incubated at 45 °C for 50 min for cDNA synthesis and followed by 90 °C for 5 min for a reverse transcriptase inactivation. Synthesized cDNA was used as a template in the real time analysis. The synthesized primers and TaqMan probes for GST  and catalase (forward primer: 5'-GAGGCCTCCTGCAGTGTTCT-3' and reverse primer: 5'-CATTAAGCCATGACGGTGCT-3') were used to obtain a large product  which is close to the entire gene size  in order to mimic real GST and catalase genes. These 923 and 2115 bp product bands were eluted from a gel and quantified using a nanodrop spectrophotometer to determine the exact copy number of the template. Templates were serially diluted from 10+, NH4+ and 20 mM Mg2+), and 11.7 µL of RNase water. PCR was then carried out in a thermocycler at 90 °C for 2 min, followed by 30 cycles of amplification at 95 °C for 60 s, 55 °C for 30 s and 70 °C for 5 min. The amplified products were then separated in a 1.5% agarose gel electrophoresis with 1× TBE buffer  at 100 V for 60 min. The gel was stained with ethidium bromide (EtBr) and illuminated under UV for detecting the expressed gene bands. A 200 bp housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward primer: 5'-GCCATCAATGACCCCTTCAT-3' and reverse primer: 5'-GCTCCTGGAAGATGGTGATG-3') were used as a reference control.The expression level of GST and catalase genes on an agarose gel was confirmed by PCR. PCR was performed in a 20 µL reaction volume, consisting of 2 µL of templates (cDNA from MCF7 cell line), 1 µL of forward and reverse primer (10 µM), 0.3 µL of enzymes (i-StarMAX™ II DNA polymerase (5 U/mL), Intron Biotech., Sungnam, Korea), 2 µL of dNTP (2.5 mM each), 2 µL of 10× reaction buffer  and 7 bp stem sequence. A FAM fluorophore and a quencher BHQ1 were labeled at 5' and 3' end, respectively . Cell lines were cultured in a 96 micro-well plate at a concentration of 5 × 104 cells/mL. After 24 h of incubation, the cells were treated with various concentrations of MgO nanoparticles and different incubation times as described earlier. MBs were delivered into the live cells with a reverse permeabilization method by activated Streptolysin O (SLO) [tris(2-carboxyethyl)phosphine (TCEP) to 2 U/mL of SLO for 30 min at 37 °C. Then, the cells were incubated for 10 min in 200 µL of serum free medium containing 0.2 U/mL of activated SLO and 3 µL of MB (1 µM) for cell permeabilization and MB delivery. After incubating with MB, the cells were resealed by adding 0.5 mL of the typical growth medium and incubated for 1 h at 37 °C before performing confocal microscopic and fluorometric analysis. The green fluorescence imaging of live cells was performed by a laser scanning confocal microscope . The culture plates were read under a microplate spectrofluorometer . The fluorescence signal of the MBs which were hybridized with a GST target gene was analyzed with excitation at 488 nm and emission detection at 530 nm.The MBs for GST gene expression were designed as 5'-CGCGATC O (SLO) . First tin vivo tests also should be performed to explore the linear relationship between gene expression and protein level in cells.In conclusion, we have evaluated the cytotoxicity of the PEG-phospholipid coated MgO nanoparticles on human cancer cell lines (HepG2) with different concentrations and incubation times. Targeting the two oxidative stress genes , we performed a quantitative analysis of gene expression at the mRNA level by using the RT-PCR and at the cellular level by using the MB technology. These results are consistent at 48 h incubation, revealing a maximum gene expression proportional to MgO concentration, whereas at 72 h, the gene expression is reduced twice the amount in the control. In addition, the increment ratio of the expressed GST and catalase gene number compared with the control was not significant, demonstrating high stability, biocompatibility, and low toxicity, suggesting that engineered MgO nanoparticles as an excellent contrast agent for magnetic resonance imaging in the field of biomedicine. For further cytotoxicity study, various primary cells and


COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI.The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish’s osteogenesis imperfecta mutation database.COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution , usually serine . We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients.The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required. COL1A1 and COL1A2 genes, located at chromosome 17q21.33 and 7q21.3, respectively [COL1A1 is more susceptible to mutation, as more α1 chains are implemented in the collagen fibrils. COL1A1 gene mutations are more pathogenic and cause OI more often than COL1A2 gene mutations. One third of glycine (Gly) substitutions in the COL1A1 gene are lethal, whereas only 1/5 of Gly pathogenic variants in the COL1A2 gene are fatal [n repetitions, where X and Y are random amino acids [Osteogenesis imperfecta (OI) is associated with high genetic heterogeneity. To date, mutations in 16 different genes have been found to cause OI phenotypes of varying severity [ectively . It was no acids .http://www.le.ac.uk/ge/collagen/). Previous studies have shown that COL1A1/2 mutations account for up to 85–90 % of all OI causative mutations, whereas only 10–15 % of OI mutations occur in non-collagenous genes [COL1A1/2 genes remain a common origin of OI [COL1A1 and COL1A2 genes among unrelated OI patients of Vietnamese origin. We applied a systematic approach to characterizing the mutation profiles of these two genes.Type 1 collagen is one of the most abundant proteins in the human body. It is a structural component of the bone, skin, tendons, cornea, and blood vessel walls and other connective tissues . OI is gus genes , 12. WhiThe study was conducted in accordance with the Helsinki Declaration and received approval from the ethical review board of Hue University Hospital  and the Ethical Review Committee on Human Research of the University of Tartu (permit no. 221/M-34). Patients were selected from the Vietnamese database of osteogenesis imperfecta patients. The database includes information on 146 OI patients from 120 OI families and also about their healthy family members. A total of 91 unrelated OI patients were included in the study. Informed written consent from the patients or their legal representatives was obtained prior to inclusion to the study. Investigators then contacted patients in order to conduct an interview, perform a clinical examination, and collect blood samples, including blood samples from parents, siblings, and close relatives. Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods, stored at −80 °C, and analyzed at the University of Tartu, Estonia.COL1A1 gene; 36 primer pairs covering the 5′ UTR and 3′ UTR regions and 52 exons of the COL1A2 gene. The PCR reaction was performed in a total volume of 20 μl, which included 4 μl of 5× HOT FIREPol® Blend Master Mix Ready to Load with 7.5 mM MgCl2 , 1 μl each of forward and reverse primer (5 pmol), and 1 μl of gDNA (50 ng). PCR reaction was performed with a Thermal Cycler  PCR machine. The PCR touchdown program was used as follows for the reaction of amplification:1 = 95.0°; 15:00 min2 = 95.0°; 0:25 min3 = 64.0°; 0:30 min4 = 72.0°; 0:40 min5 = go to 2.4 times6 = 95.0°; 0:25 min7 = 62.0°; 0:30 min8 = 72.0°; 0:40 min9 = go to 6.30 times10 = 72.0°; 5:00 min11 = 6.0°; foreverDNA samples were amplified using a polymerase chain reaction (PCR) with 25 specially designed primer pairs covering the 5′ UTR and 3′ UTR regions and 51 exons of the Amplified PCR products were electrophoresed through a 1.5 % agarose gel, to control the quality of fragments. The PCR products then purified with exonuclease I and shrimp alkaline phosphatase . Sanger sequencing reactions were performed on the purified PCR fragments using a BigDye® Terminator v3.1 Cycle Sequencing Kit . Reactions were processed on the ABI3730xl instrument.http://www.le.ac.uk/ge/collagen/). The pathogenicity of the pathogenic variants was predicted with SIFT score [Sequence reads were analyzed using Applied Biosystems’ Sequence Scanner v1.0 and aligned to the human reference genome Local Reference Genomic sequence LGR_1 and GR_2. Raw sequencing data are available from authors upon request. Sequence analysis and pathogenic variant identification were performed with Mutation Surveyor DNA variant analysis software . Prediction of mutation’s pathogenicity was performed using Alamut Visual software . Variants were checked against the osteogenesis imperfecta mutation database  .COL1A1/2 mutations, 33 with COL1A1 and 21 with COL1A2; this equated to 36.3 and 23.1 % of patients, respectively, totaling 59.4 % of the studied OI cases exhibiting collagen type I mutations. Thirty-four pathogenic variants in the COL1A1 gene  and 22 pathogenic variants in the COL1A2 gene  were identified (patients VN01 and VN47 were carriers of double pathogenic variants in both the COL1A1/2 genes)  of COL1A2 variants. All mutations were highly pathogenic, with a SIFT score of 0.0 and rarely 0.1, and located in regions of high conservation.Fifty-four patients were found to have COL1A1 gene, and 21 patients had 22 pathogenic variants of the COL1A2 gene, equating to a total of 54/91 (59.4 %) patients with COL1A1/2 pathogenic variants. Previous studies have indicated that nearly 90 % of all OI mutations appear in the COL1A1 and COL1A2 genes [In our study, we performed mutational analysis of 91 Vietnamese patients clinically diagnosed with OI (types I–IV). Thirty-three patients had 34 pathogenic variants of the A2 genes , 15. HowA2 genes –18.COL1A1 mutations and 19 of 21 missense COL1A2 pathogenic variants. Gly substitutions composed 36/56 (64.3 %) of COL1A1/2 pathogenic variants. It has been hypothesized that the majority of the clinically severe forms of OI are caused by Gly missense mutations [We identified the substitution of Gly residuals in 17 out of 23 missense utations . Howeverutations .Our research showed that out of 36 glycine substitutions, serine was the most prevalent , followed by valine , and cysteine and aspartic acid (3/36 cases each). Previous studies have suggested that glycine substitutions by cysteine often cause a greater severity of OI phenotype, and glycine substitutions by arginine were often fatal . HoweverIn our research, intronic variants were represented by seven splice site mutations; other research has reported intronic variants among 7/56 of Chinese OI patients . These mCOL1A1 gene. With respect to clinical severity, these patients showed nearly the same manifestations . However, previous studies have described OI patients with different clinical features, despite their being carriers of the c.2461G > A mutation. Current data highlights the complexity of OI genotype–phenotype correlations. It is not yet possible to predict disorder severity based only on mutational analysis data.Patients VN01, VN34, VN40, and VN49 had the same heterozygous mutation: c.2461G > A (p.Gly821Ser) in exon 37 of the COL1A1 c.2005G > A (p.Ala669Thr) pathogenic variant in exon 30. Two patients had the same pathogenic variant and level of OI severity (type IV). Similar cases of variant reoccurance have been described before by Zhang et al. and Lee et al. in both COL1A1/2 genes [Families VN88 and VN89 shared the same heterozygous /2 genes , 18. How/2 genes .COL1A1 mutations: exon 37 c.2461G > A (p.Gly821Ser) and exon 30 c.2005G > A (p.Ala669Thr) in patient VN01. Both pathogenic variants were shared by other unrelated patients among our study cohort. Patient VN47 had two heterozygous COL1A2 pathogenic variants: exon 46 c.3034G > GA (p.Gly1012Ser) and exon 41 c.2569C > CA (p.Pro857Thr). Takagi et al. reported one case of severe OI (types II–III) due to a double substitution of glycine residues in the COL1A2 gene (p.Gly208Glu and p.Gly235Asp), located on the same allele [COL1A1 gene and a mild phenotype (VN01) and moderate phenotype (VN47) based on the clinic examination.Genetic analysis revealed the presence of two heterozygous COL1A1 and 10 COL1A2 variants  were not present in Dalgleish’s OI mutation database . COLFI controls procollagen intracellular assembly and the extracellular assembly of collagen fibrils. Mutation hotspots were situated in the regions that tolerate amino acid substitutions, and pathogenic variant resulted in an altered protein, but the organisms were still able to survive. Gaps in the mutation map connected to regions with crucial functions can however lead to fatal alterations [According to our data, more OI causative pathogenic variants occurred in the erations .Fig. 2DiCOL1A1 and COL1A2 genes in patients with clinical signs of osteogenesis imperfecta were designed far from intron-exon splice sites, which allowed the identifying of splice site, missense, frameshift, and nonsense mutations in the exons of the COL1A1/2 genes. The gold standard of sequencing, the Sanger method, has an accuracy of approximately 99.9 % [COL1A1/2 pathogenic variants in the studied OI patients might have been underestimated.Sequencing primers for the performed Sanger sequencing of the y 99.9 % . HoweverWe must also take into consideration that the percentage of collagen pathogenic variants among osteogenesis imperfecta patients may vary between studies due to their different sample sizes. However, we cannot exclude the possibility that the Vietnamese population has lower rates of collagenous OI pathogenic variants, and a unique OI mutational profile with higher levels of rare non-collagenous pathogenic variants, compared to other populations.COL1A1 and COL1A2 genes among 91 Vietnamese patients with osteogenesis imperfecta. After sequencing of the COL1A1 and COL1A2 genes, we found 56 mutations in 54 patients (59.4 % of patients). Our data showed a lower number of collagen OI pathogenic variants in these Vietnamese patients compared to reported rates for other Asian OI populations. The OI mutational profile of the Vietnamese population is likely unique and is related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of patients negative for collagen OI mutations is needed in order to reveal unidentified OI genotypes from the sample.In the current study, we conducted mutational analysis of the 3′ UTR, 3′ untranslated region; 5′ UTR, 5′ untranslated region; COLFI, fibrillary collagen C-terminal domain; EDTA, ethylenediaminetetraacetic acid; gDNA, genomic DNA; OI, osteogenesis imperfecta; PCR, polymerase chain reaction