On the other hand, VitC increases iTregs generation in vitro and in vivo, however, no allograft tolerance was achieved in animals orally treated with VitC. Lastly, Tregs isolated from the draining lymph nodes of VitC-treated and transplanted mice also showed impaired suppression capacity ex vivo. Our results indicate that VitC promotes the generation and expansion of Tregs, without exhibiting CD4+ T cell-mediated allograft tolerance. These observations highlight the relevance of the nutritional status of patients when immune regulation is needed.Regulatory T cells (Tregs) are critical players of immunological tolerance due to their ability to suppress effector T cell function thereby preventing transplant rejection and autoimmune diseases. During allograft transplantation, increases of both Treg expansion and generation, as well as their stable function, are needed to ensure allograft acceptance; thus, efforts have been made to discover new molecules that enhance Treg-mediated tolerance and to uncover their mechanisms. Recently, vitamin C (VitC), known to regulate T cell maturation and dendritic cell-mediated T cell polarization, has gained attention as a relevant epigenetic remodeler able to enhance and stabilize the expression of the Treg master regulator gene Foxp3, positively affecting the generation of induced Tregs (iTregs). In this study, we measured VitC transporter (SVCT2) expression in different immune cell populations, finding Tregs as one of the cell subset with the highest levels of SVCT2 expression. Unexpectedly, we found that VitC treatment reduces the ability of natural Tregs to suppress effector T cell proliferation  Both Treg subsets play a crucial role under homeostatic and pathological settings, avoiding autoimmune diseases (in vivo or in vitro proinflammatory conditions has been related to a deficiency in suppressive capacity and gain of effector T cell functions (becoming pathogenic), which infers the importance of maintaining Treg stability (Regulatory T cells (Tregs) are a specialized CD4olerance . They cadiseases , 3, contdiseases , 6, and diseases , 8. One diseases , 10. In tability –13. Moletability , (iii) ttability , (iv) eptability –18.in vitro and in vivo generation of Tregs (in vitro studies indicate that VitC may act on T cells and dendritic cells (DCs), favoring the development of Th1-type responses over Th2  in vitro and in vivo, however, no improvement on graft survival is achieved.Vitamin C enters the cell in its physiological form through the sodium-dependent VitC transporter 2 (SVCT2), whose expression has been determined in DCs, macrophages, and circulating T cells in general –40, howein vitro studies on the role of VitC as an enhancer of Foxp3 expression on Treg, and indicates that regardless of their enhanced Foxp3 expression, VitC-treated Tregs display deficient regulatory function as seen in in vitro and ex vivo approximations.Altogether, our data support ad libitum. This study was carried out after the revision and approval of its experimental protocol in accordance with the recommendations of bioethical Committee guidelines from the Faculty of Medicine, Universidad de los Andes, and the Comisión Nacional de Ciencia y Tecnología, CONICYT.Six- to eight-week-old C57BL/6 wild-type, C57BL/6 x BALB/c (F1), Foxp3/GFP and RAG-KO mice were used (both under C57BL/6 background). All mice were maintained under pathogen free conditions, with a 12 h light/dark cycle and food and water via intraperitoneal (i.p.) injection. Tail skin (~1 cm2) from C57BL/6 (syngeneic) or F1  donors was transplanted onto the dorsal area of C57BL/6 or RAG−/− recipient mice. l-Ascorbic acid  was added daily in the water at 0.86 mg/mL, starting the day of surgery until the end of the experiment. Survival of skin allografts was evaluated twice per week and grafts were considered rejected when 80% of the original graft had disappeared or become necrotic.For skin transplantation experiments, mice were anesthetized with a mixture of ketamine at 27% and xylazine at 3% + cells were isolated from the obtained cell suspension by using mouse CD11c MicroBeads UltraPure , according to the manufacture instructions. For lymphocytes, spleens from Foxp3/GFP mice were collected in PBS 1× + 5% of FBS and mechanically disaggregated through a cell strainer. Red blood cells were lysed using RBC lysis buffer 10×  and total CD4+ T cells were purified using mouse CD4+ T cell isolation kit , according to the manufacture instructions. B cells and CD8+ T cells were sorted from the CD4− fraction based on the expression of CD8 and CD19, while CD4+ T conventional and CD4+ T regulatory (Tregs) cells were sorted from the CD4+ fraction based on the expression of CD4 and Foxp3/GFP using a BD FACSAria III .Dendritic cells were purified from wild-type C57BL/6 mouse spleen, digested in the presence of 1 mg/mL of collagenase D  and 2 µg/mL of DNAse I  in RPMI medium  supplemented with 10% FBS , 1% penicillin/streptomycin , HEPES , β-mercaptoethanol  at 50 µM. The 1 mL of this mixture was used to perfuse the organ, followed by an incubation of 45 min at 37°C in waterbath. Undigested material was filtered through a cell strainer  and CD11c+ T cells were isolated from the spleens of Foxp3/GFP mice, using mouse CD4+ T cell isolation kit , according to the manufacture instructions. 2 × 105 cells were cultured in 24-well plates, previously coated with anti-CD3  and anti-CD28  for 6 days. Culture media contained RPMI medium  supplemented with 5% FBS , HEPES , 1% penicillin/streptomycin, and β-mercaptoethanol 50 µM, in the presence of IL-2 (at 100 U/mL) and human TGF-β (at 20 ng/mL), both from Peprotech . At days 1 and 3, AA  was added at a final concentration of 200 µM, and at day 6 cells were harvested for phenotype analysis. All cell cultures were maintained at 37°C and 5% CO2.Total CD4in vivo the direct effect of VitC on naive CD4+ T cells, these were isolated from a Foxp3/GFP reporter mouse spleen and sorted based on CD4+CD62LhiCD44loFoxp3/GFP− phenotype, and intravenously (i.v.) injected into RAG-KO mice a day before skin surgery (4 × 105 cells per mouse). To test the effect of VitC on both natural and iTreg, conventional CD4+ Foxp3/GFP− T cells were sorted from a Ly5.1 Foxp3/GFP reporter mouse, and nTreg were obtained from a Ly5.2 Foxp3/GFP mouse using a mouse CD4+ CD25+ Regulatory T Cell isolation kit . A day before surgery, RAG-KO recipient mice received intravenously 1:1 mixture of CD4+ conventional T cells and Treg. Grafting was performed as previously described and AA  was delivered daily in the water at 0.86 mg/mL from the day of surgery until the end of the experiment.To evaluate in vitro assay, CD4+ Treg cells were sorted from the spleen of a Ly5.1 Foxp3/GFP reporter mouse, as described above, and incubated with VitC (at 50 µM) for 1 h before coculture with antigen presenting cells (APCs) and CD4 conventional T cells. 1 × 105 mitomycin C-treated APCs , 5 × 104 CFSE-labeled CD4+ conventional T cells and Tregs  were cultured in 96 round well-bottom plates with complete RPMI medium, in the presence of soluble anti-CD3  for 3 days. Mitomycin C was used at 50 µg/mL and CFSE at 5 µM . For ex vivo experiments, Tregs were isolated at day 20 from draining lymph nodes (DLNs) of RAG−/− transplanted mice, treated or not with VitC, using a mouse CD4+ CD25+ Regulatory T Cell isolation kit . The assay was set up as described above. Cell division was analyzed by flow cytometry to determine responder T cell proliferation (by CFSE dilution) and FlowJo used to determine division index of responder cells. Suppression was calculated with the formula% Suppression = (1 − DITreg/DITresp) ×100% .For Cell samples were stained with anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD19 (clone 6D5), anti-CD25 (clone PC61), anti-Foxp3 (clone FJK-16s), anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), and anti-CD11c (clone N418) . All antibodies were conjugated with FITC, phycoerythrin, peridinin chlorophyll protein, or allophycocyanin. All flow cytometry was performed using a BD FACS Canto II cytometer , and data were analyzed using FlowJo software .Supernatants from Treg induction assays were collected at day 6 and stored at 80°C until cytokine quantification by sandwich ELISA. Briefly, 96 well flat-bottomed plates were coated overnight with anti-IFNγ, anti-IL-17, or anti-IL-10 capture antibodies at a final concentration of 1 µg/mL. After several washing steps with PBS 1× + 0.05% Tween 20 and a blocking step with PBS 1× + 10% FBS, samples and standard curve were incubated at room temperature for 2 h. Biotinylated antibodies in conjunction with HRP–avidin were used for detecting the immobilized cytokine and tetramethylbenzidine  substrate solution was added to detect HRP activity. The reaction was stopped by adding sulfuric acid 0.16 M and absorbance measured at 450 nm using a Tecan absorbance microplate reader.in vitro cultured cells were treated for RNA isolation using TRIzol reagent , according to manufacture instructions. cDNA was prepared using iScript cDNA synthesis kit  and the expression of SVCT2 and housekeeping gene 18S was measured by real-time PCR, using HOT FIREPol EvaGreen qPCR Supermix  with Mx3000P quantitative PCR system . Primer sequences are showed in Table RNA from spleen/DLNs sorted-populations or The 500 µl of blood was collected by cardiac puncture from naive and skin transplanted C57BL/6 mice in tubes coated with heparin. Samples were centrifuged at 3,000 rpm for 15 min and plasma collected and stored at −80°C until analysis. VitC measurements were performed using the methylene blue assay under acidic conditions described by Dilgin et al. .t-test, using GraphPad Prism .Statistical significance was determined by Student’s −MHC-II+), conventional DCs (CD11c+ CD11b+), B cells (CD19+ CD3−), total CD8+ and CD4+ T cells, conventional CD4+ T cells (CD4+ Foxp3/GFP−), and Tregs (CD4+ Foxp3/GFP+), and evaluated SVCT2 expression by qRT-PCR. SVCT2 mRNA was detected in all cell subsets  , lytic activity (Granzyme and Perforin), proinflammatory cytokines (IFN-γ and IL-17), or suppressive cytokines (IL-10 and TGF-β) Figure E. Altogein vitro Tregs with TGF-β increases Foxp3 expression  , representing ~70% of total Tregs , increasing from 10.6 ± 2.5% in control cultures to 34.4 ± 3.3% , as previously reported (+ VitC); each condition at 1:1, 1:2, and 1:4 proliferating:Treg cell ratios. More clearly, Figure + VitC). The same data set demonstrates the inversely correlated frequencies of both effector T (A) and Treg (B) cells under the coculture conditions, and the unchanged Foxp3 expression on Treg cells in the cultures (C). Furthermore, Figure + T cells used in the previously described in vitro assay, and Figure + T cells is detected, excluding potential staining of proliferating Treg cells. In addition, cytokine expression and secretion was evaluated using quantitative RT-PCR and ELISA, respectively. As observed in Figure in vitro activated CD4+ T cells, favoring a milieu for iTreg differentiation and/or stabilization.Recent studies have shown that VitC supplementation during induction of pression , 36. In pression , 36. Thiin vivo using a previously standardized model to study immune tolerance based on allogeneic skin transplantation in RAG-KO mice, receiving adoptive transfer of naive CD4+ Foxp3/GFP− T cells the day before surgery  in the drinking water, for 20 days  and iTregs (Foxp3/GFP+ Ly5.1+) in response to VitC , and then isolated sufficient number of Treg to further test their suppressive activity ex vivo , as displayed in Figure Ex vivo suppression assays using Treg isolated from dLN of naive, syngeneic and allogeneic mice  confirmed that VitC does not improve Treg-mediated suppression, on the contrary, Treg isolated from VitC-treated naive and allogeneic mice are less suppressive than controls  and solid organ transplant rejection, different studies have tested the ability of small molecules to enhance the generation, expansion and function of Treg cells , 43. Curapamycin . On the apamycin , 45, 46, disease . For thein vitro, by increasing the expression levels of the master regulator gene Foxp3 through an epigenetic mechanism . Taking the above into consideration, we aimed to characterize the effect of VitC on nTreg and iTreg cells in vitro and in vivo using a mouse model for skin transplantation.Despite this new data, however, the effect of VitC on endogenous nTreg has been neglected thus far; an important matter considering that both types of Treg are known to be distinct in terms of phenotype and function , and thaWe began our study by determining the expression of VitC transporter SVCT2, which until now has not been studied in detail, although SVCT2 expression has been reported in human macrophages , murine + T cells fail to suppress T cell proliferation, which has not been described before. Since Treg cells suppression capability is mediated by several distinctive mechanisms [reviewed by Sakaguchi et al.  homing or decreased suppression capacity, as shown in ex vivo suppressive assay  cell populations under different experimental settings, but based on its special genetic background, any extrapolation of the observations made in this model toward a more clinically relevant one must be made with caution. For this reason, we also included an evaluation of VitC treatment in immune competent mice. Similar to what we observed in RAG-KO mice, VitC supplementation increased both effector and Treg cell number, with no beneficial impact on skin graft acceptance. Most importantly, Treg isolated from VitC-treated animals showed decreased suppression capacity in vivo experiments. Moreover, VitC is recognized for not being chemically stable in aqueous solution for long periods of time, due to its oxidization to dehydroascorbic acid, which led us to replace VitC-supplemented water on a daily basis. However, the rationale behind our administration route for VitC delivery was to emulate regular administration in humans in a non-invasive way. Considering the potential pitfalls of our protocol, we were able to detect an increase in VitC plasmatic levels in treated mice (Figure We acknowledge that VitC supplementation through water consumption has some drawbacks, such as our inability to accurately measure the amount of water each mouse actually ingested, which could account for the variability obtained in our data during the performance of in vitro and in vivo, without improving Treg function. On the other hand, VitC could act on effector CD4+ T cells as well, which could complement the mechanistic behind the rejection of the skin transplants observed in this study. Lastly, we believe that the nutritional status of patients, particularly regarding plasmatic vitamins levels, is an important parameter to take into consideration, as accumulating evidence shows the role of vitamins in tuning the immune response.Altogether our results show that VitC treatment acts in a distinct fashion on nTreg and iTreg, both This study was carried out after the revision and approval of its experimental protocol in accordance with the recommendations of bioethical Committee guidelines from the Faculty of Medicine, Universidad de los Andes, and the Comisión Nacional de Ciencia y Tecnología, CONICYT.KO performed experiments, analyzed data, and wrote the manuscript. MC-M performed experiments. TG-C performed experiments. KP-L designed experiments, analyzed data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to establish normal thyroglobulin (Tg) reference intervals (RIs) in regions with adequate and more than adequate iodine intake according to the National Academy of Clinical Biochemistry (NACB) guidelines and to investigate the relationships between Tg and other factors.A total of 1317 thyroid disease-free adult subjects  from 2 cities (Guangzhou and Nanjing) were enrolled in this retrospective, observational study. Each subject completed a questionnaire and underwent physical and ultrasonic examination. Serum Tg, thyroid-stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb), Tg antibody (TgAb), and urinary iodine concentration (UIC) were measured. Reference groups were established on the basis of TSH levels: 0.5 to 2.0 and 0.27 to 4.2 mIU/L.The Tg RIs for Guangzhou and Nanjing were 1.6 to 30.0 and 1.9 to 25.8 ng/mL, respectively. No significant differences in Tg were found between genders or among different reference groups. Stepwise linear regression analyses showed that TgAb, thyroid volume, goiter, gender, age, and TSH levels were correlated with Tg.In adults from regions with adequate and more than adequate iodine intake, we found that Tg may be a suitable marker of iodine status; gender-specific Tg RI was unnecessary; there was no difference between Tg RIs in regions with adequate and more than adequate iodine intake; and the TSH criterion for selecting the Tg reference population could follow the local TSH reference rather than 0.5 to 2.0 mIU/L. The National Academy of Clinical Biochemistry (NACB) has proposed guidelines for establishing such a reference; however, very few studies refer to this guideline. For this reason, the present study established proper Tg reference intervals (RIs) for 2 regions within China that have different iodine statuses.Thyroglobulin (Tg) is the most abundant protein in the thyroid gland. It is produced by thyroid follicular cells, iodized as active thyroid hormone, and released into circulation through exocytosis.2We performed a retrospective, observational study from 2010 to 2011; 1343 thyroid disease free adult residents of 2 cities, Guangzhou and Nanjing, were enrolled in the current study. Guangzhou represented adequate iodine intake and Nanjing represented more than adequate iodine intake.:(1)Age ≥18 but ≤40 years.(2)Thyroid-stimulating hormone (TSH) >0.5 mIU/L but <2.0 mIU/L.(3)Nonsmoker (including passive smoker).(4)No personal or family history of thyroid diseases.(5)No use of drugs that might influence iodine metabolism.(6)No goiter detection upon physical examination or ultrasonography.(7)Testing negative for thyroid autoantibodies .The Guangzhou participants were selected to be the reference group using the following criteria according to the NACB guidelinesThis group was defined as Guangzhou reference group A . Another reference group was designed in accordance with these criteria, except that TSH levels differed , and was defined as Guangzhou reference group B . Nanjing reference group A  and Nanjing reference group B  were similarly defined.All of the subjects completed questionnaires covering pertinent information such as age, gender, smoking, drug intake, and any personal or family history of thyroid conditions.Clinical diagnosis of goiter was performed by physical examination and ultrasonography . The goiter was defined as a visible or palpable thyroid nodule as well as a nodule appearing on the thyroid ultrasound, or a thyroid volume (TV) >25.6 mL for men and >19.4 mL for women.am after an overnight fast. All of the specimens were stored at -80°C until analysis. UICs were measured using an ammonium persulfate method based on the Sandell–Kolthoff reaction. The intra- and interassay coefficients of variation (CV) were 3% to 4% and 4% to 6% at 66 μg/L, respectively, and 2% to 5% and 3% to 6% at 230 μg/L, respectively. Serum Tg was measured on a Roche Cobas Eless 601 module immunology analyzer (Switzerland) using the electrochemiluminescence immunoassay (ECLIA) method, which was standardized against the human Tg reference material (CRM 457). This method had a LoB (limit of blank), LoD (limit of detection), and LoQ (limit of quantification) of 0.02, 0.04, and 0.1 ng/mL, respectively; the intra- and interassay CV values were 2.0% to 4.8% and 4.0% to 5.9%, respectively. Serum TSH, TPOAb, and TgAb were also measured using the ECLIA method. The intra-assay CV values were 1.57% to 4.12%, 2.42% to 5.63%, and 1.3% to 4.9%, respectively, whereas the interassay CV values were 1.26% to 5.76%, 5.23% to 8.16%, and 2.1% to 6.9%, respectively.Blood and spot urine samples were obtained from each participant at 8.00 to 10.00 2.1,11 A standard normal deviate test was used to determine whether a gender-specific RI should be calculated.,13 The Anderson–Darling method was used to assess the normality of the data distribution. Spearman rho and linear regression were calculated to evaluate correlations between Tg and other factors. To test for differences in parameters among groups, Chi-square, Mann–Whitney U test, and 1-way analysis of variance  were used. Significance was set at P  <0.05.Data were recorded using Office Excel 2007, Microsoft Co. Ltd., and analyzed using SPSS 21.0, IBM Co. Ltd.; R software 3.3.1 was used for assessing the normality of the data distribution and false discovery rate (FDR) control. Data are expressed as mean ± standard deviation (SD). The Tg RI was calculated using the logarithmic transformed Tg value as the mean ± 1.96 SD and then transformed back. Tg outliers were checked using Chauvenet criterion. A bootstrap principle-based robust method was used for reference groups consisting of fewer than 120 subjects.2.2All of the procedures involving human participants were performed in accordance with the ethical standards of the China Medical University and the 1964 Helsinki declaration, as well as its later amendments and comparable ethical standards. The research protocols were carefully explained. Informed consent was obtained from all individual participants included in the study.33.1A total of 1343 subjects participated in this study, of whom 26 individuals were excluded: 14 had Tg <0.1 ng/mL (LoQ), 8 had Tg >500 ng/mL (upper LoD), and 4 with missing data. A total of 1157 subjects  from Guangzhou and 160 subjects  from Nanjing were enrolled. The clinical characteristics of different groups are summarized in Table P = 0.091) and gender (P = 0.385) among these 4 reference groups.The distribution of Tg in each group exhibited a long-tailed positive skewness and kurtosis. The original Tg value was transformed as lg(Tg+1), which then became normally distributed. The Tg median of Guangzhou reference groups A and B was 7.5 and 8.0 ng/mL, respectively, and that of Nanjing reference groups A and B was 5.6 and 8.0 ng/mL, respectively. There were no significant differences in age (P = 0.03).In the Guangzhou population, 78 of 515 men and 137 of 642 women were classified as iodine deficient according to World Health Organization (WHO) standards . Women had a higher proportion of iodine deficiency than men   was <z∗ (3.43). The other 3 reference groups also showed this characteristic; thus, determining gender-specific Tg RI was found to be unnecessary. Tg RI was calculated using the transformed Tg value as the mean ± 1.96 SD and then transformed back , or by the t test using the transformed Tg value (P = 0.336). For this reason, the latter RI  calculated by reference group B was used, because it contained nearly 2-fold more subjects than reference group A, making it more reliable.For Guangzhou, the RIs calculated by reference groups A (n = 160) and B (n = 314) were 1.5 to 27.8 and 1.6 to 30.0 ng/mL, respectively. There was no difference between these 2 RIs, as determined by either the Mann–Whitney For Nanjing, the Tg RIs calculated using reference groups A (n = 54) and B (n = 160) were 1.3 to 28.8 and 1.9 to 25.8 ng/mL, respectively, with no differences between them. The Tg RI calculated using reference group B was also used .3.3 subjects who tested positive for TgAb (>115 IU/mL) were excluded . Correlations between Tg and other parameters were tested using the Spearman rank correlation test with FDR q-value correction. A multivariate linear regression model was also used to evaluate the relative impact of other coefficients on Tg.Subjects from Guangzhou were used for testing correlations between Tg and other factors. Because serum TgAb interferes with Tg immunoassays,P < 0.001), age (P < 0.001), TV (P < 0.01), gender (P = 0.014), body mass index (BMI) (P = 0.019), and UIC (P = 0.029) were found to be associated with Tg. Moreover, no relationship was observed between Tg and smoking, iodide-containing drug intake, TSH, TPOAb, or TgAb. In stepwise linear regression, the model had an overall adjusted R-squared value of 0.096. TgAb , TV , goiter , gender , age , and TSH  were found to be associated with Tg. BMI, smoking, iodide-containing drug intake, UIC, and TPOAb were excluded from the model.In the Spearman correlation test, goiter (4–19 immunoradiometric assay (IRMA),–23 immunoluminometric assay (ILMA), immunochemiluminescence assay (ICMA),–28 enzyme-linked immunosorbent assay (ELISA),,30 and mass spectrometry assay.–33 Among these methods, interassay variation remains the principal challenge, as results can vary from 43% to 65% in healthy subjects.,34 Using the BCR 457 Reference Material (formerly named CRM 457) only reduces interassay variation by about 14% to 27%,35,36; therefore, it is important to develop local Tg RI.This study presents the Tg RIs for 2 parts of China with different iodine statuses. The methods for Tg measurement have changed over the past several decades, and currently include radioimmunoassay (RIA),,37,38–51; however, very few of them have provided information on Tg RI (Table  reported a Tg upper reference limit (95th percentile) of 46.9 ng/mL (Tg median: 11.2 ng/mL) in 120 healthy TgAb-negative euthyroid subjects with no histories of thyroid diseases or abnormalities on thyroid ultrasonography. Bílek et al recruited a large population  with a median Tg of 17.5 ng/mL, and suggested 44.2 ng/mL as the Tg upper limits. Feldt et al established correlated reference values for age and gender  based on 143 normal subjects. Giovanella et al found higher serum Tg levels in women than in men (P = 0.0022), and provided gender-specific reference values . Among these 4 studies, 3 described participants with higher Tg levels, and only 1 study reported values similar to those reported here. These differences may actually be attributable to the methods. Luca et al and the present work both used the ECLIA method to measure Tg, which was different from the methods used by other studies . This confirms the importance of locality in developing Tg RI.Many studies have measured Tg in adultsP = 0.003), the correlation was too weak to show a significant difference between male and female subjects at all grouping levels. In addition, standard normal deviate testing did not justify establishing gender-specific Tg RI. Second, some studies that have reported gender differences in Tg failed to mention correction for other factors, which might have influenced Tg levels. Thus, the differences detected might actually be due to sampling strategies. Finally, as women were found to be more likely to have inadequate iodine intake, it is not inexplicable that women had higher Tg levels than men, resulting in a higher proportion of women exceeding the upper reference limit. Considering these facts, no gender-specific RI was developed for Tg.Studies differ on whether gender-specific RI is required. Here, we hypothesized that gender-specific Tg RI is unnecessary, primarily for the following 3 reasons. First, in this study, although gender was significantly correlated with Tg  with different TSH values. For this reason, we suggest that using a reference group with a TSH value within a reasonable reference is acceptable. This greatly reduces the difficulty in subject selection, making it more feasible and commercial for Tg reference determination.4.13) or thyroxine (T4). Although the NACB guideline for Tg RI determination does not require these parameters, there may be certain individuals who have TSH levels within the reference values but abnormal T3 or T4 levels. Second, there were more women than men in this study , because in China, women are usually more concerned about health, and as such, are willing to participate in these type of studies. However, because no significant differences in Tg levels were found between genders, this gender-specific imbalance of Tg seemed to have minor effects on our conclusions. Finally, this is a cross-sectional study; thus, the causal relationship between Tg and iodine intake could not be well established.The current study had several limitations. First, the thyroid indexes did not include serum triiodothyronine (T5Tg may be a useful marker of iodine status. Tg values are dependent on gender, but gender-specific Tg RI may be unnecessary. UIC correlates with Tg levels, but no differences exist between Tg RI in regions of adequate and more than adequate iodine intake. Finally, for the sake of simplifying subject selection for Tg RI, the TSH criterion can follow local TSH RIs other than values of 0.5 to 2.0 mIU/L.We gratefully acknowledge the contribution of physicians from Guangzhou and Nanjing. We would also like to thank the residents who participated in this study.


After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER−) breast cancer , and a single signal 3 SNP . Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis.Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region , we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen-receptor-positive (ER Genotyping was conducted with the COGS array, a custom array comprising approximately 200,000 SNPs.r2 > 0.1 (n = 424), plus a set of SNPs designed to tag all remaining SNPs with r2 > 0.9 (n = 184), but we managed to include 563 SNPs with a designability score (DS) > 0.9 and which passed QC.We examined a 1 Mb region  in which the 1000 Genomes Project cataloged 1,811 variants (March 2010 Pilot version 60 CEU project data). We aimed to genotype all 628 SNPs with minor allele frequency (MAF) > 2% and correlated with rs981782 and rs10941679 at r2 > 0.3). In European-ancestry women, 461 of these SNPs were associated with overall breast cancer risk, 489 with ER+ and 38 with ER− breast cancer risk  allele: OR = 1.12; 95% CI 1.10–1.14; p = 2.55 × 10−26; −11), and SNP3 rs200229088  but SNP2 was uncorrelated with the other two (r2 = 0.07 and 0.05).Case-control analyses were conducted on 3,365 SNPs . Within signal 2, a further 37 SNPs correlated with rs6864776 at r2 > 0.6, had likelihood ratios of <100:1 relative to rs6864776, and hence could not be excluded from being causative statistically . There are no other SNPs correlated with rs200229088 that could explain this association. All other SNPs were excluded from causality  has odds against causality > 10 million:1 versus signal 1 candidate rs10941679. Similarly, SNP rs7716600, which is an eQTL for MRPS30 expressionr2 with SNP rs10941679 = 0.77, with SNP rs6864776 = 0.05, and with SNP rs200229088 = 0.12) has odds against causality >160,000:1 versus signal 1 candidate rs10941679. These exclusions of former causal candidates highlight the need for fine-mapping studies before conducting functional analyses.The top signal, SNP1 rs10941679, is markedly more significant than any other SNP in the locus . Hence, the most parsimonious explanation is that this SNP is causally related to risk. The next most strongly associated SNP, after adjustment for signal 1 SNP rs10941679, was rs6864776, representing signal 2  = 1.09; 95% CI 1.04–1.15; p = 0.0009, conditional p = 0.0859 and per t-allele OR (rs200229088) = 1.09; 95% CI 1.02–1.15; p = 0.0065, conditional p = 0.9149   .+ breast cancer . By contrast, SNP2 rs6864776 was moderately associated with ER− but not ER+ tumors , MRPS30 (MIM: 611991), and HCN1 (MIM: 602780)—and several putative long noncoding RNAs  and breast tumors  as well as in GTEx normal breast  and breast tumors , in GTEx normal breast (p corrected = 0.002), and in TCGA  or heterozygous (A/G) for the common allele of SNP1  or with SYTO9 for lncRNAs normalized against TATA box-binding protein , we performed chromatin conformation capture 3C) assays in ERst cells .8 3C libromoters ; DNA wasC assays ll lines , and S6. the PRE A. No addRE  allele had no significant effect on the PRE enhancer activity. Although this appears to rule out an effect of this SNP on transactivation, it is possible that SNP1 affects the recruitment of key proteins required for the epigenetic modification of the enhancer, which would not be observed in a reporter assay. Another possibility is that the SNP effect may be observed only under certain biological conditions such as growth factor stimulation.The regulatory capability of the PRE, combined with the effect of SNP1, was further examined in reporter assays. Promoter-driven luciferase reporter constructs were generated by the insertion of PCR-amplified fragments containing 74 cells B and S8.80 cells . In all a) and risk (g) alleles.+ BT474, MCF7, and MDA-MB-361 or ER− MDA-MB-231 and Hs578T cells using the NE-PER nuclear and cytoplasmic extraction reagents. Biotinylated oligonucleotide duplexes were prepared by combining sense and antisense oligonucleotides, heat annealing, and slow cooling. Duplex-bound complexes were transferred onto Zeta-Probe positively charged nylon membranes by semi-dry transfer then cross-linked onto the membranes. Membranes were processed with the LightShift Chemiluminescent EMSA kit as per the manufacturer’s instructions, and signals were visualized with the C-DiGit blot scanner. For SNP1, we observed allele-specific binding by nuclear proteins only in the ER+ BT474, MCF7, and MDA-MB-361 extracts  for both the protective  suggested four proteins bound to the SNP site including FOXA1, FOXA2, CEBPB, and OCT1 . To confxpressed . FOXA1 ig-allele of signal 1 SNP rs10941679 conferred a 15% increased risk of ER+ breast cancer and higher expression levels of the MRPS30 and FGF10 genes and was the most strongly associated SNP with MRPS30 expression in this 1 Mb region. MRPS30—also called PDCD9 (Programmed Cell Death protein 9)—encodes a mitochondrial ribosomal protein involved in apoptosis.c and other pro-apoptotic proteins are released during cell death initiation.FGF10 is an extensively studied gene with compelling data suggesting its involvement in breast tumorigenesis. FGF10 is a member of the fibroblast growth factor (FGF) family and encodes a glycoprotein that specifically binds to FGFR2 (splice FGFR2IIIb) to control signaling pathways including cell differentiation, proliferation, and apoptosis.FGFR2 (MIM: 176943) have the strongest association with ER+ breast cancer susceptibility identified to date.FGF10 are highly correlated with proliferation rate of breast cancer cell lines and cancer cell invasion.FGFR2, CCND1 (MIM: 168461), and TGFB1 (MIM: 190180),In conclusion, we have provided evidence for at least three independent causal SNPs with effects on the risk of breast cancer at this locus. The minor


The tension-tension fatigue behavior of unidirectional C/SiC ceramic-matrix composite at room temperature and 800 °C under air has been investigated. The fatigue hysteresis modulus and fatigue hysteresis loss energy corresponding to different number of applied cycles have been analyzed. The fatigue hysteresis loops models for different interface slip cases have been derived based on the fatigue damage mechanism of fiber slipping relative to matrix in the interface debonded region upon unloading and subsequent reloading. The fiber/matrix interface shear stress has been estimated for different numbers of applied cycles. By combining the interface shear stress degradation model and fibers strength degradation model with fibers failure model, the tension-tension fatigue life S-N curves of unidirectional C/SiC composite at room temperature and 800 °C under air have been predicted. Ceramic materials possess high strength and modulus at elevated temperature. But their use as structural components is severely limited because of their brittleness. The continuous fiber-reinforced ceramic-matrix composites, by incorporating fibers in ceramic matrices, however, not only exploit their attractive high-temperature strength but also reduce the propensity for catastrophic failure. The carbon fiber-reinforced silicon carbide ceramic-matrix composites (C/SiC CMCs) are one of the most promising candidates for many high temperature applications, particularly as aerospace and aircraft thermostructural components ,2,3.i.e., there exist many matrix microcracks in the fabrication process [et al. [i.e., the interface frictional shear stress and fibers strength decrease with the increase of cycle number [Compared with ceramic materials and whisker-reinforced CMCs, the fatigue behavior of fiber-reinforced CMCs has many different characteristics,  process , these m process . Ruggles [et al.  investig [et al. ,8, i.e.,e number ,10,11,12e number ,14. Whene number .versus cycle number curves for different peak stresses are obtained considering interface wear. The tension-tension fatigue life S-N curves have been predicted.In this paper, the tension-tension fatigue behavior of unidirectional C/SiC composite at room temperature and 800 °C under air has been investigated. The fatigue hysteresis modulus and fatigue hysteresis loss energy have been analyzed for different numbers of applied cycles. The fatigue hysteresis loops models corresponding to different interface slipping cases have been derived based on the fatigue damage mechanism of fiber slipping relative to matrix in the interface debonded region upon unloading/reloading. The fatigue hysteresis loss energy for strain energy lost per volume during corresponding cycle is formulated in terms of interface shear stress. By comparing experimental hysteresis loss energy with theoretical computational values, fiber/matrix interface shear stress under fatigue loading has been estimated. The fiber failure probabilities The T-700™ carbon  fiber-reinforced silicon carbide matrix composites were provided by Shanghai Institute of Ceramics, People’s Republic of China. The fibers have an average diameter of 7 μm and come on a spool as a tow of 12 K fibers. The unidirectional C/SiC composite was manufactured by hot-pressing method, which offered the ability to fabricate dense composites via a liquid phase sintering method at a low temperature. The volume fraction of fibers was approximately 42%. Low-pressure chemical vapor infiltration was employed to deposit approximately 5–20 layers PyC/SiC with a mean thickness of 0.2 μm in order to enhance the desired non-linear/non-catastrophic tensile behavior. The nano-SiC powder and sintering additives were ball milled for 4 h using SiC balls. After drying, the powders were dispersed in xylene with polycarbonsilane (PCS) to form the slurry. Carbon fiber tows were infiltrated by the slurry and wound to form aligned unidirectional composite sheets. After drying, the sheets were cut to a size of 150 mm × 150 mm and pyrolyzed in argon. Then the sheets were stacked in a graphite die and sintered by hot pressing.3, and an open porosity below 5%.The dog-bone shaped specimens, with dimensions of 120 mm length, 3.2 mm thickness and 4.5 mm width in the gage section, were cut from 150 mm × 150 mm panels by abrasive water jet machining (AWJM) ,17, in wE is calculated by Equation (1).maxσ and minσ denote the fatigue peak and valley stresses, respectively; and maxε and εmin denote the fatigue peak and valley strains, respectively.The tension-tension fatigue experiments at room temperature and 800 °C under air were conducted on a MTS Model 809 servo hydraulic load-frame  equipped with edge-loaded grips. The fatigue experiments were in a sinusoidal waveform and a loading frequency of 10 Hz to investigate the evolution of stress-strain hysteresis loops and hysteresis loss energy ,19. The The direct observations of matrix multicracking at room temperature were made using HiROX optical microscope. The matrix crack densities were determined by counting the number of cracks in a length of about 15 mm. After specimens fatigue failure, the fracture surfaces were observed under optical microscope.The monotonic tensile strength is approximately 270 ± 5 MPa at room temperature. The cyclic tensile stress–strain curves are shown in σmax = 240 MPa, the specimen experienced 1,000,000 cycles without fatigue failure. The displacements corresponding to peak and valley stresses increase with the increase of applied cycles, as shown in E is calculated by Equation (1). The hysteresis modulus decreases rapidly at the initial stage of fatigue loading, as shown in i.e., from 169 to 128 GPa during the first 10 cycles. The occurrence of matrix multicracking and fiber/matrix interface debonding is the main reason for hysteresis modulus degradation during the initial cycles. When matrix cracks approach saturation and interface shear stress degrades to steady-state value, the hysteresis modulus decreases slowly due to fibers failure.When versus applied cycles curve is given in The stress-strain hysteresis loops corresponding to the 1st, 10th, 100th, 1000th, 10,000th, 100,000th and 1,000,000th cycles are illustrated in The specimen without fatigue failure was tensiled to failure at a loading speed of 10 MPa/s. After specimen tensile failure, the side and fracture surface of the failure specimen were observed under optical microscope. There exist multiple matrix cracks on the side surfaces, as shown in The tensile strength is approximately 320 ± 3 MPa at 800 °C under air. The cyclic tensile stress-strain curves are shown in σmax = 120 MPa, the specimen experienced 72215 cycles and fatigue failed. The displacement increases rapidly during the first 10 cycles due to matrix multicracking and interface debonding, then increases slowly due to interface shear stress degradation for interface wear or oxidation, before the specimen failure, the displacement increases rapidly again, mainly attributed to fibers failure, as shown in versus cycle number curve is shown in i.e., (1) at the initial stage of fatigue loading, the hysteresis modulus decreases rapidly due to matrix multicracking and interface debonding; (2) when matrix cracks approach saturation, the hysteresis modulus decreases slowly due to interface shear stress degradation; and (3) during the final 2000 cycles, the hysteresis modulus decreases rapidly due to fibers failure.When versus applied cycles curve is shown in The stress-strain hysteresis loops corresponding to 10th, 100th, 1000th, 10,000th, 50,000th, 70,000th and 72,200th cycles are shown in After specimen fatigue failure, the front and side surfaces and fracture surface were observed under optical microscope. There exist multiple matrix crackings in front and side surfaces of failure specimens, as shown in i.e., (1) the interface partially debonds and fiber completely slides relative to matrix; (2) the interface partially debonds and fiber partially slides relative to matrix; (3) the interface completely debonds and fiber partially slides relative to matrix; and (4) the interface completely debonds and fiber completely slides relative to matrix in the interface debonded region.Under fatigue loading, the hysteresis loops develop due to frictional sliding occurred along any interface-debonded region. Based on the interface slip mechanism, the hysteresis loops can be classified into four different cases, σmax = 240MPa are given in i.e., τi = 1–50 MPa, is shown in i = 27–50 MPa, i.e., the A–B part in i.e., the interface partially debonds, i.e., Ld < L/2 in i.e., y(σmin) = Ld in i = 8.8–27 MPa, i.e., the B‒C part in i.e., the interface partially debonds, i.e., Ld < L/2 in i.e., y(σmin) < Ld in i = 8.3–8.8 MPa, i.e., the C‒D part in i.e., the interface completely debonds, i.e., Ld = L/2 in i.e., y(σmin) < L/2 in i = 1–8.3 MPa, i.e., the D‒E part in i.e., the interface completely debonds, i.e., Ld = L/2 in i.e., y(σmin) = L/2 in The shape and area of hysteresis loops as a function of interface shear stress are shown in i.e., the Global Load Sharing criterion (GLS) [Tb> denotes the load carried by broken fibers; and P(T) denotes the fibers failure probability.mf denotes the fiber Weibull modulus; and σc denotes the fiber characteristic strength of a length δc of fiber [lo is the reference length; oσ is the fiber reference strength of a length of lo of fiber; and σo(N) is the fiber strength during the Nth cycle. Lee and Stinchcomb [p1 and p2 are empirical parameters. Upon first loading to fatigue peak stress, matrix multicracking and interface debonding occur. There exist partial fibers failure in the interface-debonded region and interface-bonded region. Under fatigue loading, fibers fail due to degradation of interface shear stress and fibers strength. When fibers failure probability approaches critical value, the composite would fatigue fail. When fibers begin to break, the loads dropped by broken fibers would be transferred to intact fibers at the cross-section plane. There are two dominant failure criterions presented in the literatures for modeling fibers failure, on (GLS)  and Locaon (GLS) . The twoon (GLS) .(2)σVf=of fiber .(4)σc= is the fiber/matrix interface shear stress of Nth cycle; τio is the initial interface shear stress; τimin is the steady-state interface shear stress; N is the cycle number; and ω and λ are empirical parameters.Evans et al.  developeWhen fibers fracture, fiber stress drops to zero at the break point, and the stress in the fiber builds up through interface shear stress.lf required to build the fiber stress up to its previous intact value is given by Equation (8).The slip length f(x) of the distance x of a fiber break from reference matrix crack plane, provided that a break occurs within a distance ±lf, is [The probability distribution  ±lf, is (9)f(x)=Using Equations (7)–(9), the average stress carried by broken fibers isSubstituting Equation (10) into Equation (2), it leads to the form ofT carried by intact fibers at the matrix crack plane can be determined for different peak stresses. Substituting Equations (5) and (6) and the intact fiber stress T into Equation (3), the fibers failure probability for different number of applied cycles can be determined. When fibers failure probability approaches the critical value q*, the composite would fatigue fail [Using Equations (5), (6) and (11), the stress gue fail .(12)q*=σmax = 240 MPa, the stress-strain hysteresis loops corresponding to the 1st, 10th, 100th, 1000th, 10,000th, 100,000th, 1,000,000th cycles are given in i.e., the interface partially debonds and fiber partially slides relative to matrix in the interface-debonded region upon unloading/reloading. With the increase of number of applied cycles, the interface shear stress degrades due to interface wear. When interface completely debonds, the interface shear stress decreases rapidly due to thermal residual radial tensile stress at the fiber/matrix interface. By comparing experimental hysteresis loss energy with theoretical computational value, the interface shear stress corresponding to different number of applied cycles can be estimated. The predicted interface shear stress of the 1st, 10th, 1000th, 10,000th, 100,000th and 1,000,000th cycles is shown in When versus cycle number has been simulated by Evans-Zok-McMeeking model [io = 8MPa, τimin = 0.3MPa, ω = 0.04 and λ = 1.5. The fibers strength degradation curve predicted by Lee-Stinchcomb model [p1 = 0.01 and p2 = 1.The interface shear stress ng model , as showmb model  is givenσmax = 267 and 260 MPa corresponding to different number of applied cycles is illustrated in i.e., when σmax = 267 MPa, the specimen fatigue failed after 31 cycles; and when σmax = 260 MPa, the specimen fatigue failed after 400 cycles. The fibers failure probability versus applied cycles curve can be divided into two regions as shown in i.e., (1) during the first 20 cycles, the fibers failure probability increases rapidly due to degradation of interface shear stress and fibers strength; and (2) when interface shear stress approaches steady-state value, fibers failure is mainly controlled by fibers strength degradation caused by interface wear.The fibers failure probability under i.e., (1) the A‒B part is affected by the degradation of interface shear stress and fibers strength; and (2) when interface shear stress approaches steady-state value, the B‒C part is only affected by fibers strength degradation.The experimental and predicted fatigue life S-N curves are illustrated in σmax = 120 MPa, the stress-strain hysteresis loops corresponding to the 1st, 10th, 100th, 1000th, 10,000th, 30,000th, 50,000th, 70,000th and 72,200th cycles are shown in i.e., the interface partially debonds and fiber partially slides relative to matrix in the interface-debonded region upon unloading/reloading. With the increase of applied cycles, the interface shear stress degrades due to oxidation of interphase or carbon fibers. When interface completely debonds, the interface shear stress decreases rapidly due to thermal residual radial tensile stress at the fiber/matrix interface. By comparing experimental hysteresis loss energy with theoretical computational value, the interface shear stress can be estimated for different number of applied cycles. The predicted interface shear stress of the 1st, 100th, 10,000th and 72,200th cycles is illustrated in When versus applied cycles has been simulated by Evans‒Zok‒McMeeking model [io = 6.1 MPa, τimin = 0.2 MPa, ω = 0.001 and λ = 0.8. The fibers strength degradation curve predicted by Lee-Stinchcomb model [p1 = 0.02 and p2 = 1.The interface shear stress ng model  as shownmb model  is showni.e., when σmax = 240 MPa, the specimen fatigue failed after 999 cycles; and when σmax = 150 MPa, the specimen fatigue failed after 110,962 cycles. The fibers failure probability versus applied cycles curve can be divided into two regions, as shown in i.e., (1) at the initial stage of fatigue loading, the fibers failure probability increases rapidly due to degradation of interface shear stress and fibers strength; and (2) when interface shear stress approaches steady-state value, fibers failure is mainly controlled by fibers strength degradation by oxidation.The fibers failure probability corresponding to different number of applied cycles is illustrated in i.e., (1) the A‒B part is affected by the degradation of interface shear stress and fibers strength; and (2) the B‒C part is only affected by fibers strength degradation.The experimental and predicted fatigue life S-N curves are given in (1)The hysteresis modulus decreases rapidly at the initial stage of fatigue loading due to matrix multicracking and interface debonding. When matrix cracks approach saturation, the decrease of hysteresis modulus would be mainly attributed to interface wear. However, at 800 °C in air, the hysteresis modulus decreases rapidly compared with that at room temperature, due to oxidation of interphase and carbon fibers.(2)σmax = 240 MPa; at 800 °C in air, the hysteresis loss energy degrades from 28 KPa during the 1st cycle to 1.22 KPa during the 72,200th cycle; and the interface shear stress degrades from 6.1 MPa to 0.3 MPa during the first 100 cycles under σmax = 120 MPa.At room temperature, the hysteresis loss energy degrades from 56 KPa during the 1st cycle to 8 KPa during the 1,000,000th cycle; and the interface shear stress degrades from 8 MPa to 0.4 MPa during the first 10 cycles under (3)The fatigue limit stress at room temperature was approximately 88% of tensile strength. However, there were no apparent fatigue limit stress at 800 °C in air due to oxidation of interphase or carbon fibers. The fatigue failure specimens were observed under optical microscope. The pullout fibers at 800 °C in air exhibit serious oxidation. At elevated temperature, the degradation of fibers strength due to oxidation of carbon fibers leads to the life being greatly reduced compared with that at room temperature.(4)i.e., the first part is affected by the degradation of interface shear stress and fibers strength; and the second part is only affected by fibers strength degradation.The tension-tension fatigue life S-N curves at room temperature and 800 °C under air have been predicted. The predicted fatigue life S-N curve can be divided into two regions, The tension-tension fatigue behavior of unidirectional C/SiC composite at room temperature and 800 °C under air has been investigated.


Salmonella enterica serovar Typhimurium strains has served the scientific community as a group of model organisms for basic genetic and biochemical pathway research. Here, we report the whole-genome shotgun sequences of Salmonella enterica serovar Typhimurium strains LT1, LT18, LT19, LT20, LT21, and LT22.The Lilleengen type (LT) collection of  Salmonella enterica serovar Typhimurium strains has served the scientific community as a group of model organisms, including those for basic genetic and biochemical pathway research (LT2), a mutator strain (LT7), and a source of bacteriophage  that was constructed into the default horizontal gene-transfer tool (P22HTint) used for transducing DNA from one Salmonella strain to another or backcrossing mutations into wild-type backgrounds to confirm mutation phenotypes. Inasmuch as LT2, LT7, and LT22 have contributed so much to scientific advancement in the last 65 years, the question of whether any of the other strains in the Lilleengen collection have unique characteristics or bacteriophages that could further discoveries in molecular genetics arose. Our laboratory obtained the original LT collection from the Salmonella Genetic Stock Centre , which had previously received the collection in lyophilized vials from the Lederberg Laboratory, which received it from Kaare Lilleengen  were chosen for next-generation sequencing, followed by analysis of sequence and bacteriophage content.The Lilleengen type (LT) collection of lleengen . We perfsearch LT, a mutatsequence . Six SalSalmonella genome (LT2)  was extracted from 1 mL of each culture using the Pure Link Genomic DNA kit protocol (Invitrogen). Purified gDNA was eluted with 50 µL 10 mM Tris-HCl–0.1 mM EDTA (pH 9.0), and the extracted DNA (80ng/µL) was stored at −20°C. TruSeq DNA PCR-free libraries for each gDNA sample were constructed following protocol (Illumina). Library construction targeted a 350-bp insert. Genomic libraries were combined into a single pool and sequenced in a single-lane 2 × 150 MiSeq run using standard protocols (Illumina version 1.9). We generated an average of 1,027,510 reads measuring 35 to 151 bp per LT strain, approximating 19.3-fold coverage of the reference me (LT2) , filterime (LT2)  was usedme (LT2) .All of the LT strains sequenced were unique. Each LT strain had two to four intact bacteriophages, two to six incomplete bacteriophages, and one to two questionable bacteriophage regions when submitted to bacteriophage search tool (PHASTER) analysis . As expeNDGP00000000; LT18, NDGQ00000000; LT19, NDGR00000000; LT20, NDGS00000000; LT21, NDGT00000000; and LT22, NDGU00000000. The versions described in this paper are the first versions, as follows: LT1, NDGP01000000; LT18, NDGQ01000000; LT19, NDGR01000000; LT20, NDGS01000000; LT21, NDGT01000000; and LT22, NDGU01000000.This whole-genome shotgun project has been deposited at GenBank under the following accession numbers. LT1,


The heating of the fluids used in electronic cigarettes (“e-cigarettes”) used to create “vaping” aerosols is capable of causing a wide range of degradation reaction products. We investigated formation of benzene (an important human carcinogen) from e-cigarette fluids containing propylene glycol (PG), glycerol (GL), benzoic acid, the flavor chemical benzaldehyde, and nicotine.TM “pod” system (provides no user accessible settings other than flavor cartridge choice), and two refill tank systems that allowed a range of user accessible power settings. Benzene in the e-cigarette aerosols was determined by gas chromatography/mass spectrometry. Benzene formation was ND (not detected) in the JUUL system. In the two tank systems benzene was found to form from propylene glycol (PG) and glycerol (GL), and from the additives benzoic acid and benzaldehyde, especially at high power settings. With 50:50 PG+GL, for tank device 1 at 6W and 13W, the formed benzene concentrations were 1.9 and 750 μg/m3. For tank device 2, at 6W and 25W, the formed concentrations were ND and 1.8 μg/m3. With benzoic acid and benzaldehyde at ~10 mg/mL, for tank device 1, values at 13W were as high as 5000 μg/m3. For tank device 2 at 25W, all values were ≤~100 μg/m3. These values may be compared with what can be expected in a conventional (tobacco) cigarette, namely 200,000 μg/m3. Thus, the risks from benzene will be lower from e-cigarettes than from conventional cigarettes. However, ambient benzene air concentrations in the U.S. have typically been 1 μg/m3, so that benzene has been named the largest single known cancer-risk air toxic in the U.S. For non-smokers, chronically repeated exposure to benzene from e-cigarettes at levels such as 100 or higher μg/m3 will not be of negligible risk.Three e-cigarette devices were used: the JUUL Electronic cigarettes (“e-cigarettes”) use an electrical resistance coil to vaporize mixtures of propylene glycol (PG), glycerol (GL), nicotine, and flavor chemicals. Vaporization of an e-liquid containing mostly PG and/or GL requires a temperature of ~190 to 290°C: when the ambient pressure is 1 atm, PG boils at ~190°C, GL boils at ~290°C, and PG+GL mixtures will boil between ~190 and ~290°C; the presence of other constituents besides PG and GL  will affect the boiling point.  will affect the boiling point.) Temperatures higher than the boiling point of an e-liquid are possible in the coil zone if the rate of e-liquid delivery to the coil does not keep pace with the heat delivery rate: the vicinity of the coil becomes “dry”, and the heat delivery rate surpasses the rate at which heat is carried away by evaporated liquid as “latent heat”.3. McAuley et al.[In general, e-cigarette aerosols tend to be simpler in composition than cigarette aerosols: “e-liquids” are a simpler starting matrix as compared to cigarette filler, and burning cigarettes have been reported to reach 900°C,. Neverthey et al., however2O , and two variable-power, tank-type devices. For experiments with the tank devices, the fluids used were prepared in the laboratory from PG, GL, benzoic acid, benzaldehyde, and/or nicotine . Non-labeled PG and GL and standard chemicals were obtained from Sigma-Aldrich Inc. . Determination of benzene levels in the e-cigarette aerosols was by cartridge-based adsorption/thermal desorption (ATD) followed by gas chromatography with detection by mass spectrometry (GC/MS). Three different e-cigarette devices were used in the ATD determinations: 1) JUULTM personal vaporizer and refill cartridges (“pods”)  in four different flavors  were purchased online in May 2016 ; 2) EVODTM tank-type atomizer  with 1.8 ohm resistance single horizontal coil and silica wicking material, purchased online in July, 2016; and 3) Subtank NanoTM V.1 (Kangertech) tank-type atomizer with 1.2 ohm resistance single vertical “OCC” single coil and cotton wicking material, purchased online in July, 2016. The JUULTM system has no user options other than flavor of the cartridge selected. For the EVODTM and Subtank NanoTM devices: 1) the “recommended” settings were 6W and 10 to 26W, respectively; 2) each replicate aerosol sample for gaseous benzene determination proceeded using a clean tank and a new coil so that any run-to-run changes in benzene production would not be caused by “aging” of the coil etc.; 3) at every wattage setting tested, sample collection was begun 2 h after “conditioning” the new coil; 4) conditioning occurred by taking six 50 mL puffs at 6 W for the EVOD device and 13W for Subtank NanoTM device respectively.Fully et al. [For all three devices, each ATD sample was taken as three or six 50 mL puffs without power (blanks) or with power (aerosol samples) with a puff duration of 5 s and puff-to-puff interval of 60 s.  glass fiber/cellulose acetate (GF/CA) filter (to remove the aerosol droplets), followed by a single ATD gas sampling cartridge containing 100 mg of 35/60 mesh Tenax TA and 200 mg of 60/80 mesh Carbograph 1 TD . All four different JUULTM flavors were tested. The direct “butt” connections to the filter were held in place using short pieces of flexible 0.125 in. i.d. polyvinyl chloride (PVC) tubing. The benzene levels obtained are minimum values because of the possibility of some small sorptive loss to the PVC pieces; the PVC pieces were replaced after each sample. For the method used (indoor sampling), blank levels corresponded to ~5 μg/m3. Final values given are blank-corrected; values not significantly above the blank level are reported as “not detected” (ND).Each ATD sample comprised an average benzene level for six puffs. For the JUULFor the two tank units, the device used for drawing e-cigarette puffs involved a programmable syringe pump. As above, direct “butt” connections were made using short pieces of PVC tubing. Blanks were obtained without activating the power. For vaping, flow for each sample was sequentially drawn through: 1) a GF/CA filter as above; 2) single ATD gas sampling cartridge as above; a 3) three way “T” valve connected to a 1 L Tedlar bag ; and 4) a syringe pump . Minimal “butt” connections were made as above. For verification of the total sample volume, the puffs were exhausted from the syringe pump back through the three way valve and into the Tedlar bag. Again, final values are blank-corrected, and values not significantly above the blank level are reported as ND.TM device (as above) with 50:50 PG+GL containing benzoic acid at 1% by weight. Aerosol was generated at 14 W (2 ohms resistance) using five 50 mL puffs, each drawn over five seconds with a puff interval of 1 min. In an approach similar to that described by Jensen et al. [For confirmation of the formation of benzene in the e-cigarette aerosols by nuclear magnetic resonance (NMR) spectroscopy, aerosol samples were created using an EVODValues for mg of e-liquid vaped per puff (and for the resulting TPM concentration) were estimated based on weight loss of the e-cigarette unit for each run.et al.[P(%) = 100%/(1+KpTPM) where Kp (m3/μg) is the gas-to-particle (i.e. droplet) partition coefficient, and TPM is the suspended “total particulate matter” level for the aerosol. 2) It can be estimated that Kp ≈ 10−9.7 m3/μg for benzene at 20°C for partitioning to e-cigarette aerosol particles  in glycol solutions of ~14 [8.8 μg/m3 . Then P(%) ≥ 100%/(1+10−9.7108.8) = 89%.For all the e-cigarette aerosol samples, >89% of the benzene can be deduced to have been in the gas phase as follows: 1) As discussed by Pankow et al.,17 the pw et al.,,17 usingt = 20°C, an activs of ~14 , and an For each standard sample for method calibration, an ATD cartridge was charged with 2 or 4 μL of a 1 to 100 ng/μL solution of benzene in methanol, followed by a 50 mL/min flow of nitrogen for 10 min. Each ATD sample cartridge and each standard ATD cartridge was thermally desorbed using a TurboMatrix 650 ATD unit . Prior to desorption, the ATD unit automatically amended each cartridge with 20 ng of fluorobenzene as the internal standard compound. Each ATD cartridge was thermally desorbed for 10 min at 285°C with helium desorption flow of 40 mL/min and split flow of 20 mL/min. The desorption stream was trapped at -10°C on the intermediate “air monitoring trap” (ATP). The ATP was then thermally desorbed at 295°C and 25 psi constant pressure helium with a split flow of 8 mL/min for 4 min. The non-split portion of the desorption gas stream passed onto the GC column which was mounted in an Agilent  7890A GC. The GC was interfaced to an Agilent 5975C MS operated in electron impact ionization mode. The MS scan range was 34 to 400 amu. The electron multiplier voltage was 1400 V. The fused silica capillary GC column was a model Rxi-624Sil MS  of 30 m length, 0.25 mm i.d., and 1.4 μm film thickness.TM pods were opened to obtain the e-liquid for determination of the benzoic acid and nicotine concentrations. Aliquots of 100 μL were withdrawn, diluted 1:100 with methanol, and filtered with a syringe-mounted PVDF (polyvinylidene fluoride) filter, 13 mm diameter, 0.22 μm pore size, obtained from Thermo Fisher Scientific, Inc. . Analyses proceeded by high performance liquid chromatography (HPLC) using an injection volume of 20 μL, a Waters Corp.  Model 1525 binary solvent delivery module (with Rheodyne 7725i injector), a Discovery™ C-18 column  at 40°C, and a Waters Corp. Model 2996 photodiode array detector. The mobile phase composition was maintained isocratic, 40:60 methanol:water amended with 0.1% trifluoroacetic acid (1 mL/min). The detector wavelength for benzoic acid was 228 nm, and 259 nm for nicotine. The calibration ranges for the injected standards were 0 to 1 mg/mL for benzoic acid, and 0 to 0.1 mg/mL for nicotine.JUULTM pod fluids were found to be 44.8 ± 0.6 and 61.6 ± 1.5 mg/mL respectively, corresponding to a benzoic acid/nicotine molar concentration ratio of 0.97 to 1). For comparison, as noted above, analyses in our laboratory have indicated the presence of benzoic acid in 14 commercial refill e-liquids at levels estimated to be in the range 0.02 to 2 mg/mL.The concentrations of benzoic acid and nicotine in the JUUL12C6) was confirmed by NMR for e-cigarette aerosol collected in DMSO-d6 and generated with the EVODTM device operated at 14 W (2 ohms resistance) using 50:50 PG+GL (both 12C3) containing benzoic acid (12C6) at 1% by weight. The expected strong singlet peak in DMSO-d6 at 7.37 ppm [Benzene formation  was confirmed to occur from 50:50 PG+GL (both 13C3) (see 12C3)  see  at level2C3) see .TM samples for any of the four flavors. For the other two devices, benzene gas-phase concentrations and deliveries (as μg benzene per g e-liquid vaped) are summarized in 3, respectively. For the Subtank Nano device, at 6W and 25W, the mean values were remarkably lower, namely ND and only 1.5 μg/m3, respectively.Gas-phase benzene was not found above blank levels in any of the JUUL3. Remarkably, the values at the higher power levels for the Subtank Nano device were much lower, ≤ ~100 μg/m3.Significant formation from benzoic acid and benzaldehyde was observed for both the EVOD and Subtank Nano devices at the high power settings used. With benzoic acid and benzaldehyde at 9 to 10 mg/mL, for the EVOD device, values at higher power levels were as high as 5000 μg/m3, and so a much higher risk from benzene for chronic use of tobacco cigarettes as compared to e-cigarettes. However, median ambient air concentrations of benzene in locations in the U.S. in 2013 were ~1 μg/m3 [3 will not be of negligible risk.For tobacco smoke from “regular” cigarettes, the 1999 Massachusetts Benchmark Study  reported~1 μg/m3 . Levels ~1 μg/m3 . It can S1 Text(DOCX)Click here for additional data file.


A small-molecule screen with mutantembryos identified the anti-fungal drug miconazole as a potent hemorrhagicsuppressor. Miconazole inhibited both brain hemorrhages in zebrafish andmesenteric hemorrhages in rats by decreasing matrix metalloproteinase 9(MMP9)-dependent vessel rupture. Mechanistically, miconazole downregulated thelevels of pErk and Mmp9 to protect vascular integrity in fn40amutants. Therefore, our findings demonstrate that miconazole protects bloodvessels from hemorrhages by downregulating the pERK-MMP9 axis from zebrafish tomammals and shed light on the potential of phenotype-based screens in zebrafishfor the discovery of new drug candidates and chemical probes for hemorrhagicstroke.Hemorrhagic stroke accounts for 10-15% of all strokes and is stronglyassociated with mortality and morbidity worldwide, but its prevention andtherapeutic interventions remain a major challenge. Here, we report theidentification of miconazole as a hemorrhagic suppressor by a small-moleculescreen in zebrafish. We found that a hypomorphic mutant  Summary: A phenotype-based chemical screen in zebrafish identifiesmiconazole as a novel hemorrhagic suppressor. Miconazole inhibits vessel ruptureand hemorrhages by decreasing pErk and MMP9 in zebrafish and rats. Cardiovascular diseases are the leading causes of mortality worldwide, and strokeranks among the top three. In 2012, ∼6.7 million people died of stroke,accounting for ∼17% of total mortality from non-communicable diseasesglobally . HemorrhThe current failure to develop medical treatments, which primarily rely ontarget-based drugs, suggests that the essential therapeutic targets for this diseaseare not yet discovered, or alternatively, multiple genetic factors in parallel areinvolved in this complex disease. At the same time, phenotype-based drug discoveryis gradually being recognized and actively pursued by both academic and industrialscientists using model organisms such as zebrafish . The zebfn40a, oneof the known β-pix (arhgef7b) mutantalleles -induced mutant alleles , as a hefn40a was identified as a hemorrhagic mutant from an ENU-inducedmutagenesis screen of the zebrafish genome at Massachusetts General Hospital,Boston . Hemorrhagicsites occurred most frequently in the hindbrain and occasionally in the midbrainand forebrain of the mutants at 2 days post-fertilization (dpf) (n>100), which is similar to anotherβ-pix allele, m292 (pak2a (mi149) . In contrast, erythrocytes accumulated in theintracerebral region of fn40a mutants (Movie 2). The recruitment ofcoro1a:eGFP-labeled inflammatory cells to the hemorrhagicregion in mutants  . Since t mutants H,K, acco mutants Lcompare mutants J. In addrmations , thus esrmations , the fn4fn40a mutant embryos were arrayed in each well of96-well plates, and were then subjected to administration of10 µmol/l of small molecules from 6 to 24 hpf. At24 hpf, the compounds were washed away, replaced, and embryos wereincubated with fresh egg water until the desired stages. Each compound wasreplicated in three wells. The hemorrhagic suppression efficiency was scoredbased on the hemorrhage rate for each compound. The second step was to validatethe efficacy of candidate compounds from the primary screen by using an averageof 200 mutant embryos. In the final step of validation, we tested thedose-dependent efficacy and toxicity of each candidate compound with a series ofconcentrations and more than 70 mutant embryos per condition. Through thisscreen, we isolated 7 candidate hemorrhagic suppressors  and their hemorrhagic suppression efficacy is shownby the hemorrhage rate (Table S1). Miconazole nitrate stood out of the sevencandidates because of its great efficacy on hemorrhagic suppression and leasttoxicity to embryos. Importantly, miconazole effectively suppressed brainhemorrhages in a dose-dependent manner . By applying 5 μmol/lmiconazole to fn40a mutant embryos from 6 to 24 hpf, wefound that it suppressed ∼70% of brain hemorrhages compared withlevels in DMSO-treated embryos . In addition, application of miconazole from 6 to24 hpf had more of an effect on suppression of brain hemorrhage than didtreatment from either 24 to 36 hpf or 24 to 48 hpf, and treatmentfrom 36 to 48 hpf had no inhibition effects . This suggests that miconazole plays a protectiverole in generating mature cerebral vessels before their ruptures and hemorrhagesoccur in fn40a mutants around 40 hpf, and/or thatmiconazole might take longer to reach its target and have any action invivo. Taken together, we have identified a suitable hemorrhagicstroke model in zebrafish that could be used to identify the anti-fungal drugmiconazole as a novel, potent hemorrhagic suppressor of fn40amutants.Using an in-house chemical library with diversified chemical structures of 923drug-like compounds, we performed a hemorrhagic suppressor screen. The outlineof this screen includes three major steps M. DuringO-dianisidine staining of live embryos allowed us to compare theamounts of erythrocytes in heterozygous and homozygous fn40amutants in the presence of PTU (0.003%) . To quantify the level of hemorrhage, we appliedTg(flk1:eGFP) to label vascular endothelial cells andTg(gata1:DsRed) to label erythrocytes. Consistent with thedata from O-dianisidine staining, we found that brainhemorrhage was evident in homozygous mutants compared with heterozygous siblingsin which miconazole inhibited hemorrhage . By testing five known analogues of miconazole, wefound that sulconazole and tioconazole efficiently inhibited brain hemorrhagewhile voriconazole, fluconazole or ketoconazole had little effect , suggesting that theimidazole group is essential for the activity of hemorrhagic suppression ofmiconazole. To address whether vessel patterning defects occur in homozygousmutants, we bred flk1:eGFP transgenic zebrafish into thefn40a mutant background. Theflk1:eGFP-labeled cerebral vessels were well patterned andsymmetrical in heterozygous siblings treated with DMSO or miconazole at2 dpf and 3 dpf (fn40a mutants treated with DMSO at 2 dpf(m292bubblehead ((0.003%) A,A′,C,C′(0.003%) A-B′, bra(0.003%) C′and we(0.003%) D′. We fond 3 dpf E,F,I,J. at 2 dpfG and3 dat 2 dpfK,consisheadm292 . Remarkaheadm292 D′,H,L. Ffli1a:nuc-eGFPtransgenic reporter was used to label the nuclei of both endothelial cells anderythroid cells, allowing us to easily identify proliferation and migration ofthese cells during development. In fn40a mutants, cerebralvessels were well-patterned and grew normally up to 1.5 dpf, around whichtime blood cells frequently leaked out from the central arteries to formhematomas, resulting in further inflammation and disruption of the surroundingvessels  compared withheterozygous siblings . These data suggest thatcranial vascular patterning is relatively normal but those vessels are immatureand fragile in the fn40a mutant.To gain insights into the hemorrhage processes, we first set up time-lapse3-dimensional imaging to monitor the development of brain vessels in liveembryos using a LSM700 confocal microscopy. The Fig. S4). Therefore, our data reveal the critical role ofmiconazole in protecting cerebral vessels from rupture and hemorrhage infn40a mutants.To visualize the subcellular structures of cerebral vessels and surroundingcells, we used transmission electron microscopy (TEM) and found that blood cellswere normally located inside blood vessels and did not infiltrate intosurrounding neurons in heterozygous mutants (DMSO treatment) M,Q, whicfn40a mutants. We then tested whether vascular permeabilitywas affected in this mutant by examining the leakage of DAPI injected throughthe sinus venous of wild-type embryos (fn40amutants (fn40a mutants (DMSO control) before (36 hpf) orafter (48 hpf) brain hemorrhage occurred, respectively  expression in wild-type siblings and mutants, and thiswas not affected by miconazole . On the other hand, we did not find any blood-brainbarrier structure outside the endothelium, as observed in mammals by TEM, andblood cells and plasma flowed exclusively in blood vessels with no or littleleakage of DAPI in 2.5 dpf embryos. These data confirm that miconazoleprotects these immature cerebral vessels at this early embryonic stage inzebrafish.In mammals, the neurovascular unit (NVU) maintains cerebral homeostasis . NVUincconazole A-D′, sugmmp2,mmp9, mmp13b, mmp14a,mmp14b and mmp23aa. We next tested whetherthese MMPs were affected in fn40a mutants, and if so, whetherthey were responsive to miconazole treatment. Quantitative real-time PCRrevealed that mmp9, but not the other MMP genes, wassignificantly upregulated in fn40a mutants (DMSO control), andthis was suppressed by miconazole, compared with wild-type siblings (DMSOcontrol) at 2 dpf . Importantly, elevated Mmp9 proteins wereassociated with brain vessels of homozygous mutants compared with heterozygousmutants in the absence or presence of miconazole; and like mmp9mRNA, Mmp9 protein was also suppressed by miconazole in brain vessels of thehomozygous mutant .Previous research in rodents has demonstrated that MMPs are essential componentsfor regulating vascular integrity . The incat 2 dpf M. Additiat 2 dpf N andmmpmp9 mRNA O were upsiblings N,suggesFig. S6). Consistently, MMP9 inhibitor I(3 µmol/l) effectively decreased the brain hemorrhage ratefrom 100% (in mutants with DMSO) to ∼50% (in mutants withMMP9 inhibitor I) . MMP9 inhibitor II at4 µmol/l had very low solubility in E3 medium, which mightbe the cause of its reduced efficiency in inhibiting hemorrhage . In addition, MMP9 zymography assay confirmed thatMMP9 inhibitor I, MMP9 inhibitor II and miconazole decreased Mmp9 activity inhomozygous fn40a mutants compared with DMSO-treated mutants. Taken together, our data suggest that the increasedpermeability of brain vessels and the resultant hemorrhages are at least partlydue to elevated Mmp9 in fn40a mutants, and miconazolesuppresses the hemorrhages by decreasing Mmp9 expression.MMP9 is highly conserved among species from zebrafish, mouse, rat, and rabbit tohuman (bitor I) P,Q. Mutabitor I) Q. In addMovies 5 and 6), we found that miconazole (5 or 10 mg/kg byintravenous injection) did not cause mesenteric hemorrhages (Movies 7 and 8). In contrast, pre-treatment with miconazole (5 or10 mg/kg) 30 min before uPA injection prevented mesenterichemorrhages , these pharmacological data suggest that miconazoleplays a conserved role in protecting vessels against MMP9-dependent rupture inrats. Therefore, identification of chemical suppressors such as miconazole fordeveloping cerebral vessel ruptures has important implications in decreasingadult pathological phenotypes including vessel ruptures and hemorrhages. Having found that miconazole is an effective hemorrhagic suppressor in zebrafish,we then tested whether it also played a similar role in mammals. Extending thisstudy to mammals was critical for both therapeutic utility and mechanisticstudies. To achieve this goal, we established a mesenteric hemorrhagic model inrats by using the urokinase-type plasminogen activator (uPA)-dependentactivation of MMP9, degradation of the ECM, and resultant mesenterichemorrhages. Compared with control treatment with physiological saline  includingpro-MMP9 to activated MMPs; and both plasmin and MMPs break down many componentsof the ECM A,A′,G,G′fn40a mutants but these changes were not rescued bymiconazole treatment, compared with sibling heterozygotes (data not shown),suggesting that miconazole functioned downstream to the Pix/Pak pathway.In addition, previous studies have revealed a strong correlation betweenextracellular signal-regulated kinase 1 and 2 (ERK1/2) and MMP9 at boththe transcriptional and post-transcriptional levels , pErk in thebrain vessels of the fn40a mutant increased at 36 hpf and at 48 hpf , which decreased after miconazoletreatment , suggesting that miconazole isable to suppress elevated pErk in the brain vessels of this mutant.Through a quantitative mass spectrum analysis of whole embryos, we found thatβ-Pix and five Pak-family members were downregulated inl levels . MMP9 isl levels . Throughl levels . The corl levels , we examt 24 hpf H. Importfn40amutants in a dose-dependent manner   . Althoug mutants J-K; each mutants I-K,we c mutants L. TakenAPOE(19q13), SLC25A44 (1q22), COL4A1 (13q34) andKCNK17 (6p21)  . These g defects . Other m defects , 2010. T defects . While t defects .Here, w to m292  butfn40nd pak2a , is downβ-pix in fn40a mutants  decreases Rac1 function to activate Mek1/2, which then increases thelevel of phosphorylated Erk and Mmp9 transcription/translation, leading toabnormal vascular permeability and disruption of vascular integrity. As bloodcirculation is enhanced and blood pressure increases after 24 hpf, thesefragile vessels are inclined to break down and bleed, leading to brain hemorrhagesin this mutant. By performing a chemical suppressor screen, we found that miconazolesuppressed the intracerebral hemorrhages in fn40a mutants.Miconazole is an approved antifungal agent with a fungus-specific binding siteagainst ergosterol biosynthesis and is pharmacologically described as an inhibitorof sterol demethylase and cell wall synthesis to protect vascular integrity from zebrafish to mammals. Its direct target(s) remainto be identified.Based on this study and previous work , we propynthesis . We haveClinical therapies for hemorrhagic stroke have not yet progressed beyond theintroduction of thrombin during the hematoma-forming process and neuron protectionor blood-clot surgery after the formation of a hematoma . GeneralDanio rerio) were maintained and bred as described inthe Zebrafish Book (fli1a:nuc-eGFP) (coro1a:eGFP) (flk1:eGFP) (gata1:DsRed) were purchased from Vital River, Beijing. The animals were raised and handled inaccordance with the animal protocol IMM-XiongJW-3 for this study, which wasapproved by the Institutional Animal Care and Use Committee of Peking Universitythat is fully accredited by AAALAC International.Zebrafish (ish Book . The Tg(uc-eGFP) ,Tg(coro1a:eGFP) , Tg(flk1k1:eGFP)  andTg(g1:DsRed)  zebrafis, Boston . Wild-tyfn40a mutant embryos to adults and selectedthose adult pairs that consistently produced almost 100% hemorrhagicmutant embryos for this chemical screen. This hemorrhagic suppressor screenconsists of three major steps. We started to perform a primary screen with96-well plates. After collecting, sorting and rinsing the embryos, we arrayedfive fertilized and healthy embryos before 6 hpf in each well of the96-well plates, which were filled with 200 µl E3 medium. Chemicalcompounds were dissolved in DMSO to form a stock concentration of20 mmol/l, and the compounds were diluted with E3 medium to form aworking concentration of 10 µmol/l in 96-well plates, andcompounds were applied at 6 hpf. Treated embryos were incubated in anincubator at 28°C, and compounds were washed off and replaced with freshE3 medium at 24 hpf. Each compound was repeated in 3 wells. Hemorrhagerate was calculated at 2 dpf and 3 dpf, as the percentage of thenumber of hemorrhagic mutants after compound treatment out of the totalhemorrhagic mutants used. The morphological changes/abnormalities ofmutant embryos were also documented. We set up a criterion to choose candidatecompounds wherein the compounds were capable of decreasing the hemorrhage rateto <60% but caused minimal embryonic lethality. The primary screenled us to identifying 85 candidate hemorrhagic suppressors. Second, we verifiedeach candidate compound for its efficacy with more than 200 embryos in a10 cm dish. We arrayed about 70 mutant embryos for each dish and withthree dishes for each compound. The fn40a mutant embryos werethen treated with 10 µmol/l of each compound starting from6 to 24 hpf, compounds were washed off at 24 hpf, and thehemorrhage rate/morphological abnormalities were documented at2 dpf and 3 dpf. This second verification screen resulted in 21candidate compounds. Third, we carried out the final screen on testingdose-dependent efficacy and toxicity of the 21 candidate compounds. For thosecompounds that caused some embryonic abnormalities, we set up the concentrationgradients as 0 (DMSO control), 2 µmol/l,4 µmol/l, 6 µmol/l,8 µmol/l and 10 µmol/l. For thosecompounds that caused minimal embryonic abnormalities, we first set up a widerrange of concentrations as 0 (DMSO control), 1 µmol/l,5 µmol/l, 10 µmol/l,20 µmol/l and 40 µmol/l, and thengradually narrowed down the range to the final concentration gradient from 0(DMSO control), 1 µmol/l, 2 µmol/l,3 µmol/l, 4 µmol/l to5 µmol/l. The final candidate compounds were chosen basedon their efficacy and minimal toxicity by using the narrowest concentrationgradients. About 40 mutant embryos were used for each experimentalcondition/concentration group in 6-well plates and repeated three times.After the three-step screen, we identified seven candidate hemorrhagicsuppressors and five other compounds that caused different embryonic phenotypessuch as abnormal dorsal-ventral patterning and arrhythmia.We raised homozygous fn40a mutant zebrafish embryos. Imidazole members ofsulconazole, tioconazole, voriconazole, fluconazole and ketoconazole were alsosupplied by the Chinese National Compound Library Center. Treated embryos werecollected and embedded in 1% low-melting point agarose for taking liveimages, or fixed in 4% paraformaldehyde for in situhybridization, O-dianisidine staining or fluorescenceimmunohistochemistry.An in-house small-molecule library containing 923 chemicals from the ChineseNational Compound Library Center, Beijing, China, as well as MMP9 inhibitor I, MMP9 inhibitor II , MMP9 inhibitor V, U0126 , PD0325901 , and SL-327  were applied to siblings orfn40a mutants were treated with DMSO, MMP9 inhibitorI (3 µmol/l), MMP9 inhibitor II(3 µmol/l), or miconazole (3 µmol/l)from 6 to 24 hpf, and treated embryos at 24 hpf were collected forprotein preparation with a non-reducing protein lysis buffer that contains phenylmethyl sulfonyl fluoride (Sigma-Aldrich). Proteinconcentrations were quantified by bicinchoninic acid (BCA) protein assay kit. We used each protein sample (40 mg) for MMP9zymography assay  according to the manufacturer'sprotocol. Proteinase activity was visualized as a clear band on Coomassieblue-stained gels (SDS-PAGE with substrate G from Applygen), as previouslyreported  to live, dechorionatedembryos, keeping in dark for 20 min, and washing three times withdeionized H2O as previously described  double transgenic zebrafish were bred withwild-type or fn40a mutant to generatefn40a+/−;Tg andfn40a−/−;Tg. The embryostreated with DMSO or miconazole were embedded in 1% low-melting pointagarose, and live images of brain regions at 2 dpf were taken with aZeiss LSM700 confocal microscope. The regions of interest were restricted to thehindbrain, exactly posterior to the middle cerebral vein, and the same depth ofz-stacks was used from all embryos. TheTg(gata1:DsRed) signals for labeling erythrocytes werequantified.Applying 500 μl fresh staining solution  for rat tissues,anti-ZO1 , anti-CD31 , anti-laminin , anti-collagen type IV , anti-Mmp9  for fishembryos, and anti-phospho-p44/42 MAPK (Erk1/2)  for fish embryos. The secondary antibodies wereAlexa Fluor 488 goat anti-rabbit IgG , Alexa Fluor 555 goat anti-mouse IgG , Alexa Fluor 555 goat anti-rat IgG , and Alexa Fluor 555 goat anti-rabbit IgG .invitro transcription according to a standard protocol, and themmp9 RNA probe was made as previously described (mmp9 probe fragment was amplified from zebrafish cDNAlibrary with the forward primer (5′-GGGGATTTTGCCCTGATCGTGGA-3′)and a T7-containing reverse primer(5′-TAATACGACTCACTATAGGGTTCCAGTAGCGCCCGTCCTTGA-3′). T7 RNA polymerase was used to generate theantisense mmp9 probe. Whole-mount in situhybridization was performed as described in the Zebrafish Book (Digoxigenin-labeled antisense RNA probes were synthesized by escribed . Themmpish Book .flk1:eGFP) embryos at 1.5 and 2 dpf wereanesthetized and injected with 12 nl of 0.85 mg/ml DAPIthrough the sinus venous. Embryos were incubated at 28°C for30 min, and then imaged using a laser-scanning confocal microscope at 488 nm and 405 nm.Basically, we used a protocol similar to that reported by the Watts group .Tg(flk1Embryos (50 embryos at 2 dpf or 100 embryos at 24 hpf) werecollected and rinsed. The yolk proteins were removed and 100 µlRIPA protein lysis was then applied for isolating proteins. Proteins wereresolved on 10% or 12% SDS-PAGE gels and transferred ontopolyvinylidene difluoride membrane (Bio-Rad). The primary antibodies wereanti-Mmp9 , anti-MMP9 , anti-phospho-p44/42 MAPK (Erk1/2), anti-p44/42 MAPK(Erk1/2) ,anti-α-tubulin  and anti-GAPDH . The secondary antibodies were goat anti-rabbit IgG-HRP  and goat anti-mouse IgG-HRP . The stained membranes were imaged using a Bio-Rad ChemiDoc MP ImagingSystem (USA) and the bands were analyzed using ImageJ.mmp2, mmp9,mmp13b, mmp14a, mmp14band mmp23aa are listed in Table S2 and were used to produce 100-150 bp fragments.Quantitative real-time PCR was carried out with SYBR Premix DimerEraser .Total RNA from embryos at given stages was extracted using a TRIzol protocol aspreviously described . For eacAs previously reported , the embRats were anesthetized by intramuscular injection of urethane(2 g/kg). Cannulae were inserted into the left internal jugularand femoral vein for administration of urokinase  and miconazole (5 or 10 mg/kg) respectively.Miconazole or physiological saline (NS) was injected into animals half an hourbefore imaging the baseline. After the baseline was recorded, urokinase solutionor NS was injected as quickly as possible. Rats were kept in 37°Cincubator and mesenteric membranes were kept moist with warm NS. Images andrecords were documented at 30 min intervals for a total of 2 h.Animals were placed in the right-lateral decubitus position and the mesentericmicrocirculation was observed under an inverted microscope  equipped with a color monitor  and DVD recorder. The hemorrhage spots and hemorrhagic area of each spotwere calculated by Image-Pro Plus and ImageJ. The total spot number and thehemorrhagic area of the whole mesenteric membrane are presented for each timepoint with statistic curves.t-test, one-way ANOWA or two-way ANOVA with post test wasperformed to evaluate the significance of differences between experimental andcontrol groups and data are expressed as means±s.e.m.*P<0.05 indicates a statistically significanteffect.All experiments were repeated at least three times with more than 50 zebrafishembryos, and with five or more rats. Two-tailed Student's