Different laboratory methods are used to measure serum ferritin levels as a marker of iron status in the general population. This study aimed to compare serum ferritin levels using enzyme-linked immunosorbent assay (ELISA) versus immunochemiluminescence (Cobas e411) and immunoturbidimetric (Cobas Integra 400) methods in terms of sensitivity, specificity and accuracy, and whether they can be used interchangeably. A comparative cross-sectional study enrolled one hundred and six adult Yemeni patients (33 males and 73 females) aged 18-55 years, recruited from the dermatology and cosmetic center of Hadhramout Modern Hospital, Mukalla, Yemen. Serum ferritin levels were measured using ELISA, Cobas e411, and Cobas Integra 400 methods. For method comparison, a paired-sample t-test was used. For the consistency between the three methods, they were analyzed with regression and Pearson correlation coefficient. For determining accuracy, a receiver operating curve (ROC) was used. Bias error between the methods was determined through a Bland-Altman plot analysis. Our results did not show any significant statistical difference between ELISA and Cobas e411 (52.55 ± 7.4 µg/L vs. 52.58 ± 7.5 µg/L, p = 0.967), while there were significantly higher values from Cobas Integra 400 results than Cobas e411 (56.31 ± 7.8 µg/L vs. 52.58 ± 7.5 µg/L, p < 0.001) and ELISA (52.55 ± 7.4 µg/L vs. 56.31 ± 7.8 µg/L, p < 0.001). According to the correlation coefficient and linear regression analysis, a strong association between ELISA with Cobas e411 (r = 0.993, p < 0.001) and Cobas Integra 400 results (r = 0.994, p < 0.001) were revealed. For determining accuracy, Cobas e411 and Cobas Integra 400 results showed higher sensitivity (92.0%; 90.0%) and specificity (97.7%; 99.9%) respectively. Additionally, the Bland-Altman plot analysis showed a high agreement between the ELISA and Cobas e411 methods (bias: -0.035). In contrast, there was a low agreement between the ELISA and Cobas Integra 400 methods (bias: -3.75). Similarly, the agreement between Cobas e411 and Cobas Integra 400 methods was low (bias: -3.72). Serum ferritin levels were measured by Cobas e411, and Cobas Integra 400 methods were strongly correlated with the ELISA results, with higher sensitivity, specificity, and accuracy. However, further investigations with larger samples are required for improved accuracy and more precise results, and to determine whether they can be used interchangeably.
Abstract Due to the current COVID-19 pandemic, the rapid discovery of a safe and effective vaccine is an essential issue, consequently, this study aims to predict potential COVID-19 peptide-based vaccine utilizing the Nucleocapsid phosphoprotein (N) and Spike Glycoprotein (S) via the Immunoinformatics approach. To achieve this goal, several Immune Epitope Database (IEDB) tools, molecular docking, and safety prediction servers were used. According to the results, The Spike peptide peptides SQCVNLTTRTQLPPAYTNSFTRGVY is predicted to have the highest binding affinity to the B-Cells. The Spike peptide FTISVTTEI has the highest binding affinity to the MHC I HLA-B1503 allele. The Nucleocapsid peptides KTFPPTEPK and RWYFYYLGTGPEAGL have the highest binding affinity to the MHC I HLA-A0202 allele and the three MHC II alleles HLA-DPA1*01:03/DPB1*02:01, HLA-DQA1*01:02/DQB1- *06:02, HLA-DRB1, respectively. Furthermore, those peptides were predicted as non-toxic and non-allergen. Therefore, the combination of those peptides is predicted to stimulate better immunological responses with respectable safety.
Human hepatocellular carcinoma (HCC) is the most prevalent and recurrent type of primary adult liver cancer without any effective therapy. Thus, there is an increase demands for finding new drugs and treatment strategies with selective and potent effects towards HCC. Plant-derived compounds acting as anti-cancer agents can induce apoptosis through targeting several signaling pathways. Thymoquinone (TQ), the major biologically active compound of the black seed oil (Nigella sativa) has demonstrated inhibitory activities on various cancers by targeting several pathways. In the present study, we have evaluated the molecular mechanisms that underlie the anti-proliferative, anti-metastatic, and pro-apoptotic activities exerted by TQ on liver cancer cell lineHepG2, a well-documented HCC in vitro model. Cell proliferation was determined by WST-1 assay, apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining, wound healing assay to investigate the metastasis, and the expression of target genes was assessed by Real-time RT–PCR analysis. We found that TQ significantly reduced HepG2 cell viability and induced apoptosis in a dose-dependent manner. Migration of HepG2 cells was suppressed in response to TQ. Moreover, TQ decreased the expression of several angiogenesis-related genes including versican (VCAN), growth factor receptor-bound protein 2 (Grb2), and the histone methyltransferase for lysine 27 of histone 3 (EZH2). The findings suggest that TQ exerts inhibitory effects on HCC most likely through targeting key genes involved in the invasiveness and
Zika flavivirus is suspected to cause Guillain-Barre syndrome in adults and microcephaly, along with other congenital abnormalities in infants. Presently, no vaccines or therapeutics are available. Here, we report novel compounds identified by high-throughput virtual screening of Maybridge chemical database and molecular docking studies. We selected viral enzyme NS2B/NS3 serine protease as the therapeutic target because of its important role in viral replication. We selected seven potential compounds as antiviral drug candidates because of their high GOLD fitness score, high AutoDock Vina score, or X-Score binding energy and analyzed the strength of molecular interactions between the active site amino acids and selected compounds. Our study also provides a foundation for similar studies for the search of novel therapeutics against Zika virus.
The tumor suppressor p73 is a member of p53 family and has a high degree of similarity with p53 function and structure. Like p53, p73 can also induce the expression of several genes involved in cell cycle and apoptosis. p73 expression is downregulated in many tumors by several mechanisms including the ubiquitination pathway. Thus, understanding the ubiquitin-proteasome pathway in p73 regulation will help in targeting this later and develop a new promising therapeutic strategy for cancer with p53 mutations. The aim of this study was to evaluate the effect of Thymoquinone (TQ), the major biologically active compound of the black seed oil on the expression of several E3 ubiquitin ligase enzymes known to be regulators of p73 and the related events in cancer cells with p53 mutation, such as the human acute lymphoblastic leukemia Jurkat cells, the human triple-negative breast cancer (MDA-MB-468 cells) and human promyelocytic leukemia HL60 cells. RNA-seq data showed that several E3 ubiquitin-ligase enzymes, well documented to be involved in the degradation of p73 including Itch, Pirh2, E3s Pin2, Mdm2, TRIM32 and SCFFBXO45 were downregulated in Jurkat cells. Among the target genes, Itch was significantly downregulated in TQ-treated Jurkat cells as compared with control cells. TQ-induced Itch downregulation was confirmed by real-time RT-PCR in Jurkat cells, MDA-MB-468 cells and HL60. Treating Jurkat cells with either TQ or the proteasome inhibitor MG132 induced an upregulation of p73. The present study indicates that TQ could be a promising inhibitor of the E3-ubiquitin ligase Itch leading to the upregulation of tumor suppressor p73 in cancers expressing mutant p53.
<div>Abstract<p>We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified <i>PLAG1</i> as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with <i>PLAG1</i> gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation <i>in vitro</i> and as a xenograft and promoted G<sub>1</sub> to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC<sub>50</sub> of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor.</p>Implications:<p>Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.</p></div>
Among all cancer types, colorectal cancer is the third most common in men and the second most common in women globally. Generally, the risk of colorectal cancer increases with age, and colorectal cancer is modulated by various genetic alterations. Alterations in the immune response serve a significant role in the development of colorectal cancer. In primary cancer types, immune cells express a variety of inhibitory molecules that dampen the immune response against tumor cells. Additionally, few reports have demonstrated that classical chemotherapy promotes the immunosuppressive microenvironment in both tissues and immune cells. This study assessed the expression levels of genes using RT-qPCR associated with the immune system, including interferon-γ, programmed death-1, β2-microglobulin, human leukocyte antigen-A, CD3e, CD28 and intracellular adhesion molecule 1, in patients with colorectal cancer, as these genes are known to serve important roles in immune regulation during cancer incidence. Gene expression analysis was performed with the whole blood cells of patients with colorectal cancer and healthy volunteers. Compared with the normal controls, programmed death-1was highly expressed in patients with advanced-stage colorectal cancer. Furthermore, the expression of programmed death-1 was higher in patients receiving adjuvant therapy, which suggests the therapy dampened the immune response against tumor cells. The results of the present study indicate that classical adjuvant therapies, which are currently used for patients with colorectal cancer, should be modulated, and a combination of classical therapy with anti-programmed death-1 antibody should be conducted for improved management of patients with colorectal cancer.
Abstract Thymoquinone is naturally occurring bioactive compound which showed cumulative preclinical evidence of anticancer effects. However, its exact anti-cancer activity remains to be elusive and is currently being studied. In this study, we assessed the role of thymoquinone as a potential therapy in human breast cancer cell lines in addition to its role in altering molecular clock genes expression, which in turn has been associated with tumorgenesis. Sulfa-Rhodamine-B assay (SRB) assay was used to evaluate the cytotoxic effect of thymoquinone in breast cancer cell lines (MCF-7 and T47D). Following 72 h of exposure, thymoquinone showed cytotoxic effects against MCF-7 and T47D with IC50’s of 44.4±3 µM and 152±11 µM respectively. Further investigation showed that thymoquinone showed some affect on the cell cycle distribution in both cell lines. It significantly increased the cell population in S-phase in MCF-7cells (P<0.05), while causing a significant G1 phase arrest in T47D cells (p<0.01). Interestingly, treatment of both cell lines with non-cytotoxic concentrations of thymoquinone resulted in a significant increased expression of clock genes, measured by reverse transcription–quantitative PCR (RT-qPCR). Thymoquinone appeared to increase the expression of BMAL-1 (1.4±0.1 fold), CLOCK (3.4±0.9 fold), PER-1(5.6±0.5 fold), CRY-1 (4.4±0.4 fold), CRY-2(2.2±0.6 fold) in MCF-7 cells. Similarly, thymoquinone increased the expression of BMAL-1 (1.4±0.3 fold), CLOCK (1.4±0.3 fold), PER-1(1.4±0.3 fold), CRY-1 (1.5±0.4 fold), and CRY-2 (1.8±0.1 fold) in T47D cells . In conclusion, thymoquinone possesses a potential in combating breast cancer alone. Despite the promising anti-proliferative activity of thymoquinone against breast cancer cells, the better understanding of these effects is needed to provide a novel approach for the treatment of breast cancer as a disease. Here, we postulate that the drug could be modulating clock genes expression, which ultimately can affect key molecules in cell cycle and cell proliferation. Further studies are needed to confirm this and to elucidate the exact underlying mechanism. Citation Format: Aliaa A. Alamoudi, Hanan A. Bashmail, Ghada M. Ajabnoor, Hani Choudhry, Mohammed A. Hassan, Alia M. Aldahlawi, Ahmed A. Al-abd. Thymoquinone induces the expression of clock genes in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3876.
'/%3e%3cpath fill='%23c09300' stroke-width='1.074' d='m453.16 315.06 9.613 43.755-3.158 3.155-3.3-2.439-5.451-39.018 2.296 39.018-3.157 4.016-3.157-4.016 2.296-39.018-5.45 39.018-3.303 2.44-3.154-3.156 9.611-43.755h6.314z'/%3e%3cg id='b' fill='none' stroke-linejoin='round' stroke-width='1.27'%3e%3cpath fill='%23fff' stroke-width='1.189' d='m428.48 295.84-19.08 67.705 26.255 4.018 11.188-50.924-18.363-20.8z'/%3e%3cpath d='m422.17 318.94 2.296 5.593 12.405-11.848'/%3e%3cpath d='m430.76 304.92 2.596 24.346 7.89-10.328m-3.156 4.012 4.31 14.875m1.69-5.465-8.718 13.241m2.72 13.216-2.791-13.198-2.436-13.423-5.84 7.91-2.562-9.12-8.174 8.385 4.157 15.25 5.738-9.434 3.155 9.612 5.89-9.162'/%3e%3cpath d='m414.99 361.97 5.3-7.444 3.452 11.457 4.735-8.032 3.157 9.614'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='matrix(-1 0 0 1 900 0)'/%3e%3cg stroke-linecap='round' stroke-linejoin='round' stroke-width='1.27'%3e%3cpath stroke-width='2.378' d='M450 393.81c19.943 0 39.024-1.578 50.213-4.734 4.735-.859 4.735-3.298 4.735-6.455 4.736-1.578 2.295-7.17 5.595-7.17-3.3.862-4.016-5.595-8.033-4.732 0-5.597-5.596-6.313-10.33-4.738-9.47 3.158-26.255 3.875-42.18 3.875-15.924-.717-32.567-.717-42.18-3.875-4.733-1.575-10.326-.859-10.326 4.738-4.018-.863-4.736 5.594-8.034 4.731 3.299 0 .86 5.593 5.594 7.17 0 3.158 0 5.597 4.878 6.456 11.045 3.156 30.128 4.734 50.068 4.734z'/%3e%3cpath d='M422.89 363.54c6.454.861 13.628 1.578 19.225.861 3.153 0 5.45 5.45-.863 6.313-5.593.715-14.345 0-19.08-.862-4.017-.718-12.768-2.295-18.364-3.876-5.594-2.436-1.576-7.168 1.578-6.452 4.879 1.577 11.192 3.157 17.505 4.016zm54.23 0c-6.457.861-13.628 1.578-19.081.861-3.3 0-5.596 5.45.717 6.313 5.596.715 14.347 0 19.078-.862 4.019-.718 12.77-2.295 18.364-3.876 5.597-2.436 1.58-7.168-1.577-6.452-4.878 1.577-11.19 3.157-17.501 4.016z'/%3e%3cpath d='M403.09 360.39c-4.877-.861-7.172 4.732-5.593 7.887.717-1.578 4.017-1.578 4.735-3.155.858-2.437-.718-2.437.858-4.732zm19.08 14.67c0-3.153 3.155-2.765 3.155-5.921 0-1.577-.86-4.018-2.437-4.018-1.579 0-3.158 1.578-3.158 3.156-.718 3.157 2.44 3.63 2.44 6.783zm22.71-9.08c4.73 0 4.262 6.314 1.966 9.47 0-2.295-4.017-3.156-4.017-4.732 0-2.442 3.626-2.442 2.051-4.738zm52.03-5.59c4.878-.861 7.174 4.732 5.595 7.887-.718-1.578-4.017-1.578-4.734-3.155-.86-2.437.718-2.437-.86-4.732zm-19.08 14.67c0-3.153-3.153-2.765-3.153-5.921 0-1.577.86-4.018 2.438-4.018 1.577 0 3.156 1.578 3.156 3.156.718 3.157-2.44 3.63-2.44 6.783zm-22.71-9.08c-4.732 0-4.263 6.314-1.967 9.47 0-2.295 4.015-3.156 4.015-4.732.001-2.442-3.626-2.442-2.048-4.738z'/%3e%3cpath stroke-width='.891' d='M404.67 361.97c1.58 0 4.018.718 4.735 1.577l-4.74-1.58zm7.88 2.44c.862 0 4.018.716 5.596 1.575l-5.6-1.58zm28.7 3.15c-1.578 0-4.734 0-5.593.715l5.59-.72zm-8.75 0c-.859-.862-4.016-.862-5.593 0h5.59zm62.84-5.59c-1.578 0-3.875.718-4.735 1.577l4.735-1.577zm-7.89 2.44c-.86 0-4.017.716-5.595 1.575l5.6-1.58zm-28.7 3.15c1.58 0 4.735 0 5.595.715l-5.6-.72zm8.75 0c.86-.862 4.018-.862 5.595 0h-5.6z'/%3e%3cg fill='%23c09300' stroke='none'%3e%3cpath d='M403.34 374.58c-.534-.102-.815-.525-.68-1.025.164-.609.651-.957 1.112-.794.294.104.83.645.832.839.003.222-.28.752-.402.754-.053 0-.135.046-.182.102-.1.118-.413.175-.68.124zm54.93 3.92c-.199-.074-.515-.518-.515-.724.002-.387.574-1.018.929-1.021.175-.001.655.248.834.434.325.338.254.893-.154 1.208-.201.155-.8.212-1.094.103zm.36 2.48c-.42-.142-.574-.348-.608-.814-.036-.485.012-.56.525-.84l.365-.2.347.155c.48.214.693.445.71.767.018.348-.24.704-.64.883-.352.158-.373.16-.699.05z'/%3e%3cpath d='M407.75 370.09c-.346-.01-.821.307-1.094.469-.649.152-1.404.55-2.031.125-.617-.191-1.438-.04-1.531.718.158.652 1.071 1.025 1.625.594.41-.537 1.531-.853 1.656.063-.457.692-.405 1.609-.781 2.344-.061.457-.252.892-.5 1.28-.462.047-.954-.007-1.344.282-.626.034-1.265.345-1.625.875-.403.578-.789 1.165-.906 1.875.107.725.937.933 1.562.969.667.183 1.335.424 2 .625 1.086.243 2.102.635 3.188.875 1.584.477 3.248.697 4.844 1.125.167.055.324.104.5.125.662.137 1.007-.583 1.062-1.125.329-1.16.58-2.34.906-3.5.225-.466.497-1.517-.406-1.281-.515.308-.952.773-1.594.812-.874.05-.375.945-.031 1.313.076.587-.11 1.288-.5 1.75-.52.33-1.172-.057-1.719-.188-.574.01-1.723-.283-1.187-1.062.19-.577.207-1.209.437-1.782.294-.614.336-1.285.469-1.937-.361-.777-.979.256-1.469.344-.338.234-1.605.334-.937.937.575.437.134 1.197.03 1.75-.036.77-.837.954-1.468.781-.628-.03-1.501-.497-.969-1.187.121-.575.322-1.14.438-1.719.237-.698.437-1.363.687-2.062a5.78 5.78 0 0 1 .625-1.75c.018-.686.478-1.276.47-1.97-.048-.338-.2-.461-.407-.468zm-3.594 7.25a.353.353 0 0 1 .219.031c.25.106.257.31 0 .531-.112.097-.24.191-.281.188-.468-.04-.606-.11-.625-.281-.017-.151.03-.195.343-.344a1.45 1.45 0 0 1 .344-.125zm-1.036 5.02c-.496-.466-.416-.806.317-1.353.314-.234.505-.208.863.12.553.504.575.823.087 1.255-.24.213-.346.257-.627.26-.293.003-.376-.034-.64-.282zm3.05.92a.86.86 0 0 1-.623-1.088c.129-.434.203-.477.84-.483.716-.007.854.112.861.741.004.355-.023.437-.2.6-.207.193-.605.297-.878.23zm89.11.08c-.227-.189-.273-.274-.275-.515-.003-.41.233-.712.746-.944.682-.308.96-.266 1.216.186.342.601.325.847-.084 1.252-.225.224-.268.237-.785.242-.507.003-.565-.01-.818-.221zm-85.06 1.15c-.364-.097-.552-.37-.557-.807-.004-.334.02-.387.253-.57.152-.118.39-.22.579-.245.267-.037.364-.013.58.138.55.387.667.77.348 1.14-.34.397-.662.488-1.203.344zm21.92.98c-.08-.1-.26-.16-.278-.287.032-.555.059-1.117.236-1.648.096-.523.012-1.07.194-1.579.187-.927.187-1.882.402-2.803.01-.475.055-.95.193-1.406.157-.722.013-1.476.226-2.19-.023-.267.285-.983.556-.559.394.577.912 1.045 1.421 1.516.438.252.036.624-.266.75-.416.163-.517.62-.487 1.022-.058.414-.225.806-.242 1.23-.087.622-.07 1.254-.151 1.876-.182.609-.113 1.26-.173 1.888.008.398-.182.76-.156 1.16-.048.406-.066.894-.407 1.172-.329.166-.804.138-1.068-.142zm29.7-9.8c-.482.365-.914.795-1.344 1.219-.592.449.487.71.625 1.094.157.586.148 1.21.188 1.812.179.552.258 1.138.218 1.719-.051.622-.863.491-1.218.844-.492.207-.688.69-1.063 1.03-.246.484-.312 1.077-.344 1.626.036.448-.302.95 0 1.344.04.055.086.132.125.187.102.145.343.03.5.063.504-.029 1.029.047 1.5-.157 1.21-.209 2.432-.226 3.657-.28.749.023 1.47-.238 2.218-.22.587.13.851-.506.844-.968-.332-.654-.045-1.429-.312-2.094-.235-.673-.143-1.428-.25-2.125-.015-.549-.711-.752-1.063-.344-.341.345-.893.414-1.156.844-.297.602.627.603.781 1.062.176.465.118.98.156 1.47.115.592-.529.625-.937.687-.537.2-1.29.336-1.594-.313-.19-.48-.165-.993-.219-1.5-.013-.892-.227-1.768-.28-2.656-.052-1.097-.242-2.187-.313-3.281-.01-.456-.071-1.198-.72-1.063zm-.625 8.25.313.031.03.375.032.375-.406.125a3.28 3.28 0 0 1-.407.094 2.55 2.55 0 0 1-.187-.125c-.22-.159-.164-.385.125-.688.163-.17.239-.203.5-.187zm-29.965-9.28c-.38.135-.862.693-1.25.688-.88.062-.757.919-.125 1.218.083.26.005.588.031.875.123.79-.375 1.425-.281 2.22-.044.87-.307 1.717-.344 2.593-.247.903-.25 1.832-.406 2.75-.104.823-.7.533-1.219.312-.004-.356-.027-.705-.031-1.062.216-.817-.441-1.056-1.094-1.156-.686.065-1.023-.523-.875-1.125.295-.351 1.036-.254 1.5-.313.981.204.942-1.112.438-1.562-.345-.636-1.199-.91-1.438-1.563.096-.866-.48-1.732-1.187-2.156-1.089-.076-1.91.863-2.313 1.781-.452.131-.904.267-1.344.438-.709.177-1.72 1.407-.75 1.875.545.15 2.2.528 1.47 1.28-.413.708-1.202.72-1.907.532-.724 0-1.53-.352-1.5-1.187-.148-.75-.123-1.558-.469-2.25-.148-.84-1.1-.643-1.156.125-.733.534-.636 1.397-.125 2.03.319.73.003 1.578-.313 2.25-.19.876-1.155.906-1.843 1.188-.338.131-1.629-.078-1.156.657.753.265 1.652.505 2.437.312.823-.102 1.538-.665 1.938-1.375.552-.56 1.502-.267 2.218-.281.773-.002 1.563.542 2.313.187.217-.564 1.222-1.495 1.531-.53-.053.862.71 1.325 1.5 1.218.864-.05.544.61.531 1.125.057.934.66 1.425 1.438 1.812.265.072.545.048.812 0 .748-.235 1.554-.568 1.781-1.406.304-.68.311-1.45.532-2.156.177-1.13.406-2.26.437-3.406.276-1.039.197-2.136.406-3.188.133-.782.231-1.563.313-2.344-.092-.417-.272-.487-.5-.406zm-6.781 4.156c.151.019.235.204.25.531.016.365.206.661.437.72.183.045.167.238-.03.374-.116.08-.322.127-.688.125-.578-.003-.937-.153-1.25-.469l-.188-.187.281-.188c.142-.108.399-.332.563-.5.285-.29.473-.424.625-.406zm66.031-7.876a8.365 8.365 0 0 0-1.656.25c-1.04-.065-1.563 1.027-.563 1.563.602 1.534 1.54-.317 2.5-.188 1.438.28 1.56 1.832 1.781 3 .081 1.157.415 2.282.75 3.375.963 1.07-.72 1.74-1.437.906-.611-.664-1.92-1.48-2.656-.562-.904.382-.94 1.59-1.688 1.937-1.209.368-1.361-1.119-2-1.75-.587-.824-1.71-.896-2.625-1.187-.429-.869-.205-2.346-1.094-2.938-.745.304-2.113 1.702-.968 2.344 1.117.96-.488 1.401-1 2.063-.732.814-.849 1.936-.969 2.968-1.26.696-1.504-.787-1.656-1.718-.098-1.112-1.036-.786-1.688-.375-.952.385-1.428 1.29-2.094 2-.08.647.038 1.356.032 2.03.216.812 1.202.431 1.812.376.979-.41 1.38.627.594 1.219-.532.676-2.22.322-2.063 1.406.544.063 1.111.043 1.657 0 1.09-.298 2.261-1.068 2.375-2.281.143-1.059 1.667-.866 2.5-1.094 1.04-.3 2.294-.377 2.53.937.71.745 2.25 1.584 3.032.563.795-.485 1.176-1.338 1.125-2.25-.162-.785 1.269-.813 1.625-.375.521.858 2.151.605 2.938.125.836-.617 1.017-1.705 2.218-1.719 1.783-.534 3.635-.841 5.375-1.5 1.41-.307-.21-1.198-.562-1.719-1.074-.53-2.008 1.642-3.219.563-.936-.716-.872-2.077-1.219-3.125-.237-1.358-.177-2.923-1.156-4-.672-.715-1.586-.887-2.531-.844zm-6.813 9.469c.126-.003.268.06.5.187.439.242.751.643.75.969 0 .213-.054.227-.375.375-.197.091-.42.189-.5.188-.18-.002-.624-.357-.625-.5 0-.06-.058-.287-.125-.47-.117-.32-.099-.316.063-.53.103-.138.187-.216.313-.22zm6.532.344c.372-.025.593.091.75.375.181.329.058.58-.407.78-.203.089-.407.16-.437.157-.03-.003-.198-.102-.375-.219-.27-.177-.31-.264-.313-.468-.003-.316.35-.597.782-.625zm-8.938.093c.11-.017.209.04.344.156.18.155.237.28.281.594a2.23 2.23 0 0 1 0 .625c-.048.226-.053.216-.562.219-.64.006-.7-.064-.688-.656.008-.377.012-.478.219-.688.16-.162.297-.233.406-.25zm-5.5 1.438a.57.57 0 0 1 .438.437c.065.318.008.462-.25.594-.222.113-.734.15-.875.063a.613.613 0 0 1-.188-.188c-.068-.121-.055-.18.031-.25.06-.05.126-.12.125-.156 0-.037.068-.163.157-.281a.543.543 0 0 1 .562-.22zm9.25 1.187a.651.651 0 0 1 .438.156c.185.163.179.4-.031.563-.136.104-.872.095-1.125 0-.113-.042-.156-.099-.157-.219-.002-.14.038-.227.281-.344.205-.097.416-.155.594-.156zm-17.171 4.819c-.096-.063-.238-.117-.095-.222.173-.302.518-.407.825-.52a3.436 3.436 0 0 0 1.373-1.118c.05-.333.424-.513.422-.861.003-.475.032-.962-.084-1.426-.129-.426-.452-.752-.827-.974-.256-.202-.674-.258-.768-.614-.053-.343.253-.604.36-.91.223-.421.411-.863.651-1.275.26-.288.659-.015.793.263.19.277.357.576.45.897.303.332.571.721.68 1.162.148.31.383.602.369.966 0 .416.235.787.22 1.206.014.56.013 1.145-.191 1.674-.14.294-.421.47-.607.733-.319.295-.67.579-1.08.736-.289.092-.457.426-.819.345-.514.009-1.037.079-1.547-.004.032.054-.156-.092-.125-.058zm-6.85.45c-.243-.25-.295-.355-.298-.6-.003-.248.045-.346.282-.586.439-.443.653-.453 1.41-.07.722.365.967.399 1.032.14.062-.25.478-.48.874-.481a.82.82 0 0 1 .592.203c.251.196.263.227.27.68.004.463-.002.48-.29.736-.278.244-.323.259-.666.224-.437-.043-.7-.244-.77-.588-.052-.25-.14-.31-.206-.14-.021.055-.129.122-.239.15-.204.051-.549.268-.794.5-.093.087-.255.128-.519.131-.355.003-.405-.018-.678-.299zm-19.82-8.9c-.665.007-1.38.566-1.469 1.219.152.752.777 1.49.375 2.281.278 1.013-.761 1.397-1.469.813-.427-.972-.643-2.036-1.28-2.907-.805-.227-1.155 1.057-1.72 1.5.28.755 1.096 1.433 1.188 2.375.195.915-.361 1.93-1.156 2.406-.686.655-1.626.421-2.47.563-1.023.599.753.818 1.22.844 1.047.106 2.135-.032 2.906-.813.769-.367.71-1.697 1.625-1.844 1.588-.07 3.163.244 4.75.282.85.203 2.163-.101 2.75.53.002 1.108.94 1.791 1.844 2.22.498.138 1.024-.48 1.53-.594.966-.308.673-1.502 1.25-1.969 1.651-.09 3.317-.07 4.97-.093.314.007.407-.44.625-.626-.037-.638-.06-1.272-.157-1.906-.318-.806.052-1.844-.53-2.53-.83-.227-1.537.533-2.376.593-.926.412-1.706 1.268-1.75 2.312-.548.897-1.427-.16-1.125-.906-.034-.325.07-.704-.031-1-.507-.608-1.395-.41-2.094-.5-1.018.04-2.012-.18-3.031-.156-.893-.068-1.822.078-2.688-.156-1.031-.03-.89-1.039-1.062-1.75a1.018 1.018 0 0 0-.625-.188zm2 3.688c.343.006.667.051 1 .093.43.071.89-.148 1.313 0 .976.166 1.953.127 2.937.219.26-.004.446.185.625.344.002.683 0 1.379 0 2.062-.03.258.076.598-.156.781a.7.7 0 0 1-.25.125c-.27-.07-.558-.247-.688-.5-.041-.357.018-.704.031-1.062-.007-.093.04-.237 0-.313a1.248 1.248 0 0 0-.718-.25c-.85-.029-1.682-.091-2.531-.125-.707-.044-1.434.06-2.125-.125-.269-.097-.618-.064-.782-.343-.234-.273.104-.597.344-.72a2.28 2.28 0 0 1 1-.187zm11.281.25c.159-.003.21.05.281.343.046.188.1.417.125.5.037.118-.022.156-.187.219-.494.186-.986.168-1.156-.063-.078-.104.012-.434.156-.562.29-.259.61-.437.781-.438z'/%3e%3c/g%3e%3c/g%3e%3cpath stroke-width='1.15' d='M449.98 327.23c32.642-25.106 29.843-61.883 29.843-61.883a12.72 12.72 0 0 1-2.54.258c-6.848 0-23.179-3.917-26.989-8.892-4.081 4.503-20.77 8.892-27.574 8.892-.861 0-1.722-.086-2.54-.258 0 0-2.843 36.776 29.8 61.883z'/%3e%3cpath stroke-width='.916' d='M477.22 268.04a13.55 13.55 0 0 1-.848.026c-6.205 0-20.619-3.183-26.175-7.957-5.82 4.41-20.47 7.957-26.576 7.957a4.7 4.7 0 0 1-.85-.086c-.014 1.39.059 2.832.145 4.144.323 4.9 1.183 9.819 2.488 14.552 4.108 14.895 12.516 27.553 24.572 37.155 12.066-9.61 20.484-22.28 24.6-37.186 1.308-4.732 2.17-9.65 2.496-14.55.086-1.285.157-2.692.148-4.055z'/%3e%3cpath fill='%23c09300' stroke='none' d='M439.38 265.03c-5.872 1.845-12.229 3.031-15.75 3.031a4.7 4.7 0 0 1-.844-.093c-.014 1.39.07 2.845.156 4.156a72.77 72.77 0 0 0 2.47 14.53c2.764 10.026 7.506 19.033 13.968 26.782V265.03zm20.62-.09v49.25c6.794-7.921 11.733-17.206 14.594-27.562a72.952 72.952 0 0 0 2.469-14.531c.085-1.284.166-2.7.156-4.063-.28.018-.56.032-.844.032-3.73 0-10.42-1.17-16.375-3.125z'/%3e%3cg stroke-width='1.27'%3e%3cpath stroke-width='1.218' d='M462.31 253.09c.667.04-.902-3.568-.902-3.568 1.765 1.804 8.43 2.235 8.43 2.235-4-1.766-7.998-15.096-7.528-25.721.432-10.664-1.528-14.86-3.097-16.428-2-2-8.43-3.764-12.664-4-2.392-.117-2 1.804-2 1.804-4.43-1.137-8.86-1.568-10.86-.235-1.883 1.255-2.275 7.528-.902 6.43 3.332-2.666 6.234-.235 8.233 2.666 1.764 2.549 1.647 9.763-.902 18.192-2.666 8.861-9.959 17.722-9.959 17.722 3.96 0 9.528-3.528 9.528-3.528l-1.333 5.528c4.195-2 7.528-5.097 7.528-5.097l3.999 4.195c1.333-1.764 4-4.195 4-4.195s3.332 3.529 8.429 4z'/%3e%3cpath fill='none' d='M446.12 227.57s-2.235 16.428-6.43 21.094m9.96-21.524s-.863 16.623-3.764 21.956m6.894-21.286s0 18.192 1.098 21.289m2.862-20.389s.902 15.291 4.666 20.819'/%3e%3cpath fill='%23c09300' stroke-width='.354' d='M442.08 219.61c-.196-1.45-.549-2.588-1.059-3.333-2-2.901-4.9-5.332-8.233-2.666 0 0 1.137-3.529 3.568-3.646 1.882-.118 6.155 1.411 9.92 7.841 0 0-2.784-.627-3.45-.039-1.256 1.098-.746 1.843-.746 1.843z'/%3e%3cpath fill='%23c09300' stroke-width='.354' d='M432.44 209.26c.274-.902.706-1.725 1.255-2.078 2-1.333 6.43-.902 10.86.235 0 0-.392-1.921 2-1.803 4.234.235 10.664 2 12.663 3.999.47.51 1.02 1.255 1.49 2.391h-.078c-.98-1.372-3.764-1.293-4.431-1.215-1.059.118-1.725.078-3.137.431-.666.157-1.686.353-2.235.784-.43.353-.784 1.647-1.45 1.647-1.059 0-.98-.274-1.255-.588-.353-.431-.549-1.059-.902-1.02-1.097.197-2.862-.666-5.096-2.43-2.235-1.765-3.098-2.196-5.999-2-2.862.235-3.764 1.843-3.764 1.843l.079-.196z'/%3e%3ccircle cx='448.824' cy='210.672' r='1.176' stroke='none'/%3e%3c/g%3e%3c/g%3e%3c/svg%3e)
