LEGO Creator Mystic Witch 3 in 1 40562

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Christiansen, A. et al. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum. Sci. Rep. 5, 12913 (2015). A structural bioinformatics approach was used to identify the potential targets of RKOpep. A complete overview of all cell surface proteins of five CRC cell lines, including RKO, Caco-2 and HCT 116, was provided by de Wit and collaborators 20. Using a high-resolution shotgun proteomics analysis, a total of 2609 proteins were identified in the cell surface fractions. By combining additional selection criteria including the presence in three of the cell lines in study, overexpression in carcinomas when compared to adenomas and subcellular location at the plasma membrane, about 59 candidate proteins for the RKOpep were identified. Jin, W. et al. Discovery of PSMA-specific peptide ligands for targeted drug delivery. Int. J. Pharm. 513, 138–147 (2016).

Pinheiro, C. et al. Increasing Expression of Monocarboxylate Transporters 1 and 4 Along Progression to Invasive Cervical Carcinoma. Int. J. Gynecol. Pathol. 27, 568–574 (2008). Introducing the highly anticipated LEGO 40562 - Creator 3-in-1 Halloween 2022 Limited Edition Set, a bewitching ensemble that allows you to choose from three captivating buildings: the Mystic Witch, the Creepy Cat, or the Dangerous Dragon. This spellbinding set is designed to ignite your imagination and transport you to a world of enchantment and Halloween thrills. We now have set images, release dates and prices for LEGO Creator 40562 Mystic Witch GWP October 2022. As RKO-R5-1 phage clone, the free FAM-labelled peptide RKOpep also showed specific selectivity towards RKO cells in comparison to the CCD-841-CoN normal cells. Using microscopy, it was shown for the RKO cells that the fluorescence intensity increased with an increase of FAM-RKOpep concentration, which was not verified for the CCD-841-CoN cells (Fig. 2A). The cytometry analysis also corroborated these results, clearly showing that there is no affinity to the normal cells and that the RKOpep selectively bound to RKO cells (Fig. 2B). Moreover, the FAM-RKOpep showed noteworthy specificity for other three human CRC cells, HCT 116, Caco-2 and HCT-15 (Fig. 3). In addition, the peptide specific targeting of human colorectal cancer tissues, shown by immunofluorescence staining, revealed meaningful results (Fig. 4). Visible and noticeable green fluorescent signal of FAM-RKOpep was detected in human CRC tissues and a weak signal was observed for normal-adjacent ones. This experimental data supports the potential translation of this peptide to clinical oncology. The ability of peptides to recognize human cancer tissues was also demonstrated by Liu and collaborators 33. They identified novel cancer-specific peptides selected through in vivo screening of phage display peptide libraries, that were able to target human cervical cancer tissues. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539–539 (2014).

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I wonder if the witch is a shape-shifter, and the cat and this winged-creature are her alternate forms? That would explain why they are both wearing her hat!

RKO-specific peptides were identified by BRASIL using four biopanning rounds of selection against RKO cells and a final negative selection round towards a normal colon cell line, to exclude all peptides that bind non-specifically to cancer cells. To accomplish this, a commercial combinatorial library of seven random amino acids consisting in a disulfide constrained loop was used. This type of disulfide constrained peptide library have been proved useful for the selection of bioactive peptides, disease-specific antigen mimics and/or cell-specific peptides 27, 28, 29. Huang, J., Ru, B., Li, S., Lin, H. & Guo, F.-B. SAROTUP: Scanner and Reporter of Target-Unrelated Peptides. J. Biomed. Biotechnol. 2010, 1–7 (2010). Grady, W. M. & Carethers, J. M. Genomic and Epigenetic Instability in Colorectal Cancer Pathogenesis. Gastroenterology 135, 1079–1099 (2008). Mouradov, D. et al. Colorectal Cancer Cell Lines Are Representative Models of the Main Molecular Subtypes of Primary Cancer. Cancer Res. 74, 3238–3247 (2014). To narrow this list of candidate proteins, a multiple sequence alignment for known CRC-related proteins was performed, leading to the identification of two potential targets (Supplementary Table S2). According to this homology search query, RKOpep was found to recognize MCT1, one of the 59 candidate proteins retrieved from the cell surface proteomic analysis after applying the selection criteria.

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Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–402 (1997).

The human epithelial CRC cell line RKO (ATCC CRL 2577), normal colon cell line CCD-841-CoN (ATCC CRL 1790) and CRC cell line Caco-2 (ATCC HTB-37) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Biochrom). The human CRC cell lines HCT 116 (ATCC CCL-247) and HCT-15 (ATCC CCL-225) were grown in Roswell Park Memorial Institute Medium 1640 (RPMI, Biochrom) supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrom) and 1% (v/v) penicillin-streptomycin (Biochrom). Cells were maintained at 37 °C with 5% CO 2. BRASIL MCT1 catalyzes the proton-linked movement of many monocarboxylates, such as lactate and pyruvate, across the plasma membrane, and is the major transporter involved in lactate influx into tumor cells 35, 36. In addition to the important functional role of MCT1 in tumor metabolism, increased levels of MCT1 have been implicated in disease progression and prognosis in several human malignancies 37, 38, 39 including CRC 40. MCT1 has a pivotal role in CRC maintenance and supports its use as a biomarker in primary and metastatic CRC 41, which makes it a promising target for the novel RKOpep peptide herein identified. Ferro, S. et al. Characterization of acetate transport in colorectal cancer cells and potential therapeutic implications. Oncotarget 7 (2016).Interestingly, this upcoming LEGO October 2022 GWP is a Creator 3 in 1 set. Usually, they are other types of LEGO GWP like 18+ LEGO IDEAS or just LEGO Creator. The expression level of MCT1 in normal and CRC cell lines was assessed by western blot (Fig. 6). The levels of expression of all CRC cell lines are in good agreement with the binding affinity of the RKOpep to them, as showed by the microscopy and cytometry results. Moreover, the specific recognition of human colorectal cancer tissues by the RKOpep can also be correlated with the overexpression of MCT1 at the plasma membrane of primary CRC tumors, as shown by the immunohistochemistry experiments performed by Martins and coworkers 41. Park, S. J. et al. An overview of MCT1 and MCT4 in GBM: small molecule transporters with large implications. Am. J. Cancer Res. 8, 1967–1976 (2018).