Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.

• Keywords
• CD44 isoforms, RNA splicing, cancer markers, colon polyps, colorectal precancerosis,
• MeSH
• Hyaluronan Receptors genetics   metabolism   MeSH
• Colon metabolism   pathology   MeSH
• Colorectal Neoplasms diagnosis   genetics   metabolism   MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Colonic Polyps metabolism   pathology   MeSH
• Prognosis MeSH
• Protein Isoforms MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hyaluronan Receptors MeSH
• CD44 protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• Protein Isoforms MeSH

Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.

• MeSH
• Biocompatible Materials metabolism   MeSH
• Cholestyramine Resin metabolism   MeSH
• Membrane Fluidity * MeSH
• Fluorescence Polarization MeSH
• Lab-On-A-Chip Devices MeSH
• Liposomes chemical synthesis   MeSH
• Microfluidics instrumentation   methods   MeSH
• Nanostructures * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biocompatible Materials MeSH
• Cholestyramine Resin MeSH
• Liposomes MeSH

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd+3 prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.

• MeSH
• Gadolinium DTPA * adverse effects   toxicity   MeSH
• Fibrinolytic Agents MeSH
• Phosphatidylethanolamines * adverse effects   toxicity   MeSH
• Hepatocytes drug effects   MeSH
• Inflammasomes MeSH
• Contrast Media * MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Liposomes * MeSH
• Magnetic Resonance Imaging * MeSH
• Macrophages drug effects   MeSH
• Nanoparticles MeSH
• Drug Carriers * MeSH
• NLR Family, Pyrin Domain-Containing 3 Protein MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gadolinium DTPA * MeSH
• Fibrinolytic Agents MeSH
• Phosphatidylethanolamines * MeSH
• gadolinium phosphatidylethanolamine-DTPA MeSH Browser
• Inflammasomes MeSH
• Contrast Media * MeSH
• Liposomes * MeSH
• NLRP3 protein, human MeSH Browser
• Drug Carriers * MeSH
• NLR Family, Pyrin Domain-Containing 3 Protein MeSH

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

• Keywords
• ABD scaffold, binding protein, combinatorial library, fibrin, fibrinogen Bβ chain, liposome, metallochelation, thrombus imaging, thrombus targeting,
• Publication type
• Journal Article MeSH