Expression and Impact of C1GalT1 in Cancer Development and Progression

C1GalT1 (T-synthase) is one of the key glycosyltransferases in the biosynthesis of O-linked mucin-type glycans of glycoproteins. It controls the formation of Core-1 disaccharide Galβ1,3GalNAcα- (Thomsen–Friedenreich oncofetal antigen, T or TF antigen) and Core-1-associated carbohydrate structures. Recent studies have shown that C1GalT1 is overexpressed in many cancers of epithelial origin including colon, breast, gastric, head and neck, pancreatic, esophageal, prostate, and hepatocellular cancer. Overexpression of C1GalT1 is often seen to also be associated with poorer prognosis and poorer patient survival. Change of C1GalT1 expression causes glycosylation changes of many cell membrane glycoproteins including mucin proteins, growth factor receptors, adhesion molecules, and death receptors. This leads to alteration of the interactions of these cell surface molecules with their binding ligands, resulting in changes of cancer cell activity and behaviors. This review summarizes our current understanding of the expression of C1GalT1 in various cancers and discusses the impact of C1GalT change on cancer cell activities in cancer development and progression.

Characterization of Host Cell Potential Proteins Interacting with OsHV-1 Membrane Proteins

The interaction between viral membrane associate proteins and host cellular surface molecules should facilitate the attachment and entry of OsHV-1 into host cells. Thus, blocking the putative membrane proteins ORF25 and ORF72 of OsHV-1 with antibodies that have previously been reported to subdue OsHV-1 replication in host cells, especially ORF25. In this study, prey proteins in host hemocytes were screened by pull-down assay with recombinant baits ORF25 and ORF72, respectively. Gene Ontology (GO) analysis of these prey proteins revealed that most of them were mainly associated with binding, structural molecule activity and transport activity in the molecular function category. The protein–protein interaction (PPI) network of the prey proteins was constructed by STRING and clustered via K-means. For both ORF25 and ORF72, three clusters of these prey proteins were distinguished that were mainly associated with cytoskeleton assembly, energy metabolism and nucleic acid processing. ORF25 tended to function in synergy with actins, while ORF72 functioned mainly with tubulins. The above results suggest that these two putative membrane proteins, ORF25 and ORF72, might serve a role in the transport of viral particles with the aid of a cytoskeleton inside cells.

Exosomal targeting and its potential clinical application

Exosomes are extracellular vesicles secreted by a variety of living cells, which have a certain degree of natural targeting as nano-carriers. Almost all exosomes released by cells will eventually enter the blood circulation or be absorbed by other cells. Under the action of content sorting mechanism, some specific surface molecules can be expressed on the surface of exosomes, such as tetraspanins protein and integrin. To some extent, these specific surface molecules can fuse with specific cells, so that exosomes show specific cell natural targeting. In recent years, exosomes have become a drug delivery system with low immunogenicity, high biocompatibility and high efficacy. Nucleic acids, polypeptides, lipids, or small molecule drugs with therapeutic function are organically loaded into exosomes, and then transported to specific types of cells or tissues in vivo, especially tumor tissues, to achieve targeting drug delivery. The natural targeting of exosome has been found and recognized in some studies, but there are still many challenges in effective clinical treatments. The use of the natural targeting of exosomes alone is incapable of accurately transporting the goods loaded to specific sites. Besides, the natural targeting of exosomes is still an open question in disease targeting and efficient gene/chemotherapy combined therapy. Engineering transformation and modification on exosomes can optimize its natural targeting and deliver the goods to a specific location, providing wide use in clinical treatment. This review summarizes the research progress of exosomal natural targeting and transformation strategy of obtained targeting after transformation. The mechanism of natural targeting and obtained targeting after transformation are also reviewed. The potential value of exosomal targeting in clinical application is also discussed. Graphical abstract

Synthesis, Surface Modification and Purification of Silicon and Germanium Quantum Dots for Biological Applications

<p>Quantum dots have applications in biomedical fields such as bio-imaging and drug delivery systems. This thesis describes research on silicon and germanium nanoparticles (quantum dots) synthesis and surface modification for biological applications. Purification methods of these quantum dots were also explored. In chapter 6 the application of silica nanoparticles into dry eye diagnosis was studied. The purpose of this research is to contribute the application of nanotechnology into biological fields. The crystalinity of the quantum dots was characterised by Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction analysis (SAED). The molecules on the surface of the quantum dots were characterised by Fourier Transform Infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). Silicon quantum dots were synthesised with a microemulsion system and various types of molecules were attached on the surface of the silicon quantum dots. However, some of the capping molecules which have oxygen atoms tend to form bonds between oxygen and silicon. Therefore, in the later chapter (chapter 4) various chemical reactions were conducted on the molecules attached to the silicon quantum dots. The silicon quantum dots were capped with diene molecules and one of the double bonds was left on the terminal end. The terminal end double bonds were converted to the functional groups which contain oxygen atoms to form peptide bonds. In this way it was confirmed that it can reduce the risk of oxygen atoms to be attached on the surface of the silicon quantum dots. The molecules on the surface of the silicon quantum dots were characterised mainly by FTIR and ¹H NMR. Optical properties and cyto-toxicity of these silicon quantum dots were also measured and analysed depending on the surface molecules. Two synthetic approaches were taken to produce germanium quantum dots. The first approach was the microemulsion system at room temperature. Different combinations of the surfactant and capping molecules were tested. For the second approach, high temperature bench top system was applied. In this method the bio-friendly molecules which have high boiling points were chosen as capping agents. The surface molecules were characterised by FTIR spectroscopy. In chapter 6 the synthesis of dye molecules conjugated silica nanoparticles was described. The purpose of this research is to produce biologically safe nanoparticles which can be applied in dry eye diagnosis. Three different dyes were used to conjugate with the silica nanoparticles. Only fluorescein isothiocyanate (FITC) succeeded in conjugating with the nanoparticles. Optical properties of this sample were measured and compared with the free dye molecule. Also the sample was applied in human eyes to analyse the tear film layer. An overall conclusion and future plans for the research were given in the last chapter.In this chapter, ideas of overcoming the problems and improving the techniques conducted in the research were described.</p>

Synthesis, Surface Modification and Purification of Silicon and Germanium Quantum Dots for Biological Applications

<p>Quantum dots have applications in biomedical fields such as bio-imaging and drug delivery systems. This thesis describes research on silicon and germanium nanoparticles (quantum dots) synthesis and surface modification for biological applications. Purification methods of these quantum dots were also explored. In chapter 6 the application of silica nanoparticles into dry eye diagnosis was studied. The purpose of this research is to contribute the application of nanotechnology into biological fields. The crystalinity of the quantum dots was characterised by Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction analysis (SAED). The molecules on the surface of the quantum dots were characterised by Fourier Transform Infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). Silicon quantum dots were synthesised with a microemulsion system and various types of molecules were attached on the surface of the silicon quantum dots. However, some of the capping molecules which have oxygen atoms tend to form bonds between oxygen and silicon. Therefore, in the later chapter (chapter 4) various chemical reactions were conducted on the molecules attached to the silicon quantum dots. The silicon quantum dots were capped with diene molecules and one of the double bonds was left on the terminal end. The terminal end double bonds were converted to the functional groups which contain oxygen atoms to form peptide bonds. In this way it was confirmed that it can reduce the risk of oxygen atoms to be attached on the surface of the silicon quantum dots. The molecules on the surface of the silicon quantum dots were characterised mainly by FTIR and ¹H NMR. Optical properties and cyto-toxicity of these silicon quantum dots were also measured and analysed depending on the surface molecules. Two synthetic approaches were taken to produce germanium quantum dots. The first approach was the microemulsion system at room temperature. Different combinations of the surfactant and capping molecules were tested. For the second approach, high temperature bench top system was applied. In this method the bio-friendly molecules which have high boiling points were chosen as capping agents. The surface molecules were characterised by FTIR spectroscopy. In chapter 6 the synthesis of dye molecules conjugated silica nanoparticles was described. The purpose of this research is to produce biologically safe nanoparticles which can be applied in dry eye diagnosis. Three different dyes were used to conjugate with the silica nanoparticles. Only fluorescein isothiocyanate (FITC) succeeded in conjugating with the nanoparticles. Optical properties of this sample were measured and compared with the free dye molecule. Also the sample was applied in human eyes to analyse the tear film layer. An overall conclusion and future plans for the research were given in the last chapter.In this chapter, ideas of overcoming the problems and improving the techniques conducted in the research were described.</p>

Cladribine Alters Immune Cell Surface Molecules for Adhesion and Costimulation: Further Insights to the Mode of Action in Multiple Sclerosis

Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.

Shedding of Trypanosoma cruzi Surface Molecules That Regulate Host Cell Invasion Involves Phospholipase C and Increases Upon Sterol Depletion

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.

Identification of Cell Surface Molecules That Determine the Macrophage Activation Threshold Associated With an Early Stage of Malignant Transformation

The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for the stage of transformation from normal to fully malignant cells that first provides a threat (danger) signal requiring a response. We co-cultured human macrophages with a unique set of genetically related human cell lines that recapitulate successive stages in breast cancer development: MCF10A (immortalized, normal); MCFNeoT (benign hyperplasia); MCFT1 (atypical hyperplasia); MCFCA1 (invasive cancer). Using cytokines-based assays, we found that macrophages were inert towards MCF10A and MCFNeoT but were strongly activated by MCFT1 and MCFCA1 to produce inflammatory cytokines, placing the threshold for recognition between two premalignant stages, the earlier stage MCFNeoT and the more advanced MCFT1. The cytokine activation threshold paralleled the threshold for enhanced phagocytosis. Using proteomic and transcriptomic approaches, we identified surface molecules, some of which are well-known tumor-associated antigens, that were absent or expressed at low levels in MCF10A and MCFNeoT but turned on or over-expressed in MCFT1 and MCFCA1. Adding antibodies specific for two of these molecules, Annexin-A1 and CEACAM1, inhibited macrophage activation, supporting their role as cancer “danger signals” recognized by macrophages.

Bsh coordinates neuronal fate specification with synaptic connectivity

It is widely accepted that neuronal fate is initially determined by spatial and temporal cues acting in progenitors, followed by transcription factors (TFs) that act in post-mitotic neurons to specify their functional identity (e.g. ion channels, cell surface molecules, and neurotransmitters). It remains unclear, however, whether a single TF can coordinately regulate both steps. The five lamina neurons (L1-L5) in the Drosophila visual system, are an ideal model for addressing this question. Here we show that the homeodomain TF Brain-specific homeobox (Bsh) is expressed in a subset of lamina precursor cells (LPCs) where it specifies L4 and L5 fate, and suppresses homeodomain TF Zfh1 to prevent L1 and L3 fate. Subsequently, in L4 neurons, Bsh initiates a feed forward loop with another homeodomain TF Apterous (Ap) to drive recognition molecule DIP-β expression, which is required for precise L4 synaptic connectivity. We conclude that a single homeodomain TF expressed in both precursors and neurons can coordinately generate neuronal fate and synaptic connectivity, thereby linking these two developmental events. Furthermore, our results suggest that acquiring LPC expression of a single TF, Bsh, may be sufficient to drive the evolution of increased brain complexity.

Remodeling the Tumor Myeloid Landscape to Enhance Antitumor Antibody Immunotherapies

Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called ‘phagocytosis checkpoints’. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of ‘don’t eat me signals’ or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance.