3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes.

We present novel workflows for Q-FISH nanoscopy with the potential for prognostic applications and resolving novel chromatin compaction changes. DNA-fluorescence in situ hybridization (DNA-FISH) is a routine application to visualize telomeres, repetitive terminal DNA sequences, in cells and tissues. Telomere attrition is associated with inherited and acquired diseases, including cancer and cardiomyopathies, and is frequently analyzed by quantitative (Q)-FISH microscopy.

Calpain cleavage of Junctophilin-2 generates a spectrum of calcium-dependent cleavage products and DNA-rich NT-fragment domains in cardiomyocytes.

Calpains are calcium-activated neutral proteases involved in the regulation of key signaling pathways. Junctophilin-2 (JP2) is a Calpain-specific proteolytic target and essential structural protein inside Ca release units required for excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in heart failure has been reported, the precise molecular identity of the Calpain cleavage sites and the (patho-)physiological roles of the JP2 proteolytic products remain controversial.

Biallelic variants in YRDC cause a developmental disorder with progeroid features.

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

Background: Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets.

Results: We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined.

The glycosphingolipid P₁ is an ovarian cancer-associated carbohydrate antigen involved in migration.

Background: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid.

Methods: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array.

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array.

Glycan-based suspension array (SGA) is an "in-house" developed multi-target immunoassay, employing commercially available fluorescent microbeads as a solid support for unique chemically synthesized glycopolymers which capture naturally occurring human anti-glycan antibodies. SGA is a sensitive and reliable tool for the high-throughput screening of anti-glycan antibody alterations characteristic for a vast number of human diseases including cancer. However, unspecific background binding, for instance binding of non-target antibodies, is a common obstacle in such immunoassays.

Tumor-associated glycans and their role in gynecological cancers: accelerating translational research by novel high-throughput approaches.

Glycans are important partners in many biological processes, including carcinogenesis. The rapidly developing field of functional glycomics becomes one of the frontiers of biology and biomedicine. Aberrant glycosylation of proteins and lipids occurs commonly during malignant transformation and leads to the expression of specific tumor-associated glycans.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer.

No benefit from combining HE4 and CA125 as ovarian tumor markers in a clinical setting.

Objective: About 70% of epithelial ovarian cancer patients (EOC) are diagnosed at advanced stage with a five-year survival rate of only 30%. Whilst CA125 detects peritoneally-spread disease, it has limited sensitivity for early cancers, many of which are potentially curable.

Methods: We compared the new commercially available tumor marker HE4 with CA125 individually, in combination, within the risk of malignancy index (RMI) and the newly defined risk of malignancy algorithm (ROMA).

Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a printed glycan array.

Epithelial ovarian cancer has the highest mortality rate among gynecological cancers. Altered glycosylation is associated with oncogenic transformation producing tumor-associated carbohydrate antigens. We investigated the potential of natural occurring antiglycan antibodies in the diagnosis of ovarian cancer by using printed glycan array.

Multiplex suspension array for human anti-carbohydrate antibody profiling.

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum.

Solid-phase assays for study of carbohydrate specificity of galectins.

We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA.

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting.

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells.

Design of the blood group AB glycotope.

Although the nature of the blood groups A and B has been comprehensively studied for a long time, it is still unclear as to what exactly is the epitope that is recognized by antibodies having AB specificity, i.e. monoclonal and polyclonal antibodies which are capable of interacting equally well with the antigens GalNAcalpha 1-3(Fucalpha 1-2)Gal (A trisaccharide) and Galalpha 1-3(Fucalpha 1-2)Gal (B trisaccharide), but do not react with their common fragment Fucalpha 1-2Gal.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker.

High molecular weight neoglycoconjugates for solid phase assays.

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc).

Uncharged P-selectin blockers.

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x).

P-selectin blocking potency of multimeric tyrosine sulfates in vitro and in vivo.

P-selectin blocking potency was investigated using synthetic monomeric and polymeric anionic compounds containing sulfate groups such as O-sulfotyrosine (sTyr) and/or sulfated Lewis structures. A non-carbohydrate-containing polyacrylamide conjugate sTyr-PAA (80% mol of sTyr) was a remarkably potent inhibitor of P-selectin binding in vitro, having an IC(50) value of 6 ng/mL (equivalent to 10 nM calculated on the basis of sTyr residues or 0.1 nM calculated by the mass of the macromolecule).

Assembly of p-selectin ligands on a polymeric template.

High-affinity receptor-ligand interactions frequently involve molecular interactions at two distinct sites. A derivatized polyacrylic-based polymer was synthesized to allow substitution with multiple ligands (e.g.

[Effects of mannose derivatives on development of selectin-dependent peritoneal inflammation in rats and mice].

The inhibitory effect of mannose-containing oligosaccharides on model carbohydrate ligand interaction with E-, P- and L-selectins in vitro, as well as on the ability of these compounds to block the leukocyte extravasation in rat and mouse peritonitis in vivo was studied. The monomeric and polymeric compounds, 4-nitrophenylthiomannoside, phenylmannoside, conjugated with polyacrylic acid, and alpha-mannose, conjugated with polyacrylamide, inhibited the binding of the model ligand to P- and L-selectins (but not to E-selectin). Intravenous injection of these compounds was found to cause a dose-dependent reduction of neutrophil accumulation in rat peritoneum.

[Inhibitory activity of monomeric and polymeric selectin ligands].

The ability of tetrasaccharides (SiaLex, SiaLea, HSO3Lex), their conjugates with polyacrylamide (40 kDa), and several other monomeric and polymeric substances to block selectins has been compared with that of polysaccaride fucoidan. Two assay systems were used: one was constructed on the base of recombinant E-, P-, and L-selectins; the other was a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 microM with the recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker.

[The tissue localization and secretion of mucin-D in Drosophila melanogaster].

Glycoprotein with biochemical characteristics that allow us to classify it as a glycoprotein of mucin-type was isolated from cultured embryonic cells of Drosophila melanogaster. This is the first finding of mucin-type glycoprotein in insects. Using high-affinity monoclonal antibodies against a carbohydrate epitope, we demonstrated that the accumulation of this glycoprotein in the culture fluid of Drosophila cell line and cultured cells of other insects was inhibited by secretion inhibitors.