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Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be realized by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real-time imaging of three different endocytic ligands—folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single-particle tracking of Gal3- or STxB-functionalized QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs, which bear a unique stoichiometry of endocytic ligands, represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics. Quantum dots encapsulated inside DNA icosahedra that display a single endocytic ligand are used to track compartmental dynamics along endocytic pathways.

Background/Aims: Myocardial reperfusion has the potential to salvage the ischemic myocardium after a period of coronary occlusion. Reperfusion, however, can cause a wide spectrum of deleterious effects. Galectin-3 (GAL-3), a beta galactoside binding lectin, is closely associated with myocardial infarction (MI), myocardial fibrosis and heart failure. In our study, we investigated its role in ischemia-reperfusion injuries (IR) as this phenomenon is extremely relevant to the early intervention after acute MI. Methods: C57B6/J wild type (WT) mice and GAL-3 knockout (KO) mice were used for murine model of IR injury in the heart where a period of 30 minutes ischemia was followed by 24 hours of reperfusion. Heart samples were processed for immunohistochemical and immunofluorescent labeling, morphometric analysis, western blot and enzyme-linked immunosorbent assay to identify the apoptotic, inflammatory and oxidative stress role of GAL-3. Results: Our results show that there was a significant increase in GAL-3 levels in the heart which shows GAL-3 is playing a role in the ischemia reperfusion injury. Troponin I was also significantly higher in GAL-3-KO group than wild type. Our study shows that GAL-3 is associated with an increase in the antioxidant activity in the IR injured myocardium. Antioxidant enzymes superoxide dismutase, glutathione and catalase were found to be significantly raised in the GAL-3 wild type IR as compared to the GAL-3 KO IR group. A significant increase in apoptotic activity is seen in GAL-3 KO IR group as compared with GAL-3 wild IR group. Conclusion: Our study shows that GAL-3 can affect the redox pathways, controlling cell survival and death, and plays a protective role on the myocardium following IR injury.

Background Wisteria floribunda agglutinin (WFA) + Mac-2-binding protein (M2BPGi) is a novel serum marker for liver fibrosis. Although an elevated serum level of M2BPGi can predict development of hepatocellular carcinoma (HCC), the effect of M2BPGi on HCC remains unclear. There are no reports about the association of M2BPGi with HCC aggressiveness. We aimed to clarify the significance of M2BPGi in HCC. Methods The protein expression of M2BPGi and galectin-3, a ligand of M2BP, and the mRNA expression of M2BP were evaluated in surgically resected human HCC samples. M2BPGi-regulating signals in HCC cells were investigated using transcriptome analysis. The effects of M2BPGi on HCC properties and galectin-3/mTOR signaling were evaluated. Results M2BPGi and galectin-3 proteins co-localised in HCC cells, while M2BP mRNA was detected in cirrhotic liver stromal cells. mTOR signaling was upregulated in M2BPGi-treated HCC cells. Moreover, M2BPGi treatment induced tumour-promoting effects on HCC in vitro by activated mTOR signaling. In addition, M2BPGi bound to galectin-3 to induce membranous galectin-3 expression in HCC cells. In vivo, M2BPGi enhanced the growth of xenografted HCC. Conclusions M2BPGi is produced in stromal cells of the cirrhotic liver. Furthermore, M2BPGi enhances the progression of HCC through the galectin-3/mTOR pathway.

Biomarkers may play an important role in identifying patients at risk for cancer therapy cardiotoxicity. Our objectives were to define the patterns of change in biomarkers with cancer therapy and their associations with cardiotoxicity. In a multicenter cohort of 78 breast cancer patients undergoing doxorubicin and trastuzumab therapy, 8 biomarkers were evaluated at baseline and every 3 months over a maximum follow-up of 15 months. These biomarkers, hypothesized to be mechanistically relevant to cardiotoxicity, included high-sensitivity cardiac troponin I (hs-cTnI), high-sensitivity C-reactive protein (hsCRP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), growth differentiation factor 15 (GDF-15), myeloperoxidase (MPO), placental growth factor (PlGF), soluble fms-like tyrosine kinase receptor-1 (sFlt-1), and galectin 3 (gal-3). We determined if biomarker increases were associated with cardiotoxicity at the same visit and the subsequent visit over the entire course of therapy. Cardiotoxicity was defined by the Cardiac Review and Evaluation Criteria; alternative definitions were also considered. Across the entire cohort, all biomarkers except NT-proBNP and gal-3 demonstrated increases by 3 months; these increases persisted for GDF-15, PlGF, and hs-cTnI at 15 months. Increases in MPO, PlGF, and GDF-15 were associated with cardiotoxicity at the same visit [MPO hazard ratio 1.38 (95% CI 1.10-1.71), P = 0.02; PlGF 3.78 (1.30-11.0), P = 0.047; GDF-15 1.71 (1.15-2.55), P = 0.01] and the subsequent visit. MPO was robust to alternative outcome definitions. Increases in MPO are associated with cardiotoxicity over the entire course of doxorubicin and trastuzumab therapy. Assessment with PlGF and GDF-15 may also be of value. These findings motivate validation studies in additional cohorts.

Galectins (gal) are members of the mammalian β-galactoside-binding proteins and recognize Galβ1-4GlcNAc and Galβ1-4GalNac (Thomsen-Friedenreich antigen (TF)) sequences of several cell surface oligosaccharides. In this study, gal-1, -2, -3 and -13 were investigated systematically in the trophoblast and decidua compartment of intrauterine growth restriction (IUGR) placentas and normal third trimester control placentas and stratified by fetal gender and gestational age. Within this study, 29 third trimester placentas after delivery were analyzed. Fetal gender was equally divided within both groups, and immunohistochemical staining was analyzed according to fetal gender and gestational age. Double immune-fluorescence with trophoblast-specific markers was used to identify galectin-expressing cells at the feto-maternal interface in the decidua. Gal-3 was significantly downregulated only in the extravillous trophoblast of IUGR placentas. In contrast, expressions of gal-2 and gal-13 were downregulated in both villous and extravillous trophoblast cells of IUGR placentas. In addition, gal-2 and gal-13 showed a highly correlated expression scheme in the placenta. There are significant gender-specific expression patterns for single prototype galectins with downregulation of gal-2 and gal-13 of male gender placentas in cases of IUGR. Gal-3 as the chimera type galectin shows only little gender-specific differences in expression, which disappear in IUGR cases.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

Purpose Parathyroid carcinoma (PC) is an endocrine malignancy with a poor prognosis. However, the diagnosis of PC is still a difficult problem. A model with immunohistochemical (IHC) staining of 5 biomarkers has been reported from limited samples for the differential diagnosis of PC. In the present study, a series of IHC markers was applied in relatively large samples to optimize the diagnostic model for PC. Methods In this study, 44 patients with PC, 6 patients with atypical parathyroid tumors and 57 patients with parathyroid adenomas were included. IHC staining for parafibromin, Ki-67, galectin-3, protein-encoding gene product 9.5 (PGP9.5), E-cadherin, and enhancer of zeste homolog 2 (EZH2) was performed on formalin-fixed, paraffin-embedded tissue samples. The effects of clinical characteristics, surgical procedure, and IHC staining results of tumor tissues on the diagnosis and prognosis of PC were evaluated retrospectively. Results A logistic regression model with IHC results of parafibromin, Ki-67, and E-cadherin was created to differentiate PC with an area under the curve of 0.843. Cox proportional hazards analysis showed that negative parafibromin staining (hazard ratio: 3.26, 95% confidence interval: 1.28–8.34, P  = 0.013) was related to the recurrence of PC. Conclusion An IHC panel of parafibromin, Ki-67 and E-cadherin may help to distinguish PC from parathyroid neoplasms. Among the 6 IHC markers and clinical features examined, the risk factor related to PC recurrence was parafibromin staining loss.

Background Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model. Methods We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. Results Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin–glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30–40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. Conclusions Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.

AimHeart failure (HF) affects millions of older individuals in both developed and low/middle-income countries. Serum galectin-3 levels have been shown to have prognostic value. However, its use as a diagnostic biomarker has not been explored. The aim was to establish a saliva galectin-3 reference range and to demonstrate the potential diagnostic utility of salivary and serum galectin-3 levels in assessing HF.MethodsBlood and saliva samples were collected from age-matched healthy controls (n=51) and patients with HF (n=63). Customised immunoassays were developed to quantify salivary galectin-3 levels. The diagnostic performances of these assays were evaluated by receiver operator characteristic (ROC) curves analysis.ResultsThe galectin-3 concentrations were significantly elevated in saliva and serum samples of patients with HF compared with controls (p<0.001 and p<0.0001, respectively). Using ROC curve analysis, both serum and salivary galectin-3 gave area under the curve (AUC)=0.86 and AUC=0.73, respectively. There was also a significant correlation (r=0.4, p<0.01) between serum and salivary galectin-3 levels.ConclusionsFor the first time, we have quantified galectin-3 levels in human saliva and have demonstrated potential clinical utility in diagnosing HF. Further, larger multicentre clinical trials are needed before salivary galectin-3 levels can be implemented in a clinical setting.