Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an Enzyme-based Immunoassay

BACKGROUND: Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. METHODS: To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. RESULTS: We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025±0.081, 0.023±0.006 and 0.001±0.0002, respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013±0.003%) in comparison to healthy controls. CONCLUSION: The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets

Preparation of a Microporous Polyurethane Film with Negative Surface Charge for siRNA Delivery via Stent

Polyurethane (PU) and polyethylene glycol (PEG) were used to prepare a porous stent-covering material for the controlled delivery of small interfering RNA (siRNA). Microporous polymer films were prepared using a blend of polyurethane and water-soluble polyethylene glycol by the solution casting method; the PEG component was extracted in water to make the film microporous. This film was dipped in 2% poly(methyl methacrylate-co-methacrylic acid) solution to coat the polymer film with the anionic polyelectrolyte. The chemical components of the film surface were characterized by Fourier Transform Infrared (FTIR) spectroscopy and its structural morphology was examined by scanning electron microscopy (SEM). The effect of the negatively charged surface after attachment of a fluorescein isothiocyanate- (FITC-) labeled siRNA-polyethyleneimine complex onto the microporous polyurethane film and the controlled release of the complex from the film was investigated by fluorescence microscopy. Fluorescence microscopy showed the PU surface with intense fluorescence by the aggregates of the FITC-labeled-siRNA-PEI complex (measuring up to few microns in size); additionally, the negatively charged PU surface revealed broad and diffuse fluorescence. These results suggest that the construction of negatively charged microporous polyurethane films is feasible and could be applied for enhancing the efficiency of siRNA delivery via a stent-covering polyurethane film

Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab′)2 were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies in the immunoassays compared to randomly bound IgG, particularly, in the low antibody density range. The observed improvements could have resulted from controlled molecular orientation and also from flexibility, offering conditions suitable for binding complex formations

Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy

Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ

Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration.

Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration

Venous Hemangioma of Parapharyngeal Space with Calcification

A hemangioma of the parapharyngeal space (PPS) is an extremely rare tumor and is responsible for 0.5-1% of all tumors occurring in the PPS. We report a case of PPS venous hemangioma in a 49-year-old woman presenting with diffuse swelling in the submandibular region. A preoperative computed tomography (CT) scan showed a cystic mass with multiple calcifications in the PPS. The calcific nodules were round and about 2 mm in diameter. The hemangioma was completely resected via a transcervical approach. During surgery, we found several calcific nodules, which represented phleoboliths or areas of thrombosis with dystrophic calcification. Despite its rarity, a venous hemangioma of the PPS should be considered in a differential diagnosis when a cystic mass with calcification is found by CT scan. To our knowledge, this is the first reported case of a PPS venous hemangioma; we describe its pathognomonic findings on imaging

The Expression of AGO2 and DGCR8 in Idiopathic Sudden Sensorineural Hearing Loss

ObjectivesThe microRNAs have been implicated in the development and function of the inner ear, especially in contribution to hearing. However, the impact of idiopathic sudden sensorineural hearing loss (SSNHL) on expression of miRNA biogenesis-related components has not been established. To investigate the regulations of microRNA (miRNA) biogenesis-related components, argonaute 2 (AGO2) and DiGeorge syndrome critical region gene 8 (DGCR8) mRNA expression in SSNHL and to evaluate the value of clinical parameters on their expression.MethodsThirty-seven patients diagnosed with SSNHL and fifty-one healthy volunteers were included in this study. We measured mRNA expression levels of AGO2 and DGCR8 in whole blood cells but erythrocytes of patients with SSNHL and controls, using reverse transcription and real-time polymerase chain reaction analysis.ResultsThe mRNA expression level of AGO2 is upregulated in SSNHL. The expression level of AGO2 was significantly correlated with that of DGCR8 in both patients with SSNHL and controls. Expression level of AGO2 in SSNHL was correlated with white blood cell counts.ConclusionThis study demonstrated for the first time that the AGO2 mRNA expression level was upregulated in SSNHL, suggesting its important role in pathobiology of SSNHL development