Activity of glucose oxidase functionalized onto magnetic nanoparticles

BACKGROUND: Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. METHODS: Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. RESULTS: The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles CONCLUSION: Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological application

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

Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics

Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision

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

Ultrasound-guided nanobubbles for targeted drug delivery

In a large number of biological and environmental applications, ultrasound (US)-powered micro- and nano-motors have attracted considerable attention. However, their applications in biological settings have been limited due to the incompatibility of metallic motors and the lack of precision guiding. Here, we demonstrate that cellulosic polymer nanobubbles (200-800nm) can be propelled, aligned, accelerated, and assembled in solution using Doppler ultrasound beam (DUB) and simultaneously imaged using low-frequency ultrasound. Results show that by utilizing Doppler ultrasound beam (DUB), nanobubbles accumulation at a pre-determined site can be enhanced. Moreover, bubbles’ trajectory and velocity can be also be manipulated. Related parameters associated with nanobubble accumulation and velocity change such as bubble size, ultrasound intensity, frequency, and beam angle were investigated using a factorial design of experiments. Frequency and intensity of the DUB were found to be significant for nanobubble accumulation whereas beam intensity was significant in changing the velocity of nanobubbles. The precise beam steering using the Doppler ultrasound and simultaneous ultrasound imaging contrast enhancement offered by nanobubbles holds considerable promise for various applications

Fundamental Characterization of Oxygen Nanobubbles

A hypoxic environment is created by tumors’ incredible growth rate. Hypoxia provides radioresistance to the tumors, thus making radiation treatment less effective. The issue is that increasing the radiation leads to increased side effects in patients. Our goal for the oxygen-filled nanobubble is to deliver oxygen to the tumor to lessen radioresistance and make radiation treatment more efficient. However, we need preliminary research to understand and improve the nanobubbles before further research and implementation. To do this, we synthesized different batches of nanobubbles to optimize the production method and find the best container and temperature to store nanobubbles. We measured the oxygen release profile of the nanobubbles and obtained Transmission Electron Microscope (TEM) images and Dynamic Light Scattering (DLS) data to characterize the nanobubbles. The nanobubbles’ peak oxygenation happened 4-5 days after synthesis, and 3 mL SKS glass vials were optimal for storing the nanobubbles. We have not chosen the best synthesis technique or storage temperature yet, due to inadequate TEM images and inconclusive oxygen profile data. We will eventually conduct a proof-of-concept experiment on mice to see if nanobubbles improve irradiation. The optimal storing condition aids our chances for a successful experiment by limiting undesired oxygen release before entering the mouse’s body

Optogenetic regulation of site-specific subtelomeric DNA methylation

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest

Sensor technologies for the detection and monitoring of endocrine-disrupting chemicals

Endocrine-disrupting chemicals (EDCs) are a class of man-made substances with potential to disrupt the standard function of the endocrine system. These EDCs include phthalates, perchlorates, phenols, some heavy metals, furans, dimethoate, aromatic hydrocarbons, some pesticides, and per- and polyfluoroalkyl substances (PFAS). EDCs are widespread in the environment given their frequent use in daily life. Their production, usage, and consumption have increased many-fold in recent years. Their ability to interact and mimic normal endocrine functions makes them a potential threat to human health, aquatics, and wild life. Detection of these toxins has predominantly been done by mass spectroscopy and/or chromatography-based methods and to a lesser extent by advanced sensing approaches such as electrochemical and/or colorimetric methods. Instrument-based analytical techniques are often not amenable for onsite detection due to the lab-based nature of these detecting systems. Alternatively, analytical approaches based on sensor/biosensor techniques are more attractive because they are rapid, portable, equally sensitive, and eco-friendly. Advanced sensing systems have been adopted to detect a range of EDCs in the environment and food production systems. This review will focus on advances and developments in portable sensing techniques for EDCs, encompassing electrochemical, colorimetric, optical, aptamer-based, and microbial sensing approaches. We have also delineated the advantages and limitations of some of these sensing techniques and discussed future developments in sensor technology for the environmental sensing of EDCs

Nanobubbles Provide Theranostic Relief to Cancer Hypoxia

Hypoxia is a common motif among tumors, contributing to metastasis, angiogenesis, cellular epigenetic abnormality, and resistance to cancer therapy. Hypoxia also plays a pivotal role in oncological studies, where it can be used as a principal target for new anti-cancer therapeutic methods. Oxygen nanobubbles were designed in an effort to target the hypoxic tumor regions, thus interrupting the hypoxia-inducible factor-1α (HIF-1α) regulatory pathway and inhibiting tumor progression. At less than 100nm, oxygen nanobubbles act as a vehicle for site-specific oxygen delivery, while also serving as an ultrasound contrast agent for advanced imaging purposes. Through in vitro and in vivo studies, it was shown the reversal of 5mC hypomethylation was achieved in the hypoxia-afflicted regions. An obvious increase in the oxygen concentration within hypoxic regions was also observed, implying adequate oxygen dissociation from the nanobubbles to the hypoxic tumor microenvironment. These implications suggest nanobubbles can be used as a means for epigenetic regulation, ultrasound imaging, and cancer therapeutics, thus having a significant impact on new-age cancer treatment methods in oncology