Shannon E. Weigum

Nano-bio-chip sensor platform for examination of oral exfoliative cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve ≤0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival. Cancer Prev Res; 3(4); 518–28. ©2010 AACR.

Functional melatonin receptors and metabolic coupling in cultured chick astrocytes

Melatonin is an important hormone regulating circadian clocks in birds, but the specific cellular sites of action are not completely known. The present study was designed to determine whether astrocytes derived from chick brain contained functional melatonin receptors. Primary cell cultures of diencephalon astrocytes that express glial fibrillary acidic protein (GFAP), but not neuron‐specific enolase (NSE) immunoreactivity, were employed to determine the cellular distribution and physiological role for the three known receptor subtypes. Saturation and Scatchard analysis of 2‐[125I]iodomelatonin binding demonstrated melatonin receptor binding with a high affinity and a pharmacological profile similar to that obtained from brain. In situ hybridization for receptor subtypes revealed Mel1A and Mel1C receptor mRNA, but not Mel1B. Administration of pharmacological levels of melatonin acutely inhibited forskolin‐stimulated 2‐deoxyglucose (2DG) uptake, while rhythmic administration of physiological levels of melatonin gradually imposed a rhythm in 2DG uptake and of the release of both lactate and pyruvate into the medium. These results indicate that (1) there are functional Mel1A and Mel1C melatonin receptors in astrocyte‐rich cultures, and (2) rhythmic administration of melatonin plays an important role in the regulation of astrocytic metabolic activity. Together, the data suggest that the circadian secretion of melatonin probably plays a role in the global metabolic economy of the avian brain through rhythmic regulation of metabolism in astrocytes. GLIA 39:268–278, 2002.

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA

ABSTRACT We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 103 purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection.

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

BackgroundNuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there.ResultsUsing confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence.ConclusionWe therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

GFAP and nuclear lamins share an epitope recognized by monoclonal antibody J1-31

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.

A new bio-nanochip sensor aids oral cancer detection.

A diagnostic cytology-on-a-chip technique rapidly detects pre-malignant and malignant cells with high sensitivity and specificity.

Microchip-based enumeration of human white blood cells.

The advent of flow cytometry has considerably changed the ways in which medical testing is conducted. However, the cost of flow cytometers, their large size, and their maintenance needs make them scarce in resource-poor settings and available almost only in clinical pathology laboratories in developed countries. Because cell enumeration is a basic and crucial support of diagnosis, prognosis, and treatment, an alternative cell-counting method that would potentially be cost-effective, portable, and suitable for use in resource-poor settings is warranted. We describe here a protocol for conducting cell-counting experiments in a simple microfluidic structure. This protocol describes how to build a simple microfluidic cell and perform a total white blood cell (WBC) count through capture and immunolabeling of the WBCs with an anti-CD45 antibody.

Amplification-Free Detection of Cryptosporidium parvum Nucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies

Abstract:   This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a redshift in the wavelength of light scattered by the gold nanoparticles. The limit of detection was between 4 × 105 and 4 × 106 copies of RNA per microliter reaction mix, when a short synthetic target or full-length in vitro transcribed target was employed. With total nucleic acids purified from C. parvum oocysts spiked into 100-mg stool, as few as 670 oocysts/μl reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

Targeting hepatocellular carcinoma with aptamer-functionalized PLGA/PLA-PEG nanoparticles

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, particularly in regions where chronic Hepatitis B and C infections are common. Nanoparticle assemblies that incorporate high-affinity aptamers which specifically bind malignant hepatocellular carcinoma cells could be useful for targeted drug delivery or enhancing contrast with existing ablation therapies. The in vitro interactions of a tumor-specific aptamer, TLS11a, were characterized in a hepatoma cell line via live-cell fluorescence imaging, SDS-PAGE and Western Blotting techniques. Cell surface binding of the aptamer-AlexaFluor®546 conjugate was found to occur within 20 minutes of initial exposure, followed by internalization and localization to late endosomes or lysosomes using a pH-sensitive LysoSensor™ Green dye and confocal microscopy. Aptamer-functionalized polymer nanoparticles containing poly(lactic-co-glycolic acid) (PLGA) and poly(lactide)-b-poly(ethylene glycol) (PLA-PEG) were then prepared by nanoprecipitation and passively loaded with the chemotherapeutic agent, doxorubicin, yielding spherical nanoparticles approximately 50 nm in diameter. Targeted drug delivery and cytotoxicity was assessed using live/dead fluorescent dyes and a MTT colorimetric viability assay with elevated levels of cell death found in cultures treated with either the aptamer-coated and uncoated polymer nanoparticles. Identification and characterization of the cell surface protein epitope(s) recognized by the TLS11a aptamer are ongoing along with nanoparticle optimization, but these preliminary studies support continued investigation of this aptamer and functionalized nanoparticle conjugates for targeted labeling and drug delivery within malignant hepatocellular carcinomas.