Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

Functional consequences of polyamine synthesis inhibition by L-alpha-difluoromethylornithine (DFMO): cellular mechanisms for DFMO-mediated ototoxicity

L-Alpha-difluoromethylornithine (DFMO) is a chemopreventive agent for colon cancer in clinical trials. Yet, the drug produces an across-frequency elevation of the hearing threshold, suggesting that DFMO may affect a common trait along the cochlear spiral. The mechanism for the ototoxic effects of DFMO remains uncertain. The cochlear duct is exclusively endowed with endocochlear potential (EP). EP is a requisite for normal sound transduction, as it provides the electromotive force that determines the magnitude of the receptor potential of hair cells. EP is generated by the high throughput of K(+) across cells of the stria vascularis, conferred partly by the activity of Kir4.1 channels. Here, we show that the ototoxicity of DFMO may be mediated by alteration of the inward rectification of Kir4.1 channels, resulting in a marked reduction in EP. These findings are surprising given that the present model for EP generation asserts that Kir4.1 confers the outflow of K(+) in the stria vascularis. We have proposed an alternative model. These findings should also enable the rational design of new pharmaceuticals devoid of the untoward effect of DFMO.     

Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling

Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characterized the thermally induced and GuHCl-induced unfolding features of SARS_S1b using circular dichroism (CD), tryptophan fluorescence, and stopped-flow spectral techniques. For the thermally induced unfolding at pH 7.4, the apparent activation energy (E(app)) and transition midpoint temperature (Tm) were determined to be 16.3 +/- 0.2 kcal/mol and 52.5 +/- 0.4 degrees C, respectively. The CD spectra are not dependent on temperature, suggesting that the secondary structure of SARS_S1b has a relatively high thermal stability. GuHCl strongly affected SARS_S1b structure. Both the CD and fluorescent spectra resulted in consistent values of the transition middle concentration of the denaturant (Cm, ranging from 2.30 to 2.45 M) and the standard free energy change (deltaG(o), ranging from 2.1 to 2.5 kcal/mol) for the SARS_S1b unfolding reaction. Moreover, the kinetic features of the chemical unfolding and refolding of SARS_S1b were also characterized using a stopped-flow CD spectral technique. The obvious unfolding reaction rates and relaxation times were determined at various GuHCl concentrations, and the Cm value was obtained, which is very close to the data that resulted from CD and fluorescent spectral determinations. Secondary and three-dimensional structural predictions by homology modeling indicated that SARS_S1b folded as a globular-like structure by beta-sheets and loops; two of the four tryptophans are located on the protein surface, which is in agreement with the tryptophan fluorescence result. The three-dimensional model was also used to explain the recently published experimental results of S1-ACE-2 binding and immunizations.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.     

Lack of increased genetic damage in 1,3-butadiene-exposed Chinese workers studied in relation to EPHX1 and GST genotypes

1,3-Butadiene (BD) is an important industrial chemical and pollutant. Its ability to induce genetic damage and cause hematological malignancies in humans is controversial. We have examined chromosome damage by fluorescence in situ hybridization (FISH) and mutations in the HPRT gene in the blood of Chinese workers exposed to BD. Peripheral blood samples were collected and cultured from 39 workers exposed to BD (median level 2 ppm, 6 h time-weighted average) and 38 matched controls in Yanshan, China. No difference in the level of aneuploidy or structural changes in chromosomes 1, 7, 8, and 12 was detected in metaphase cells from exposed subjects in comparison with matched controls, nor was there an increase in the frequency of HPRT mutations in the BD-exposed workers. Because genetic polymorphisms in glutathione S-transferase (GST) enzymes and microsomal epoxide hydrolase (EPHX1) may affect the genotoxic effects of BD and its metabolites, we also related chromosome alterations and gene mutations to GSTT1, GSTM1 and EPHX1 genotypes. Overall, there was no effect of variants in these genotypes on numerical or structural changes in chromosomes 1, 7, 8 and 12 or on HPRT mutant frequency in relation to BD exposure, but the GST genotypes did influence background levels of both hyperdiploidy and HPRT mutant frequency. In conclusion, our data show no increase in chromosomal aberrations or HPRT mutations among workers exposed to BD, even in potentially susceptible genetic subgroups. The study is, however, quite small and the levels of BD exposure are not extremely high, but our findings in China do support those from a similar study conducted in the Czech Republic. Together, these studies suggest that low levels of occupational BD exposure do not pose a significant risk of genetic damage.     

Dietary linolenic acid and fasting glucose and insulin: the National Heart, Lung, and Blood Institute Family Heart Study

Objective: To assess whether dietary linolenic acid is associated with fasting insulin and glucose. Research Methods and Procedures: In a cross‐sectional design, we studied 3993 non‐diabetic participants of the National Heart, Lung, and Blood Institute Family Heart Study 25 to 93 years of age. Linolenic acid was assessed through a food frequency questionnaire, and laboratory data were obtained after at least a 12‐hour fast. We used generalized linear models to calculate adjusted means of insulin and glucose across quartiles of dietary linolenic acid. Results: From the lowest to the highest sex‐specific quartile of dietary linolenic acid, means ± standard error for logarithmic transformed fasting insulin were 4.06 ± 0.02 (reference), 4.09 ± 0.02, 4.13 ± 0.02, and 4.17 ± 0.02 pM, respectively (trend, p < 0.0001), after adjustment for age, sex, energy intake, waist‐to‐hip ratio, smoking, and high‐density lipoprotein‐cholesterol. When dietary linolenic acid was used as a continuous variable, the multivariable adjusted regression coefficient was 0.42 ± 0.08. There was no association between dietary linolenic acid and fasting glucose (trend p = 0.82). Discussion: Our data suggest that higher consumption of dietary linolenic acid is associated with higher plasma insulin, but not glucose levels, in non‐diabetic subjects. Additional studies are needed to assess whether higher intake of linolenic acid results in an increased insulin secretion and improved glucose use in vivo.     

ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia

ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Here, we show that forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia.