Reproducible Enhancement of Fluorescence by Bimetal Mediated Surface Plasmon Coupled Emission for Highly Sensitive Quantitative Diagnosis of Double‐Stranded DNA

Plasmonic enhancement of fluorescence from SYBR Green I conjugated with a double‐stranded DNA (dsDNA) amplicon is demonstrated on polymerase chain reaction (PCR) products. Theoretical computation leads to use of the bimetallic (Au 2 nm–Ag 50 nm) surface plasmons due to larger local fields (higher quality factors) than monometallic (Ag or Au) ones at both dye excitation and emission wavelengths simultaneously, optimizing fluorescence enhancement with surface plasmon coupled emission (SPCE). Two kinds of reverse Kretschmann configurations are used, which favor, in signal‐to‐noise ratio, a fluorescence assay that uses optically dense buffer such as blood plasma. The fluorescence enhancement (12.9 fold at maximum) with remarkably high reproducibility (coefficient of variation (CV) < 1%) is experimentally demonstrated. This facilitates credible quantitation of enhanced fluorescence, however unlikely to obtain by localized surface plasmons. The plasmon‐induced optical gain of 46 dB due to SPCE‐active dye molecules is also estimated. The fluorescence enhancement technologies with PCR enables LOD of the dsDNA template concentration of ≈400 fg µL^?1 (CV < 1%), the lowest ever reported in DNA fluorescence assay to date. SPCE also reduces photobleaching significantly. These technologies can be extended for a highly reproducible and sufficiently sensitive fluorescence assay with small volumes of analytes in multiplexed diagnostics.     

Correlation of near infrared spectroscopy cerebral blood flow estimations and microsphere quantitations in newborn piglets

We compared cerebral blood flow (CBF) estimated using transmission mode near infrared spectroscopy (NIRS) and a modification of the Fick principle with CBF quantitations by radioactive microspheres (MSs) in newborn piglets. Thirteen piglets were studied during steady state, ischemia, and during two reflow periods. NIRS and MS flows were not significantly different during any measurement period. NIRS flows were compared to total brain blood flows and to regional brain blood flows quantitated with MSs and correlated best with temporal cortical flows. Linear regression analysis of the NIRS flows plotted against MS-quantitated temporal cortical flows showed r = 0.71. Thus, CBFs obtained with NIRS were not significantly different from, showed the same directional changes, and correlated acceptably with flows quantitated by MSs.     

Glucose effectiveness, peripheral and hepatic insulin sensitivity, in obese and lean prepubertal children

Vascular injuries in lumbar disk surgery, although rare, are serious complications which may be overlooked due to a broad range of clinical manifestations. It is important that surgeons and radiologists be aware of these potentially fatal complications and develop an appropriate symptom-based diagnostic paradigm. We reviewed 8099 consecutive cases of lumbar disk surgery, performed over a 14-year period at a single institution, for postoperative vascular complications. We identified four patients (0.05%) with lumbar disk surgery-related vascular complications: intraoperative lacerations of the abdominal aorta and median sacral artery, an arteriovenous fistula between the left common iliac artery and vein detected 19 days postdiskectomy, and a partially thrombosed aortic aneurysm with an arteriovenous fistula between the aneurysm and the inferior vena cava, diagnosed 11 months after surgery. The majority of cases in the literature of vascular injury in lumbar disk surgery were reported prior to 1965. Diagnostic approaches described in that period do not reflect the great range of diagnostic techniques available today. Angiography remains the gold standard for diagnosis and guidance as to surgical repair. However, a high index of suspicion based on clinical signs and/or the use of sonography or CT is important in the detection of these complications.     

Light deprivation accelerates recovery from frontal cortical neglect: Relation to locomotion and striatal fos expression.

Rats given unilateral medial agranular (AGm) cortex ablations show neglect for contralateral multimodal stimuli, symptoms that are reversed by 48 hr of light deprivation. To address processes that contribute to this restorative effect, both the rats' locomotion and basal ganglia c-fos expression were studied. AGm-lesioned rats showed less activity in continuous darkness than in normal (12 hr light/12 hr dark) cycles, and the reduced locomotion correlated with the extent of their subsequent behavioral recovery. The AGm ablation reduced the numbers of amphetamine-stimulated Fos-immunoreactive nuclei in the ipsilateral dorsolateral striatum, where the AGm innervation is normally densest. Light deprivation also reduced Fos in this striatal region and attenuated the lesion-induced hemispheric Fos asymmetry. A restored balance of activity between the 2 hemispheres, especially the basal ganglia, appears central to the action of light deprivation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.     

Purification and characterization of a lipid-mobilizing factor associated with cachexia-inducing tumors in mice and humans

A scheme is described for the purification of a lipid-mobilizing factor from a cachexia-inducing murine tumor (MAC16) using a combination of ion exchange (Mono Q), exclusion (Superose), and hydrophobic (C8) chromatography. This process yields an active material with an apparent molecular weight of 24,000 with an overall purification of 3,500 from the tumor homogenate and representing 0.005% of the total protein present. The material tends to aggregate to high molecular mass, is acidic (pI < 4), and displays heterogeneity of charge as evidenced by a broad elution profile on ion exchange and exclusion chromatography and multiple peaks on hydrophobic columns. The purified material was heat and alkali (pH 10.4) labile and activity could be completely inhibited by sulfatase, suggesting that the negative charge could arise from sulfate residues. There was no evidence that the material possessed triglyceride lipase activity. Animals transplanted with the MAC16 tumor and with a delayed weight loss contained in their serum antibodies that recognized a M(r) 24,000 band on Western blots. This material copurified with the lipid-mobilizing factor. Such antibodies were not present in the serum of mice transplanted with the MAC13 tumor, which does not induce cachexia, suggesting that the antibodies were directed to the induction of cachexia rather than the tumor itself. Urine from patients with cancer cachexia also contained a lipid-mobilizing factor which adhered to DEAE-cellulose and gave an apparent M(r) of 24,000 by exclusion chromatography. Western blotting using serum from MAC16 tumor-bearing animals showed the presence of a band of M(r) 24,000 in such fractions, which was not detected using serum from mice bearing the MAC13 tumor. This band was not present in Western blots of urine from normal subjects. The fact that serum from mice bearing the MAC16 tumor can detect the human lipid-mobilizing activity suggests a high degree of structural similarity between the two and raises the possibility that cachexia in humans may be caused by the same species as in the mouse.     

Bond strengths and SEM evaluation of Clearfil Liner Bond 2

PURPOSE: To evaluate a dental adhesive system that uses a single conditioning/primer agent. MATERIALS AND METHODS: Twenty-five flat enamel and dentin bonding sites were prepared to 600 grit on human molar teeth. The Clearfil Liner Bond 2 adhesive system was used to bond Clearfil AP-X composite to both enamel and dentin. After 24 hours of water storage, shear bond strengths were determined using an Instron testing machine. Fifty V-shaped cavity preparations were prepared in human molar teeth with an enamel and cementum margin. Composite restorations were placed using the new adhesive system. The teeth were stored for 24 hours, thermocycled, stained with AgNO3 , sectioned and examined for microleakage. SEM examinations were also completed to evaluate the effects of the treatment steps on enamel and dentin surfaces. RESULTS: Mean shear bond strengths for the experimental adhesive to enamel and dentin were 28.2 +/- 4.9 and 19.4 +/- 3.1 MPa. A t-test revealed that the enamel bond strength was significantly greater (P<0.05) than the dentin strength. No marginal leakage was observed from the enamel margins of the restorations. Three restorations showed minimal leakage from the cementum margins. SEM examinations showed resin penetration into both the conditioned enamel and dentin surfaces. The adhesive system produced high bond strengths to both enamel and dentin, exhibited very minimal microleakage and was easy to use.     

Thermal unfolding of small proteins with SH3 domain folding pattern

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34 degrees C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39 degrees C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39 degrees C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39 degrees C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal-glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons.