Probing Membrane Receptor-Ligand Specificity with Surface- and Tip- Enhanced Raman Scattering.

The specific interaction between a ligand and a protein is a key component in minimizing off-target effects in drug discovery. Investigating these interactions with membrane protein receptors can be quite challenging. In this report, we show how spectral variance observed in surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) can be correlated with ligand specificity in affinity-based assays. Variations in the enhanced Raman spectra of three peptide ligands (i.e., cyclic-RGDFC, cyclic-isoDGRFC, and CisoDGRC), which have different binding affinity to αvß3 integrin, are reported from isolated proteins and from receptors in intact cancer cell membranes. The SERS signal from the purified proteins provides basis spectra to analyze the signals in cells. Differences in the spectral variance within the SERS and TERS data for each ligand indicate larger variance for nonspecific ligand-receptor interactions. The SERS and TERS results are correlated with single particle tracking experiments of the ligand-functionalized nanoparticles with purified receptors on glass surfaces and living cells. These results demonstrate the ability to elucidate protein-ligand recognition using the observed vibrational spectra and provide perspective on binding specificity for small-molecule ligands in intact cell membranes, demonstrating a new approach for investigating drug specificity.

Chemical Analysis of Morphological Changes in Lysophosphatidic Acid-Treated Ovarian Cancer Cells.

Ovarian cancer (OvCa) cells are reported to undergo biochemical changes at the cell surface in response to treatment with lysophosphatidic acid (LPA). Here we use scanning electron microscopy (SEM) and multiplex coherent anti-Stokes Raman scattering (CARS) imaging via supercontinuum excitation to probe morphological changes that result from LPA treatment. SEM images show distinct shedding of microvilli-like features upon treatment with LPA. Analysis of multiplex CARS images can distinguish between molecular components, such as lipids and proteins. Our results indicate that OvCa429 and SKOV3ip epithelial ovarian cancer cells undergo similar morphological and chemical responses to treatment with LPA. The microvilli-like structures on the surface of multicellular aggregates (MCAs) are removed by treatment with LPA. The CARS analysis shows a distinct decrease in protein and increase in lipid composition on the surface of LPA-treated cells. Importantly, the CARS signals from cellular sheddings from MCAs with LPA treatment are consistent with cleavage of proteins originally present. Mass spectrometry on the cellular sheddings show that a large number of proteins, both membrane and intracellular, are present. An increased number of peptides are detected for the mesenchymal cell line relative to the epithelial cell indicating a differential response to LPA treatment with cancer progression.

Tracking Bulk and Interfacial Diffusion Using Multiplex Coherent Anti-Stokes Raman Scattering Correlation Spectroscopy.

Multiplex coherent anti-Stokes Raman scattering correlation spectroscopy (CARS-CS) is shown as a label-free, chemically specific approach for monitoring the molecular mobility of particles in solution and at interfaces on the millisecond time scale. The CARS spectral range afforded by broadband excitation facilitates a quantitative measurement for the number of particles in the focal volume, whereas the autocorrelation of spectral data elucidates dynamic events, such as diffusion. The measured diffusion coefficients for polymer beads ranging from 100 nm to 1.1 µm in diameter are on the order of 10(-8)-10(-9) cm(2)/s, in good agreement with predicted Stokes-Einstein values. Diffusion at different interfaces shows particles are fastest in bulk medium, marginally slower at the liquid/glass interface, and 1.5-2 times slower rate at the air/liquid interface. Multivariate curve resolution analysis of distinct spectral features in multiplex CARS measurement distinguishes different composition lipid vesicles in a mixture diffusing through the focal volume. The observed diffusion is consistent with results obtained from single particle tracking experiments. This work demonstrates the utility of multiplex CARS correlation spectroscopy for monitoring particle diffusion from different chemical species across diverse interfaces.

BDNF and NT-3 regulation of trkB and trkC mRNA levels in the developing chick spinal cord.

In this study we investigated the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 on mRNA levels of TrkB and TrkC receptors. We used an expression system to supply developing chicks with the neurotrophic factor and then analysed the receptor mRNA levels at embryonic day 8 (E8), E10 and E15 using semi-quantitative RT-PCR. In control chicks, maximal expression levels of both receptors were observed at E10. Treatment with BDNF resulted in significant down-regulation of TrkB mRNA levels (P<0.05) at E10 but not E8 or E15. Treatment with NT-3 showed down-regulation of trkB levels at all developmental stages. TrkC mRNA levels were down-regulated at all developmental stages with NT-3 treatment and at E10 and E15 with BDNF treatment. For both receptors the down-regulation was greater in NT-3-treated chicks than those treated with BDNF. Thus, our data indicate that neurotrophin receptor mRNA levels in the spinal cord are regulated by neurotrophic factors during embryonic development.

Empyema management: twelve years' experience since the introduction of video-assisted thoracoscopic surgery.

We reviewed empyema management at our center since video-assisted thoracoscopic surgery (VATS) was introduced. Between 1991 and 2003, 58 patients (34 male, 24 female) ages 1 day to 17 years (median, 6 years) were identified. The median interval from the onset of symptoms to presentation was 7 days; 31% of patients received inpatient care at another hospital, and 24% were from northern communities, including the Arctic. Symptoms included fever (100%), cough (66%), shortness of breath (46%), chest pain (38%), and abdominal pain (17%). Most patients (96%) had pneumonia. The most common organisms were Streptococcus pneumoniae (22%), group A streptococcus (8%), and Mycobacterium tuberculosis (3%). No organisms were identified in 31% of patients. One empyema was trauma-related and one was due to complications of esophageal stricturoplasty. All patients received intravenous antibiotics (median, 17 days). Three patients had thoracentesis, 17 had chest tubes, 3 had thoracentesis and chest tubes, 26 had VATS, 6 had thoracotomy, and 3 had minithoracotomy. Median operative times were 86 minutes for VATS, 90 minutes for thoracotomy, and 75 minutes for minithoracotomy. Median total length of stay (LOS) was 15 days for VATS, 20.5 days for thoracotomy, and 21 days for minithoracotomy. The median preoperative LOS was longer for thoracotomy (10 days) than for VATS (5 days) or minithoracotomy (1 day). The median postoperative LOS was 14 days for VATS, 9.5 days for thoracotomy, and 8 days for minithoracotomy. Our experience shows that VATS is a safe and effective approach to managing children with empyema. The shorter preoperative LOS for VATS is likely due to earlier surgical referral and intervention using less invasive surgical procedures.

Laparoscopic versus open reduction of intussusception in children: a single-institution comparative experience.

BACKGROUND: The role of laparoscopic surgery in treating intussusception has been controversial. This study reviews our institution's experience with the laparoscopic approach (LAP) compared to the open surgical approach (OPEN). METHODS: Retrospective analysis of all patients undergoing surgery for intussusception at our center from January 2002 to February 2006. Statistical assessment included Student's t test and chi2 analysis. RESULTS: A total of 41 patients required operation for intussusception (18 LAP, 23 OPEN). Mean age was 22 months for LAP and 11 months for OPEN (P = .17). In the LAP group, 28% (5/18) were converted to an open procedure. Operative times and complications were not significantly different. Pathologic lead points were found in 33% (6/18) LAP and 35% (8/23) OPEN patients (P = 1.0). Time to full feeds was significantly shorter (LAP vs OPEN: 3.4 +/- 2.7 vs 5.6 +/- 3.4 days, P = .02). Length of stay was shorter (LAP vs OPEN: 4.8 +/- 3.5 vs 9.1 +/- 7.5 days, P = .03). CONCLUSIONS: Intussusception can be treated safely and effectively using a LAP with a significant decrease in time to full feeds and length of stay. The LAP should be considered as the initial approach for stable patients with intussusception requiring operative intervention.

Unusual case of mesenteric venous thrombosis associated with oral contraceptive use in an adolescent girl.

A 16-year-old girl presented with hematochezia and abdominal pain. The patient developed a small bowel obstruction that required a laparotomy, which showed a segment of grossly infarcted small bowel with 3 perforations, localized abscesses, and creeping fat. Resection and an end-to-end anastomosis were performed. Pathologic examination showed underlying mesenteric venous thrombosis with no evidence of Crohn's disease. Hematology workup showed no evidence of a congenitally inherent hypercoagulable state. The use of oral contraceptives was the only predisposing factor identified.