Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410–670 nm. We developed a new approach to antibody conjugation, using Huisgen–Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340–380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.     

MRI as a method of evaluation and predicting mandibular growth based on temporomandibular joint

BackgroundCondylar cartilage acts as a centre of craniofacial complex growth and magnetic resonance imaging (MRI) is the most suitable examination to differentiate tissues and identify pathological conditions of TMJ. Condylar growth disturbance can alter the maxillofacial morphology, including occlusal relationship, and the affected children may not present symptoms. Although TMJ in children is similar to that in adults, the anterior condylar inclination is straight and increases with age, presenting slight condylar flattening and slight contrast enhancement.ObjectiveThe present work aimed to review the literature focussing to assess how MRI and the development of TMJ can contribute to orthopaedic/orthodontic diagnosis in growing patients.Main resultsMRI can also complement the predictive diagnosis of growth in which the image of a double contour of the condyle reveals proliferation of cartilage cells with immature ossification. Considering that orthodontic therapy usually begins after eruption of permanent teeth, the moment of alternation of the double contour-like structure is close or coincident with the eruption of upper second molars.ConclusionClinically, the advantage of diagnosis of double contour is not to use any ionizing radiation related to scan in these subjects. Therefore, MRI could be a choice for the follow-up of growing patients.     

Lung protective ventilation during pulmonary resection in children: a prospective,single-centre,randomised controlled trial

Background

Perioperative ventilatory strategies for lung protection in children are underexplored. This study evaluated the effects of lung protective ventilation (LPV) on postoperative clinical outcomes in children requiring one-lung ventilation (OLV) for pulmonary resection.

Expression of immunohistochemical markers according to histological type in patients with early gastric cancer

Objective. We compared the biological characteristics of early gastric cancer (EGC) using immunohistochemical (IHC) staining among histological types. Materials and methods. IHC staining results were analyzed in 86 EGCs resected with endoscopic submucosal dissection to identify mucin phenotype and biological characteristics. Results. The histological type was classified as tubular adenocarcinoma (TAC), mixed adenocarcinoma (MAC), or poorly cohesive carcinoma (PCC). Significant differences in MUC-2 (34.4% vs. 10.7%, p < 0.05) and MUC-5AC (59.4% vs. 85.7%, p < 0.05) expression were observed between TAC and PCC. The poorly cohesive component of MAC showed stronger immunoreactivity to CD10 (46.2% vs. 14.3%, p < 0.05) but weaker reactivity to MUC-5AC (57.7% vs. 85.7%, p < 0.05), compared to that of PCC. E-cadherin and β-catenin expression levels significantly decreased in the poorly cohesive component of MAC (15.4% vs. 90.6%, p < 0.05; 7.7% vs. 90.6%, p < 0.05, respectively) and PCC (10.7% vs. 90.6%, p < 0.05; 14.3% vs. 90.6%, p < 0.05, respectively), compared to TAC. However, vascular endothelial growth factor expression significantly increased in the poorly cohesive component of MAC (42.3% vs. 9.4%, p < 0.05) and PCC (39.3% vs. 9.4%, p < 0.05), compared to TAC. Conclusion. IHC analysis showed that EGC histological types differ in terms of mucin phenotype and biological characteristics. The poorly cohesive components showed decreased E-cadherin and β-catenin expression levels and increased vascular endothelial growth factor expression. These characteristics may contribute to the poor prognosis of patients with MAC and PCC.     

Effect of graded running on esophageal motility and gastroesophageal reflux in fed volunteers

The effects of different grades of running on esophageal motility and gastroesophageal reflux in the fed state were evaluated. We studied healthy volunteers (male: 12, age: 27 +/- 5 yr) using ambulatory esophageal manometry, pH catheter and portable digital data recorder. Each exercise was performed 30 min after meal, with 20 min of rest between exercises. Subjects exercised on a treadmill at 40% and 70% maximal heart rate. The number of gastroesophageal reflux episodes, the duration of esophageal acid exposure and percent time pH below 4 were significantly (p < 0.01) increased during exercise at 70% maximal heart rate. The frequency of contraction (contraction/min) (p < 0.05), frequency of repetition (p < 0.01), percent of simultaneous contraction (p < 0.01), percent of above 100 mmHg amplitude (p < 0.05), and frequency of 2-peak contraction (p < 0.01) were significantly increased during exercise at 70% maximal heart rate. However, median amplitude and median duration showed no significant changes between each exercise session. Postprandial running exercises induce gastroesophageal reflux, which correlates with exercise intensity. These effects are mediated by disorganized esophageal motility.