An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability.

Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signalling molecules bind to NRPs through a carboxy (C)-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumour-homing CendR peptides penetrate through tumour tissue and have shown utility in enhancing drug delivery into tumours. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signalling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway.

Identification of Inhibitors of triacylglyceride accumulation in muscle cells: comparing HTS results from 1536-well plate-based and high-content platforms.

Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual-emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the National Institutes of Health library. The screening data from the plate reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate reader assay is a powerful high-throughout screening platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.

Computerized decision support system for intraoperative analysis of margin status in breast conservation therapy.

Background. Breast conservation therapy (BCT) is the standard treatment for breast cancer; however, 32-63% of procedures have a positive margin leading to secondary procedures. The standard of care to evaluate surgical margins is based on permanent section. Imprint cytology (IC) has been used to evaluate surgical samples but is limited by excessive cauterization thus requiring experienced cytopathologist for interpretation. An automated image screening process has been developed to detect cancerous cells from IC on cauterized margins. Methods. IC was prospectively performed on margins during lumpectomy operations for breast cancer in addition to permanent section on 127 patients. An 8-slide training subset and 8-slide testing subset were culled. H&E IC automated analysis, based on linear discriminant analysis, was compared to manual pathologist interpretation. Results. The most important descriptors, from highest to lowest performance, are nucleus color (23%), cytoplasm color (15%), shape (12%), grey intensity (9%), and local area (5%). There was 100% agreement between automated and manual interpretation of IC slides. Conclusion. Although limited by IC sampling variability, an automated system for accurate IC cancer cell identification system is demonstrated, with high correlation to manual analysis, even in the face of cauterization effects which supplement permanent section analysis.

Quantitative comparison of surgical margin histology following excision with traditional electrosurgery and a low-thermal-injury dissection device.

BACKGROUND: This study is the first to examine in vivo the effect of thermal injury in breast conservation pathology in a direct comparison of traditional electrosurgery and an alternative low-thermal-injury device. METHODS: A prospective study of 20 consecutive subjects with biopsy-proven invasive ductal carcinoma (IDC) tumors 1 cm was conducted. Following excision, incisions were made into the tumor with the two devices. Thermal injury depth, margin distance, tissue type, and histological effect were compared on the same breast tissue cut with each excision instrument. A probability evaluation of close and positive margin cases for the true tumor margins was conducted. RESULTS: Compared to traditional electrosurgery, the low-thermal-injury instrument reduced collagen denaturation depth from 435 to 102 µm (77%), fused tissue depth from 262 to 87 µm (67%), and distortion depth from 1,132 to 774 µm (30%). CONCLUSIONS: Based on analysis of the close subset of the true margins, using the traditional electrosurgical device in place of the low-thermal-injury device would have resulted in 48% of the close margin samples being negatively converted to false-positive, and in 11% converting from close to false-negative. The methodology of this work may be readily applied to larger, more definitive studies.

Comparative healing of human cutaneous surgical incisions created by the PEAK PlasmaBlade, conventional electrosurgery, and a standard scalpel.

BACKGROUND: The authors investigated thermal injury depth, inflammation, and scarring in human abdominal skin by comparing the histology of incisions made with a standard "cold" scalpel blade, conventional electrosurgery, and the PEAK PlasmaBlade, a novel, low-thermal-injury electrosurgical instrument. METHODS: Approximately 6 and 3 weeks before abdominoplasty, full-thickness incisions were created in the abdominal pannus skin of 20 women, using a scalpel (scalpel), the PlasmaBlade, and a conventional electrosurgical instrument. Fresh (0-week) incisions were made immediately before surgery. After abdominoplasty, harvested incisions were analyzed for scar width, thermal injury depth, burst strength, and inflammatory response. RESULTS: Acute thermal injury depth was reduced 74 percent in PlasmaBlade incisions compared with conventional electrosurgical instrument (p < 0.001). Significant differences in inflammatory response were observed at 3 weeks, with mean CD3 response (T-lymphocytes) 40 percent (p = 0.01) and 21 percent (p ≈ 0.12) higher for the conventional electrosurgical instrument and PlasmaBlade, respectively, compared with the scalpel. CD68 response (monocytes/macrophages) was 52 percent (p = 0.05) and 16 percent (p ≈ 0.35) greater for a conventional electrosurgical instrument and the PlasmaBlade, respectively. PlasmaBlade incisions demonstrated 65 percent (p < 0.001) and 42 percent (p < 0.001) stronger burst strength than a conventional electrosurgical instrument, with equivalence to the scalpel at the 3- and 6-week time points, respectively. Scar width was equivalent for the PlasmaBlade and the scalpel at both time points, and 25 percent (p = 0.01) and 12 percent (p = 0.15) less than for electrosurgery, respectively. CONCLUSIONS: PlasmaBlade incisions demonstrated reduced thermal injury depth, inflammatory response, and scar width in healing skin compared with electrosurgery. These results suggest that the PlasmaBlade may provide clinically meaningful advantages over conventional electrosurgery during human cutaneous wound healing. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.(Figure is included in full-text article.).

Quantitative automated image analysis system with automated debris filtering for the detection of breast carcinoma cells.

OBJECTIVE: To develop an intraoperative method for margin status evaluation during breast conservation therapy (BCT) using an automated analysis of imprint cytology specimens. STUDY DESIGN: Imprint cytology samples were prospectively taken from 47 patients undergoing either BCT or breast reduction surgery. Touch preparations from BCT patients were taken on cut sections through the tumor to generate positive margin controls. For breast reduction patients, slide imprints were taken at cuts through the center of excised tissue. Analysis results from the presented technique were compared against standard pathologic diagnosis. Slides were stained with cytokeratin and Hoechst, imaged with an automated fluorescent microscope, and analyzed with a fast algorithm to automate discrimination between epithelial cells and noncellular debris. RESULTS: The accuracy of the automated analysis was 95% for identifying invasive cancers compared against final pathologic diagnosis. The overall sensitivity was 87% while specificity was 100% (no false positives). This is comparable to the best reported results from manual examination of intraoperative imprint cytology slides while reducing the need for direct input from a cytopathologist. CONCLUSION: This work demonstrates a proof of concept for developing a highly accurate and automated system for the intraoperative evaluation of margin status to guide surgical decisions and lower positive margin rates.

Automated microscopy to evaluate surgical specimens via touch prep in breast cancer.

BACKGROUND: Breast conservation therapy is the standard treatment for breast cancer; however, 20-50% of operations have a positive margin leading to secondary procedures. The standard of care to evaluate surgical margins is based on permanent section. Imprint cytology (touch prep) has been used to evaluate surgical samples, but conventional techniques require an experienced cytopathologist for correct interpretation. An automated image screening process has been developed to discern cancer cells from normal epithelial cells. This technique is based on cellularity of the imprint specimen and does not require expertise in cytopathology. METHODS: A rapid immunofluorescent staining technique coupled with automated microscopy was used to classify specimens as cancer vs. noncancer based on the density of epithelial cells captured on touch prep of tumor cross-sections. The results of the automated analysis vs. a manual screen of ten 20x fields were compared to the pathology interpretation on permanent section. RESULTS: A total of 34 consecutive cases were analyzed: 10 normal cases, and 24 cancer cases. The cross-section specimens for invasive cancer were correctly classified in at least 65% of the cases by using manual microscopy and at least 83% by using automated microscopy. The manual and automated microscopy correlated well for measurements of epithelial cell density (R(2)=0.64); however, the automated microscopy was more accurate. CONCLUSIONS: This preliminary study using an automated system for intraoperative interpretation does not require a cytopathologist and shows that rapid, low-resolution imaging can correctly identify cancer cells for invasive carcinoma in surgical specimens. Therefore, automated determination of cellularity in touch prep is a promising technique for future margin interpretation of breast conservation therapy.

Comparative gas sensing in cobalt, nickel, copper, zinc, and metal-free phthalocyanine chemiresistors.

The sensitivities of metallophthalocyanine (MPcs: M = Co, Ni, Cu, Zn, and H(2)) chemiresistors to vapor phase electron donors were examined using 50 nm MPc films deposited on interdigitated electrodes. Sensor responses were measured as changes in current at constant voltage. Analytes were chosen to span a broad range of Lewis base and hydrogen bond base strengths. The MPc sensor responses were correlated exponentially with binding enthalpy. These exponential fits were consistent with the van't Hoff equation and standard free energy relationships. Sensor recovery times were found to depend exponentially on binding enthalpy, in agreement with the Arrhenius equation. Relative sensitivities of all MPcs were compared via two-way ANOVA analysis. Array response patterns were differentiated via linear discriminant analysis, and analyte identification was achieved over a range of concentrations with 95.1% classification accuracy for the strong binding analytes. The ability to distinguish among different analytes, regardless of their concentration, through normalization of the responses to a reference sensor is particularly noteworthy.