Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D; community with critical information on the tools, technologies, and trends that drive the biotech industry.
Issue link: http://gen.epubxp.com/i/155541
Drug Discovery laboratory animals. Dr. Sweedler's team utilized creative cellisolation and sample-preparation methods to inventory the neuropeptides. To determine what neuropeptides affect circadian rhythm, they isolated the small region of the brain responsible for controlling this behavior and performed a comprehensive peptidomic analysis to inventory its neuropeptide content. This research found 102 endogenous In addition, the overlap between the cells was measured by using the Co-localization wizard in the IDEAS 5.0 software. The Co-localization wizard measures the Bright Detail Similarity between two images (the cross-correlation of the smaller fluorescence features), in Ch07_DAPI Ch11_Actin Ch07_DAPI 45053 Ch07_DAPI Ch11_Actin clock." The neuropeptides released from this area were collected and measured as a function of time of day and electrical stimulation. Both known circadian-rhythm-related neuropeptides and peptides with unknown roles in circadian rhythms were identifed. "Coupling this information with metabolic profling generates an amazing informational map of mammalian circadian physiology," adds Dr. Sweedler. "We develop Figure 2: DAPI shown in purple, Valley mask shown in blue, and actin signal shown in red. A) Cell conjugates with no organized immunological synapse. B) Cell conjugates with an organized immunological synapse. Ch11_Actin B. Organized Immunological Synapse Ch11_Actin 4e4 _ Ch01BF 30999 31156 2e4 _ No Immune Synapse Ch01BF 1e4 _ Ch11_actin CD19/CD3 1.2 _ 0.9 _ 0.6 _ 0.3 _ 1633 0_ 0 1.5 Bright Detail Similarity CD3/CD19 By plotting the Bright Detail Intensity of the actin in the Valley mask vs the Bright Detail Similarity of the CD3 and CD19, the number of cells with an organized immunological synapse were quantified, as shown in Figure 3. The percent of T cells in the Immune Synapse gate was 8.4% for the SEB-treated sample versus 1.6% for the control (no SEB) sample. Ch11_actin CD19/CD3 30424 3e4_ this case CD3 and CD19. Higher Bright Detail Similarity scores are associated with greater overlap between the cells and an organized immunological synapse. Conclusion Immune Synapse Immune Synapse _ Polymerization and concentration of actin at the immunological synapse results in a high local pixel intensity. A "Valley" mask operation on the DAPI image defined the region of contact between cell conjugates, as shown in Figure 2. The actin intensity was then quantified within the Valley mask. Ch07_DAPI 27586 Bright Detail Int_Valley_Actin A FlowSight imaging flow cytometer equipped with the Quantitative Imaging (QI) option was used to assess the frequency of conjugates with an organized immunological synapse. Image analysis was completed using image-based algorithms available in the IDEAS 5.0 image analysis software package. Figure 1 demonstrates how to isolate cell conjugates using the IDEAS software. A. No Organized Immunological Synapse _ The SEB-loaded APCs were incubated with human T cells purified from peripheral blood. After incubation the cells were fixed, permeabilized and labeled with CD3-PE-Texas Red (T cells, orange), CD19-AF488 (Raji B cells, green), phalloidin-AF647 (actin, red) and DAPI (nuclear stain, purple). peptides, including 33 that were previously unidentifed, and 12 posttranslational modifcations (including amidation, phosphorylation, pyroglutamylation, and acetylation). "Next, we looked at circadian neuropeptides in the functional context, by following peptide release from living neurons," continues Dr. Sweedler. They prepared thin slices of the areas of the rodent brain containing its "biological 4531 10204 Figure 3: Scatter plot of Bright Detail Intensity of the Valley mask on the actin signal vs the Bright Detail Similarity of the CD3+ signal to the CD19+ signal. Blue squares represent the control sample and Pink triangles represent the SEB treated sample. Gates representing the cells with an organized immunological synapse and no organized immunological synapse are shown. Representative images from the no synapse gate and immune synapse gate are shown. Imaging flow cytometry combines the quantitative power of large sample sizes common to flow cytometry with the information content of microscopy. This study utilized the FlowSight and its companion IDEAS data analysis software to demonstrate in an objective and statistically robust manner that there is a significant increase in number of cell conjugates with an organized immunological synapse when the APCs have been loaded with SEB. Author: Haley Pugsley, PhD is an Applications Scientist in the Biology Group at Amnis Corporation, part of EMD Millipore, in Seattle, WA, USA. Sensitive: Industry-leading fluorescence sensitivity Fast: Up to 2,000 - 5,000 cells per second Powerful: Up to 12 images of every cell directly in flow, including brightfield, darkfield, and nine fluorescence channels Easy: Simple user interface with real-time plotting, graphical gating, and powerful compensation and analysis wizards Flexible: Broad range of field upgradable options including lasers, AutoSampler and more Efficient: Up to 95% sample utilization for high yields with rare cells INSPIRE® software simplifies data acquisition Discover how Amnis can accelerate your research at www.amnis.com © 2012 Amnis Corporation. Amnis, ImageStream, FlowSight and INSPIRE are registered trademarks of Amnis Corporation. Genetic Engineering & Biotechnology News | capabilities to manipulate neurons, use these approaches to learn about the chemistry in individual cells, and determine how this relates to higher order processes such as animal behavior. As our techniques are animal independent, the next step is to apply these discoveries to human health." Tracking Changes in Protein Isoforms Gary Nelsestuen, Ph.D., professor at the University of Minnesota in Minneapolis, maintains that a growing body of evidence points to the absence of a single new clinical test that has resulted from proteomics studies. "Most 'bottom-up' methods are expensive and suffer from inconsistent protein detection. Perhaps, instead of striving to identify as many proteins as possible, we should focus on approaches that analyze just a few proteins but with excellent predictive value," he says. Dr. Nelsestuen's work focuses on MALDI-TOF analysis of intact protein isoforms. While seemingly low-tech, this approach allows the screening of a large number of samples in a reproducible and costeffcient manner. Profling of a 5,000 blood/urine sample set representing a wide variety of disease states revealed important information about biological variations in normal populations. "We found that isoform ratios are consistent for each individual," continues Dr. Nelsestuen. "At the same time, even if the individual ratio falls within the 'normal' range, a person may be still very ill. This necessitates comparison of each individual to his/ her own baseline to improve the ability to detect change in health status." Change of isoform ratio related to health outcomes is illustrated by the analysis of glycoisoforms of intact apolipoprotein C3 (ApoC3). A 1.8-fold change in the glyco-isoform ratios correlated with obesity, specifcally among subjects eligible for bariatric surgery. Bariatric surgery resulted in the rapid change of isoform distribution to that of nonobese individuals, after which the distribution was stable in each individual. Similarly, glyco-isoform ratios were indicative of chronic hepatitis C, liver cirrhosis, and sepsis. The information provided by glyco-isoform ratio changes may provide important, novel information for diagnostic, prognostic, and therapy response to metabolic conditions. However, it requires monitoring of each patient over time. "Our method was suffcient to provide clinically important information for monitoring kidney transplants," says Dr. Nelsestuen. "The health of transplanted kidneys is often monitored by frequent biopsies. A non-invasive urine analysis for anomalous protein biomarkers would provide signifcant advantage over current clinical practice." Remarkably, two ubiquitous isoforms of saposin B, thought to be an activator of lipid degradation, were low or absent in patients with advanced kidney disease, while a number of other components appeared. The team is combining this data with metabolomics profling to identify early diagnostic biomarkers. GENengnews.com | September 1, 2013 | 23