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Drug Discovery 22 | Central nervous system neuropeptides are involved in many physiological processes such as circadian rhythms, pain, hunger, feeding, and body-weight regulation. Some neuropeptides undergo functionally important posttranslational modifcations. Coupled with their small size (3–50 amino "To do that, we develop analytical methods to follow chemical and quantitative changes in neuropeptides alongside with their distribution and dynamics." Neuropeptides impact many signaling pathways and are highly conserved in the animal kingdom. This allows for discovery and validation to be done on well-defned neuronal networks in the simpler nervous systems of marine invertebrates and other ADV E RTO R I A L Figure 1: Gating strategy to isolate conjugates for immunological synapse analysis. 0.9 _ _ _ _ _ 1e3 1.5e3 Step 3: Plot Aspect Ratio vs Area of the CD19 signal from the double positive population to isolate doublets with only one CD19+ cell. Double Positive 0.6 _ 0.4 _ 0.2 _ 0_ 0 100 200 300 _ 1e4 _ Single CD3+ _ Aspect Ratio _CD3_PE-TR 1e5 _ 1_ 0.8_ _ 1.5e3 Area_BF 1e3 _ 0_ -1e3 _ 500 _ 1e3 _ 500 Step 1: Plot the Aspect Ratio vs Area of the brightfield image to identify doublets. Cytometry without limits 0 Area_CD19_AF488 _ _ _ 0 In this experiment, Raji B cells were loaded with Staphylococcal enterotoxin B (SEB) to make APCs. Single CD19+ 0.6 _ Doublets 0.30 _ Quantifying the frequency of organized immunological synapse 0.9 _ 0.30 _ 0.6 _ 400 Area _CD3_PE-TR -1e3 0 1e3 1e4 _ _ -1e4 _ _ Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging via confocal or conventional fluorescence microscopy. However, immunological synapses are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. In this study, the Amnis® FlowSight® imaging flow cytometry platform was employed to collect imagery of large numbers of cells to assess the percentage of T cells involved in an organized immunological synapse. Aspect Ratio _CD19_AF488 Measuring Immunological Synapse Using the FlowSight Imaging Flow Cytometer _ The holy grail of protein profling is to determine comprehensively the identity, abundance, and isoforms of proteins in a large number of clinical samples, maintains Jun Qu, Ph.D, associate professor at the University of Buffalo, SUNY. "Biomedical discovery necessitates comparison of multiple samples to overcome known problems with individual variability. However, accurate and reliable profling of multiple samples remains challenging," he explains. "We developed a highly reproducible and quantitative method that can detect proteomic signatures despite a highly variable biological background." The "precipitation/on-pellet-digestion" method relies on a unique detergent cocktail to achieve a near complete extraction, including that of membrane proteins. Addition of organic solvents, like acetone, denatures and aggregates the pellet, which is notoriously diffcult to re-solubilize. "Our sample prep philosophy is the simpler, the better," continues Dr. Qu. "We let the enzymes digest the proteins right on the pellet. This method produces a clean peptide mixture suitable for LC-MS analysis." The team designed a unique separation column that features low void volume, large loading capacity, and high separation effciencies. Peptides are separated on a heated, 100-cm-long column during a seven-hour run. The team determined that a label-free MS approach was the most cost-effcient method. It provided excellent accuracy for relative protein quantitation when well controlled. The process was previously applied to several clinically important questions, such as fnding underlying causes of hibernating myocardium and chronic obstructive pulmonary disease. The most recent research focused on HIV. "Five percent of HIV carriers never progress to a disease state," says Xiaomeng Shen, a Ph.D. candidate in Dr Qu's lab. "Proteomic comparison of normal progressors vs. long-term nonprogressors (LTNPs) provides highly valuable insights for directing therapeutic efforts." Comparison of 10 individuals from each cohort resulted in the identifcation of 78 differentially expressed proteins. Subsequent annotation by Ingenuity Pathway Analysis software pointed to several signaling pathways that may account for viral control mechanisms in LTNPs. "We were excited to fnd the SAMHD1 protein in the interferon pathway, which was recently implicated as a critical player in viral restriction," remarks Dr. Qu, who notes that it would take many years to generate the same Cell-to-Cell Communication acids) and low concentrations in the CNS, these neuropeptides pose an analysis challenge. Measurements based on MS are well suited for neuropeptide research, simultaneously deducing structural and quantitative information about low-quantity compounds. "We are interested in knowing how the brain works," says Jonathan Sweedler, Ph.D., professor at the University of Illinois, UrbanaChampaign. _ _ Reliable Proteomic Analysis amount of data by the traditional methods. Aspect Ratio _BF with an increased risk of developing pancreatic cancer, either genetically or due to smoking or diabetes. A similar approach was used to detect breast cancer signatures. To capture a larger percentage of the proteome, researchers used two types of functionalized beads and combined the resulting data. Meanwhile, the research continues to improve the method to make it ready for clinical use. Continued from page 21 Intensity _CD3_PE-TR Protein Profling 1e5 Intensity _CD19_AF488 Step 2: Find cells that are positive for both CD3 (orange) and CD19 (green). Step 4: Plot the Aspect Ratio vs Area of the CD3 signal from the doublets with only one CD19+ cell to isolate doubles with only one CD3+ cell. This final population consists of doublets with only one CD19+ Raji B cell and one CD3+ T cell. Extend the Range of Your Research The Amnis® FlowSight® and ImageStreamX Mark II cytometers combine the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. This unique combination enables a broad range of applications that would be impossible using either technique alone. ImageStreamX Mark II September 1, 2013 | GENengnews.com | Genetic Engineering & Biotechnology News FlowSight The FlowSight cytometer provides up to 4 lasers and 12 images of every cell for visual verification and quantitative image analysis. The ImageStreamX Mark II delivers high-resolution imagery at up to 60X magnification for more demanding applications, as well as additional lasers, options, and capabilities.