Genetic Engineering & Biotechnology News

DEC 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | DECEMBER 2017 | 17 rates of cellular proliferation. Using XF tech- nology to noninvasively measure glycolytic rate, activation of T cells after stimulation with anti-CD3/anti-CD28 beads is evident within minutes (Figure 1). This rapid in- crease in glycolytic rate has been observed in both CD4 + and CD8 + T-cell subsets. Conventionally, the progression of T-cell activation is measured in terms of changes in cell size/morphology, interleukin/interferon expression, and/or lactate efflux. The in- crease in glycolytic rate observed in this study is directly correlated with these approaches. Importantly, modulating T-cell activation by adding inhibitors of the glycolytic pathway also results in decreased activation when measured using alternative approaches. Quantify Every Phase of Macrophage Activation Macrophages and dendritic cells also ex- hibit an immediate glycolytic response to pathogenic stimuli. Similarly, as in the acti- vated T-cell example above, glycolysis is re- quired to support the high energy and bio- synthetic demands of activated macrophages. However, in some macrophage cell types this activation is biphasic. The initial phase consists of a sustained increase in glycolytic rate with little to no impact on mitochon- drial function. The second phase, occurring several hours later, is typified by a further in- creased glycolytic rate and a marked decrease in mitochondrial function. An example of this finding in the RAW 264.7 macrophage cell line is shown in Figure 2. In work by the lab of Edward Pearce, Ph.D., of the Max Planck Institute of Im- munobiology and Epigenetics, in Freiburg, Germany, this second phase was shown to be dependent on the production of nitric oxide. 2 Nitric oxide is a well-known inhibitor of mi- tochondrial function, and in activated macro- phages is produced by the enzyme-inducible nitric oxide synthase. Increased expression of this enzyme requires new protein synthesis— the timing of which contributes to the multi- phasic nature of the response of macrophages to activation stimuli. By correlating this real- time functional information to orthogonal bi- ological data, we have gained greater insight into the causes and metabolic implications of macrophage activation. Beyond Activation: Metabolic Fuels Drive Immune-Cell Fate There are now several examples of long- lived cell types selectively choosing to oxidize fatty acids as a fuel source. While the under- lying reason for this remains poorly under- stood, it has become clear that this "choice" to burn fat is, in fact, a requirement for the long-lived cell phenotype. Nowhere is this more evident than in the long-lived memory T-cell subpopulation that is retained follow- ing pathogen clearance. Now researchers are using this knowledge to identify mechanisms that drive T cell longevity to translate it to the clinical research setting. CAR-T cells are T cells which have been transduced to express a synthetic cell-surface receptor. The extracellular portion of this syn- thetic receptor is targeted to a specific antigen of interest, and the intracellular portion is comprised of several signaling domains which translate the ligation of the external portion to intracellular activity. Recent work from the lab of Carl June, M.D., at the University of Penn- sylvania has demonstrated that the intracellu- lar signaling component is a key regulator of the long-lived central memory phenotype. 3 It is not yet clear if fatty acid oxidation is sup- porting this long-lived phenotype in the CAR- T cells. However, there is evidence from the lab of Erika Pearce, Ph.D., at the Max Planck In- stitute, that fatty acid metabolism is critical not just for the function of this cell type, but for the creation of these cells in the first place. Without Real-Time Kinetics, What Are Investigators Missing? The vignettes highlighted here demonstrate that both short- and long-term kinetic analy- ses provide valuable information and mecha- nistic detail that would be missed by using any other approach. Researchers are increasingly focused on early events in immune-cell acti- vation, where the response to an inflamma- tory signal can be tuned to impact overall cell function. In research areas such as immuno- oncology, increased activation is connected to improved cell expansion and overall cellular health; whereas in the field of immunosup- pression, the converse is desired. As the relevance of immune-cell function grows, researchers are continuously seeking tools that can enable a more refined study of the kinetics of these dynamic cell types. Agilent XF technology is uniquely poised to offer a robust, real-time view of activation, enabling the study of compounds or treat- ments modulating effector function. OMICS Assay Tutorial Insights Genomics & Proteomics Understanding the molecular intricacies of cancer is the gateway to unlocking new therapeutics and treatment strategies. With that approach in mind, SeqLL announced recently that it has installed a true Single Molecule Sequencing (tSMS) Instrument at the Harvard-MGH Cancer Center to support oncology research conducted by David Ting, M.D., assistant pro- fessor of medicine at Harvard Medical School, who is a medical oncologist with a focus on gastrointestinal malignancies. Dr. Ting and his laboratory utilize RNA- sequencing and RNA in situ hybridization technolo- gies to better understand the complex transcriptional landscape of cancers. The laboratory has employed these technologies to identify noncoding RNA (ncRNA) sequences that are differentially expressed in cancer versus normal tissues. This work led to the identification of repeat ncRNAs that were aberrantly expressed across all major epithe- lial cancers and had opened the door to a new area of research that can identify novel biomarkers and thera- peutic targets. "SeqLL is extremely pleased that Dr. Ting has cho- sen the tSMS platform to continue his research into pancreatic cancer," noted Elizabeth Reczek, Ph.D., CEO of SeqLL. "David is conducting groundbreaking research, and SeqLL is excited to be able to provide the level of accuracy, reproducibility and unbiased molecule counting capabilities necessary for this work." Accurate and reproducible RNA transcriptome analysis has traditionally been challenging for next- generation sequencing platforms due to errors as- sociated with reverse transcription, amplification, and manipulations during library preparation. The detection of low-fold changes and identification of rare transcripts remains difficult on these platforms, particularly when using FFPE and other degraded or low-input sample types. SeqLL tSMS technology sequences individual strands of first strand cDNA or RNA directly, without amplification or library prep, providing accurate RNA sequence information that reflects the true information content of each sample without introduced bias or sample loss. n Harvard-MGH Cancer Center Gets Single-Molecule Sequencing Instrument Brian P. Dranka, Ph.D. (brian.dranka@ agilent.com), is manager of biology, cell analysis division; and Luke Dimasi (luke. dimasi@agilent.com) is manager of global product marketing and technical support, cell analysis division, at Agilent Technologies. Website: www.agilent.com/ chem/immunology. References available online. Figure 1. Rapid detection of T cell activation using Agilent XF technology. Human naïve CD4+ T cells were measured using an Agilent Seahorse XFp Analyzer. Activating anti-CD3/CD28 beads were injected as indicated at the arrow. Proton Efflux Rate (PER) was monitored for an additional 2 hours. Figure 2. Discrimination of multiple phases of macrophage activation. Murine immortalized macrophages (RAW 264.7) were measured using an Agilent Seahorse XFe96 Analyzer. A mixture of LPS (100 ng/mL) and IFNγ (20 ng/mL) were injected at the arrow. Proton Efflux Rate was monitored for an additional 9.5 hours.

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