“Pixelated Spatial Gene Expression Analysis from Tissue”
Anurup Ganguli, a Ph.D. student in bioengineering, 3D Micro- and Nanosystems Group
We present a technique that performs on-chip picoliter real-time reverse transcriptase loop mediated isothermal amplification (RT-LAMP) reactions on a histological tissue section without any analyte purification while preserving the native spatial location of the nucleic acid molecules. We demonstrate this method by amplifying TOP2A messenger RNA (mRNA) in a prostate cancer xenograft with 100µm spatial resolution and by visualizing the variation in threshold time of amplification across the tissue. The on-chip reaction was validated by mRNA fluorescence in situ hybridization (mFISH) from cells in the tissue section. The entire process, from tissue loading on microchip to results from RT-LAMP can be carried out in less than two hours. We anticipate that this technique with its ease of use, fast turnaround, and quantitative molecular outputs, would become an invaluable tissue analysis tool for researchers and clinicians in the biomedical arena.
“cFLIP Inhibits Interferon Expression Controlled by the Regulatory Factor, IRF7”
Lauren T. Gates-Tanzer, a Ph.D. student in microbiology, Bioacoustics Research Laboratory
IFNa is important for anti-viral and anti-cancer defenses. However, over-production is associated with autoimmune disorders. Thus, the cell must precisely up- and down-regulate IFNa to achieve immune system homeostasis. The cellular FLICE-like inhibitory protein (cFLIP) is reported to inhibit IFNa production. However, the mechanism for this antagonism remained unknown. The goal was to identify this mechanism. Here, we examined the signal transduction events that occur during TLR9-induced IRF7 activation. cFLIPL inhibited the expression of IRF7-controlled natural or synthetic genes in several cell lines, including those with abundant IRF7 protein levels (e.g., dendritic cells). cFLIPL inhibited IRF7 phosphorylation, however cFLIPL-IRF7 interactions were not detectable, implying that cFLIPL acted upstream of IRF7 dimerization. Interestingly, cFLIPL co-immunoprecipitated with IKKa, and these interactions correlated with a loss of IKKa-IRF7 interactions. Thus, cFLIP appears to bind to IKKa to prevent IKKa from phosphorylating and activating IRF7. To the best of our knowledge, this is the first report of a cellular protein that uses this approach to inhibit IRF7 activation. Perhaps this cFLIP property could be engineered to minimize the deleterious effects of IFNa expression that occur during certain autoimmune disorders.
“Biosynthesis of D-amino Acid-containing Peptides in the Nervous System"
Hua-Chia Tai, chemistry, Cellular and Molecular Foundations of Intelligent Behavior
Neuropeptides act as cell-to-cell signaling molecules in the regulation of various physiological processes. Classical neuropeptides are produced from protein precursors made via ribosomal synthesis, and many of these peptides undergo post-translational modifications that are crucial for their bioactivity. Importantly, post-translational conversion of an L-amino acid residue into a D-residue in a neuropeptide can substantially alter the shape, modify the bioactivity and change the metabolism of the resulting D-amino acid-containing peptide (DAACP). Current research into L/D peptide isomerization is limited because this modification is not easily detected in mass spectrometry-based peptidomics due to the lack of an associated mass shift. We previously developed protocols to screen for DAACPs in a non-targeted manner and used them to identify three novel DAACPs in Aplysia californica. Our results suggest that the peptide isomerase responsible for the L-to-D modification in this animal works only on the second residue from the N-terminus but is promiscuous in terms of peptide length as well as the specific amino acid to isomerize. Here we describe efforts to identify and characterize this enzyme through activity-guided protein purification using a liquid chromatography-mass spectrometry-based assay. This is the first characterization of a neuronal enzyme that catalyzes L/D-peptide isomerization.
