Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
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In JoVE (1)
Other Publications (6)
Articles by Todd Huffman in JoVE
Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University
The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
Other articles by Todd Huffman on PubMed
Insulin-stimulated Phosphorylation of Lipin Mediated by the Mammalian Target of Rapamycin
The phosphorylation of a previously uncharacterized protein of apparent M(r) approximately 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy (fld) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121-124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent M(r) from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.
Selective Attention Affects Human Brain Stem Frequency-following Response
Selective attention modifies long-latency cortical event-related potentials. Amplitudes are typically enhanced and/or latencies reduced when evoking stimuli are attended. However, there is controversy concerning the effects of selective attention on short-latency brain stem evoked potentials. The objective of the present study was to assess possible attention effects on the brain stem auditory frequency-following response (FFR) elicited by a periodic tone. Young adult subjects heard a repetitive auditory stimulus while detecting infrequent target stimuli in either an auditory or visual detection task. Five channels of high frequency electroencephalographic (EEG) activity were recorded along the scalp midline with the center electrode positioned at the vertex. The FFR was elicited by the repetitive tone during both tasks. There were significant individual differences in the electrode sites yielding maximum response amplitudes, but overall FFR amplitudes were significantly larger during the auditory attention task. These results suggest that selective attention in humans can modify signal processing in sensory (afferent) pathways at the level of the brain stem. This may reflect top-down perceptual preprocessing mediated by extensive descending (efferent) pathways that originate in the cortex. Overall, the FFR appears to be a robust indicator of early auditory neural processing and shows effects not seen in brain stem auditory evoked response studies employing transient (click) acoustic stimuli.
The Arrest of Biological Time As a Bridge to Engineered Negligible Senescence
Biological systems can remain unchanged for several hundred years at cryogenic temperatures. In several hundred years, current rapid scientific and technical progress should lead to the ability to reverse any biological damage whose reversal is not forbidden by physical law. We therefore explore whether contemporary people facing terminal conditions might be preserved well enough today for their eventual recovery to be compatible with physical law. The ultrastructure of the brain can now be excellently preserved by vitrification, and solutions needed for vitrification can now be distributed through organs with retention of organ viability after transplantation. Current law requires a few minutes of cardiac arrest before cryopreservation of terminal patients, but dogs and cats have recovered excellent brain function after 16-60 min of complete cerebral ischemia. The arrest of biological time as a bridge to engineered negligible senescence, therefore, appears consistent with current scientific and medical knowledge.
Insulin Controls Subcellular Localization and Multisite Phosphorylation of the Phosphatidic Acid Phosphatase, Lipin 1
Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.
Telemedicine for Audiology Screening of Infants
Distortion product otoacoustic emissions (DPOAE) and automated auditory brainstem response (AABR) screening were conducted in infants at a distant hospital using remote computing. Eighteen males and twelve females ranging in age from 11-45 days were tested. Both DPOAE and AABR data were recorded using an integrated test system which was connected to the computer network at the Utah Valley Regional Medical Center. Using a broadband Internet connection, an examiner at Utah State University, 200 km away, could control the DPOAE and the ABR equipment. Identical hearing screening results were obtained for face-to-face and telemedicine trials with all infants. The DPOAE means for face-to-face and telemedicine trials were not significantly different at any frequency. In an analysis of variance, there was no significant difference for the test method (F = 0.8, P > 0.05). These results indicate that remote computing is a feasible telemedicine method for providing DPOAE and ABR hearing screening services to infants in rural communities.
Multiscale Exploration of Mouse Brain Microstructures Using the Knife-edge Scanning Microscope Brain Atlas
Connectomics is the study of the full connection matrix of the brain. Recent advances in high-throughput, high-resolution 3D microscopy methods have enabled the imaging of whole small animal brains at a sub-micrometer resolution, potentially opening the road to full-blown connectomics research. One of the first such instruments to achieve whole-brain-scale imaging at sub-micrometer resolution is the Knife-Edge Scanning Microscope (KESM). KESM whole-brain data sets now include Golgi (neuronal circuits), Nissl (soma distribution), and India ink (vascular networks). KESM data can contribute greatly to connectomics research, since they fill the gap between lower resolution, large volume imaging methods (such as diffusion MRI) and higher resolution, small volume methods (e.g., serial sectioning electron microscopy). Furthermore, KESM data are by their nature multiscale, ranging from the subcellular to the whole organ scale. Due to this, visualization alone is a huge challenge, before we even start worrying about quantitative connectivity analysis. To solve this issue, we developed a web-based neuroinformatics framework for efficient visualization and analysis of the multiscale KESM data sets. In this paper, we will first provide an overview of KESM, then discuss in detail the KESM data sets and the web-based neuroinformatics framework, which is called the KESM brain atlas (KESMBA). Finally, we will discuss the relevance of the KESMBA to connectomics research, and identify challenges and future directions.