Creating physical 3D stereolithograph models of brain and skull

The human brain and skull are three dimensional (3D) anatomical structures with complex surfaces. However, medical images are often two dimensional (2D) and provide incomplete visualization of structural morphology. To overcome this loss in dimension, we developed and validated a freely available, semi-automated pathway to build 3D virtual reality (VR) and hand-held, stereolithograph models. To evaluate whether surface visualization in 3D was more informative than in 2D, undergraduate students (n = 50) used the Gillespie scale to rate 3D VR and physical models of both a living patient-volunteer's brain and the skull of Phineas Gage, a historically famous railroad worker whose misfortune with a projectile tamping iron provided the first evidence of a structure-function relationship in brain. Using our processing pathway, we successfully fabricated human brain and skull replicas and validated that the stereolithograph model preserved the scale of the VR model. Based on the Gillespie ratings, students indicated that the biological utility and quality of visual information at the surface of VR and stereolithograph models were greater than the 2D images from which they were derived. The method we developed is useful to create VR and stereolithograph 3D models from medical images and can be used to model hard or soft tissue in living or preserved specimens. Compared to 2D images, VR and stereolithograph models provide an extra dimension that enhances both the quality of visual information and utility of surface visualization in neuroscience and medicine.     

SHG nanoprobes: advancing harmonic imaging in biology

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.     

hERG1a/1b heteromeric currents exhibit amplified attenuation of inactivation in variant 1 short QT syndrome

Potassium channels encoded by hERG (human ether-à-go-go-related gene) underlie the cardiac rapid delayed rectifier K⁺ current (I_Kr) and hERG mutations underpin clinically important repolarization disorders. Virtually all electrophysiological investigations of hERG mutations have studied exclusively the hERG1a isoform; however, recent evidence indicates that native I_Kr channels may be comprised of hERG1a together with the hERG1b variant, which has a shorter N-terminus. Here, for the first time, electrophysiological effects were studied of a gain-of-function hERG mutation (N588K; responsible for the ‘SQT1’ variant of the short QT syndrome) on current (I_hERG1a/1b) carried by co-expressed hERG1a/1b channels. There were no significant effects of N588K on I_hERG1a/1b activation or deactivation, but N588K I_hERG1a/1b showed little inactivation up to highly positive voltages (?+80 mV), a more marked effect than seen for hERG1a expressed alone. I_hERG1a/1b under action potential voltage-clamp, and the effects on this of the N588K mutation, also showed differences from those previously reported for hERG1a. The amplified attenuation of I_hERG inactivation for the N588K mutation reported here indicates that the study of co-expressed hERG1a/1b channels should be considered when investigating clinically relevant hERG channel mutations, even if these reside outside of the N-terminus region.     

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

Fluoxetine is one of the most commonly prescribed medications for many behavioral and neurological disorders. Fluoxetine acts primarily as an inhibitor of the serotonin reuptake transporter (SERT) to block the removal of serotonin from the synaptic cleft, thereby enhancing serotonin signals. While the effects of fluoxetine on behavior are firmly established, debate is ongoing whether inhibition of serotonin reuptake is a sufficient explanation for its therapeutic action. Here, we provide evidence of two additional aspects of fluoxetine action through genetic analyses in Caenorhabditis elegans. We show that fluoxetine treatment and null mutation in the sole SERT gene mod-5 eliminate serotonin in specific neurons. These neurons do not synthesize serotonin but import extracellular serotonin via MOD-5/SERT. Furthermore, we show that fluoxetine acts independently of MOD-5/SERT to regulate discrete properties of acetylcholine (Ach), gamma-aminobutyric acid (GABA), and glutamate neurotransmission in the locomotory circuit. We identified that two G-protein–coupled 5-HT receptors, SER-7 and SER-5, antagonistically regulate the effects of fluoxetine and that fluoxetine binds to SER-7. Epistatic analyses suggest that SER-7 and SER-5 act upstream of AMPA receptor GLR-1 signaling. Our work provides genetic evidence that fluoxetine may influence neuronal functions and behavior by directly targeting serotonin receptors.FLUOXETINE is a selective serotonin reuptake inhibitor (SSRI) and has made a major impact on the treatment of many behavioral disorders. The empirical action of SSRIs is blocking the serotonin reuptake transporter (SERT). SERT is localized in the plasma membrane and transports extracellular serotonin (5-HT) into the cytoplasm (Blakely et al. 1991; Hoffman et al. 1991), this being the major mechanism of terminating 5-HT signaling. Consequently, SSRIs are thought to exert therapeutic effects by blocking SERT from removal of 5-HT in the synaptic clef, thereby increasing the level of 5-HT signals (Schatzberg and Nemeroff 2004). However, several observations point to additional actions of SSRIs on the 5-HT system and neuronal functions. First, knockout of SERT in mouse caused a marked reduction of 5-HT in the brain (Bengel et al. 1998). Second, a variety of studies with cultured mammalian cells and mouse brain slices showed that SSRIs and tricyclic antidepressant agents (TCAs) have high affinities to many 5-HT receptor subtypes and act as agonists or antagonists depending on particular receptors being tested (Ni and Miledi 1997; Kroeze and Roth 1998; Eisensamer et al. 2003). Third, genetic analyses of the nematode Caenorhabditis elegans in our laboratory and others showed that fluoxetine and the TCA imipramine (Tofrani) could influence behavior independent of SERT function (Weinshenker et al. 1995; Ranganathan et al. 2001; Dempsey et al. 2005). In this study, we carried out a systematic survey of SSRIs treatment in C. elegans to gain new insights into actions of SSRIs on the 5-HT system and other neurotransmitter systems.In both vertebrates and invertebrates, 5-HT functions as a neuromodulator to either facilitate or inhibit synaptic transmission of other neurotransmitters (Fink and Gothert 2007). Modulation of synaptic activity by 5-HT signaling underscores the synaptic plasticity involved in stress responses, learning, adaptation, and memory (Kandel 2001; Zhang et al. 2005). The role of 5-HT in C. elegans was initially identified through pharmacological experiments showing that exogenous 5-HT can promptly induce changes in a variety of behaviors, including feeding, egg laying, and locomotion (Avery and Horvitz 1990; Weinshenker et al. 1995; Nurrish et al. 1999). The relevance of these behaviors to endogenous 5-HT has since been validated through studies of mutants of 5-HT signaling. Importantly, multiple 5-HT receptors may function in distinct cells synergistically or antagonistically to regulate a specific behavior (Carnell et al. 2005; Dernovici et al. 2007; Murakami and Murakami 2007; Hapiak et al. 2009). In nearly all tested paradigms, fluoxetine and imipramine induce behavioral changes similarly to exogenous 5-HT (Weinshenker et al. 1995; Nurrish et al. 1999), implying that fluoxetine regulates 5-HT inputs to these neural circuits. However, the tryptophan hydroxylase gene tph-1 is required for 5-HT biosynthesis in C. elegans (Sze et al. 2000), mod-5 encodes its sole SERT (Ranganathan et al. 2001), and yet fluoxetine could stimulate egg laying and inhibit locomotion in mod-5 and tph-1 mutants (Weinshenker et al. 1995; Choy and Thomas 1999; Ranganathan et al. 2001; Dempsey et al. 2005). These findings provided a basis for further investigation into genes and synaptic functions regulated by 5-HT and the impact of fluoxetine on 5-HT signaling.Here we present genetic evidence of multifaceted effects of fluoxetine on the 5-HT system and its downstream targets in C. elegans. We show that fluoxetine treatment and loss of MOD-5/SERT function do not simply increase presynaptic 5-HT signals. Rather, they may eliminate 5-HT in specific neurons. Furthermore, fluoxetine acts independently of SERT to regulate 5-HT serotonin receptors and their downstream targets involved in acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glutamate neurotransmission.     

Genomic Deletion Marking an Emerging Subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.     

Human tribbles, a protein family controlling mitogen-activated protein kinase cascades

Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.     

Basolateral sorting of transforming growth factor-alpha precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants

In polarized Madin-Darby canine kidney (MDCK) cells, newly synthesized transforming growth factor-alpha precursor (proTGFalpha) is directly sorted to the basolateral cell surface where it is sequentially cleaved and released into the basolateral conditioned medium (Dempsey, P.J., Coffey, R.J., J. Biol. Chem. 269 (1994) 16878-16889). In the present study, the role of the proTGFalpha cytoplasmic domain in basolateral sorting has been investigated using deletional and site-directed mutagenesis, as well as chimeric analyses of different TGFalpha constructs stably expressed in MDCK cells. The loss of polarized secretion of a proTGFalpha secretory mutant (TGFsec88) indicated that the proTGFalpha transmembrane and/or cytoplasmic domains contain essential basolateral sorting information. Using reporter chimeras with two apically sorted membrane proteins, p75 neurotrophin growth factor receptor and placental alkaline phosphatase, we show that the proTGFalpha cytoplasmic domain contains dominant basolateral sorting information. Analysis of proTGFalpha cytoplasmic domain truncation and internal deletion mutants, together with site-directed mutagenesis studies within the full-length proTGFalpha cytoplasmic domain, revealed redundant basolateral sorting motifs. Importantly, the C-terminal type I PDZ-binding motif was not required for basolateral sorting as determined by the integrity of basolateral sorting in deletion mutants lacking this motif. ProTGFalpha basolateral sorting may have important consequences for ligand presentation and spatial compartmentalization of epidermal growth factor receptor signaling networks in polarized epithelial cells.     

Aryl[a]pyrrolo[3,4-c]carbazoles as selective cyclin D1-CDK4 inhibitors

The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.