protaTETHER - A Method for the Incorporation of Variable Linkers in Protein Fusions Reveals Impacts of Linker Flexibility in a PKAc-GFP Fusion Protein

Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression

Infrared light excites cells by changing their electrical capacitance

Optical stimulation has enabled important advances in the study of brain function and other biological processes, and holds promise for medical applications ranging from hearing restoration to cardiac pace making. In particular, pulsed laser stimulation using infrared wavelengths >1.5 μm has therapeutic potential based on its ability to directly stimulate nerves and muscles without any genetic or chemical pre-treatment. However, the mechanism of infrared stimulation has been a mystery, hindering its path to the clinic. Here we show that infrared light excites cells through a novel, highly general electrostatic mechanism. Infrared pulses are absorbed by water, producing a rapid local increase in temperature. This heating reversibly alters the electrical capacitance of the plasma membrane, depolarizing the target cell. This mechanism is fully reversible and requires only the most basic properties of cell membranes. Our findings underscore the generality of pulsed infrared stimulation and its medical potential

SMF-1, SMF-2 and SMF-3 DMT1 Orthologues Regulate and Are Regulated Differentially by Manganese Levels in C. elegans

Manganese (Mn) is an essential metal that can exert toxic effects at high concentrations, eventually leading to Parkinsonism. A major transporter of Mn in mammals is the divalent-metal transporter (DMT1). We characterize here DMT1-like proteins in the nematode C. elegans, which regulate and are regulated by Mn and iron (Fe) content. We identified three new DMT1-like genes in C. elegans: smf-1, smf-2 and smf-3. All three can functionally substitute for loss of their yeast orthologues in S. cerevisiae. In the worm, deletion of smf-1 or smf-3 led to an increased Mn tolerance, while loss of smf-2 led to increased Mn sensitivity. smf mRNA levels measured by QRT-PCR were up-regulated upon low Mn and down-regulated upon high Mn exposures. Translational GFP-fusions revealed that SMF-1 and SMF-3 strongly localize to partially overlapping apical regions of the gut epithelium, suggesting a differential role for SMF-1 and SMF-3 in Mn nutritional intake. Conversely, SMF-2 was detected in the marginal pharyngeal epithelium, possibly involved in metal-sensing. Analysis of metal content upon Mn exposure in smf mutants revealed that SMF-3 is required for normal Mn uptake, while smf-1 was dispensable. Higher smf-2 mRNA levels correlated with higher Fe content, supporting a role for SMF-2 in Fe uptake. In smf-1 and smf-3 but not in smf-2 mutants, increased Mn exposure led to decreased Fe levels, suggesting that both metals compete for transport by SMF-2. Finally, SMF-3 was post-translationally and reversibly down-regulated following Mn-exposure. In sum, we unraveled a complex interplay of transcriptional and post-translational regulations of 3 DMT1-like transporters in two adjacent tissues, which regulate metal-content in C. elegans

Rebound Discharge in Deep Cerebellar Nuclear Neurons In Vitro

Neurons of the deep cerebellar nuclei (DCN) play a critical role in defining the output of cerebellum in the course of encoding Purkinje cell inhibitory inputs. The earliest work performed with in vitro preparations established that DCN cells have the capacity to translate membrane hyperpolarizations into a rebound increase in firing frequency. The primary means of distinguishing between DCN neurons has been according to cell size and transmitter phenotype, but in some cases, differences in the firing properties of DCN cells maintained in vitro have been reported. In particular, it was shown that large diameter cells in the rat DCN exhibit two phenotypes of rebound discharge in vitro that may eventually help define their functional roles in cerebellar output. A transient burst and weak burst phenotype can be distinguished based on the frequency and pattern of rebound discharge immediately following a hyperpolarizing stimulus. Work to date indicates that the difference in excitability arises from at least the degree of activation of T-type Ca2+ current during the immediate phase of rebound firing and Ca2+-dependent K+ channels that underlie afterhyperpolarizations. Both phenotypes can be detected following stimulation of Purkinje cell inhibitory inputs under conditions that preserve resting membrane potential and natural ionic gradients. In this paper, we review the evidence supporting the existence of different rebound phenotypes in DCN cells and the ion channel expression patterns that underlie their generation

Effects of oxygen concentration on the proliferation and differentiation of mouse neural stem cells in vitro.

BACKGROUND AND PURPOSE: Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. METHODS: NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. RESULTS: The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. CONCLUSIONS: These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation

Highly enantioselective copper(I)-catalyzed conjugate addition of 1,3-diynes to a,b-unsaturated trifluoromethyl ketones

[EN] The conjugate diynylation of a,b-unsaturated trifluoromethyl ketones is carried out in the presence of a low catalytic load (2.5 mol%) of a copper(I)–MeOBIPHEP complex, triethylamine and a terminal 1,3-diyne. Pre-metalation of the terminal 1,3-diyne with stoichiometric or higher amounts of dialkylzinc reagent is not required. The corresponding internal diynes bearing a propargylic stereogenic center are obtained with good yields and excellent enantioselectivities.Financial support from the Ministerio de Economia y Competitividad (MINECO-Gobierno de Espana) and FEDER (EU) (CTQ2013-47494-P) and from Generalitat Valenciana (ISIC2012/001) is gratefully acknowledged. A.S.-M. thanks the MINECO for a predoctoral grant (FPI program). Access to NMR and MS facilities from the Servei Central de Suport a la Investigacio Experimental (SCSIE)-UV is also acknowledged.Sanz-Marco, A.; Blay, G.; Muñoz Roca, MDC.; Pedro, J. (2015). 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Highly Enantioselective Copper(I)-Catalyzed Conjugate Addition of Terminal Alkynes to 1,1-Difluoro-1-(phenylsulfonyl)-3-en-2-ones: New Ester/Amide Surrogates in Asymmetric Catalysis. Chemistry - A European Journal, 20(3), 668-672. doi:10.1002/chem.201303920Nishimura, T., Guo, X.-X., Uchiyama, N., Katoh, T., & Hayashi, T. (2008). Steric Tuning of Silylacetylenes and Chiral Phosphine Ligands for Rhodium-Catalyzed Asymmetric Conjugate Alkynylation of Enones. Journal of the American Chemical Society, 130(5), 1576-1577. doi:10.1021/ja710540sNishimura, T., Sawano, T., & Hayashi, T. (2009). Asymmetric Synthesis of β-Alkynyl Aldehydes by Rhodium-Catalyzed Conjugate Alkynylation. Angewandte Chemie International Edition, 48(43), 8057-8059. doi:10.1002/anie.200904486Fillion, E., & Zorzitto, A. K. (2009). Enantioselective Rhodium-Catalyzed Conjugate Alkynylation of 5-Benzylidene Meldrum’s Acids with TMS-acetylene. Journal of the American Chemical Society, 131(41), 14608-14609. doi:10.1021/ja905336pBlay, G., Cardona, L., Pedro, J. R., & Sanz-Marco, A. (2012). Enantioselective Zinc-Mediated Conjugate Addition of Terminal Alkynes to Enones. Chemistry - A European Journal, 18(41), 12966-12969. doi:10.1002/chem.201201765Blay, G., Muñoz, M. C., Pedro, J. R., & Sanz-Marco, A. (2013). Enantioselective Synthesis of 4-Substituted Dihydrocoumarins through a Zinc Bis(hydroxyamide)-Catalyzed Conjugate Addition of Terminal Alkynes. Advanced Synthesis & Catalysis, 355(6), 1071-1076. doi:10.1002/adsc.201201120Cui, S., Walker, S. D., Woo, J. C. S., Borths, C. J., Mukherjee, H., Chen, M. J., & Faul, M. M. (2010). Practical Asymmetric Conjugate Alkynylation of Meldrum’s Acid-Derived Acceptors: Access to Chiral β-Alkynyl Acids. Journal of the American Chemical Society, 132(2), 436-437. doi:10.1021/ja909105sKwak, Y.-S., & Corey, E. J. (2004). 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Chemical Communications, 47(48), 12873. doi:10.1039/c1cc15968bLiu, T.-L., Zhang, H.-X., Zheng, Y., Yao, Q., & Ma, J.-A. (2012). Catalytic enantioselective addition of terminal 1,3-diynes to N-sulfonyl aldimines: access to chiral diynylated carbinamines. Chemical Communications, 48(100), 12234. doi:10.1039/c2cc37290hZhang, F.-G., Ma, H., Zheng, Y., & Ma, J.-A. (2012). Zinc-mediated enantioselective addition of terminal 1,3-diynes to N-arylimines of trifluoropyruvates. Tetrahedron, 68(37), 7663-7669. doi:10.1016/j.tet.2012.05.086Zhang, F.-G., Ma, H., Nie, J., Zheng, Y., Gao, Q., & Ma, J.-A. (2012). Enantioselective Diynylation of Cyclic N-Acyl Ketimines: Access to Chiral Trifluoromethylated Tertiary Carbinamines. Advanced Synthesis & Catalysis, 354(8), 1422-1428. doi:10.1002/adsc.201100926Nie, J., Guo, H.-C., Cahard, D., & Ma, J.-A. (2011). Asymmetric Construction of Stereogenic Carbon Centers Featuring a Trifluoromethyl Group from Prochiral Trifluoromethylated Substrates. Chemical Reviews, 111(2), 455-529. doi:10.1021/cr100166aCahard, D., Xu, X., Couve-Bonnaire, S., & Pannecoucke, X. (2010). Fluorine & chirality: how to create a nonracemic stereogenic carbon–fluorine centre? Chem. Soc. Rev., 39(2), 558-568. doi:10.1039/b909566gKirk, K. L. (2008). Fluorination in Medicinal Chemistry: Methods, Strategies, and Recent Developments. Organic Process Research & Development, 12(2), 305-321. doi:10.1021/op700134jMa, J.-A., & Cahard, D. (2008). Update 1 of: Asymmetric Fluorination, Trifluoromethylation, and Perfluoroalkylation Reactions. Chemical Reviews, 108(9), PR1-PR43. doi:10.1021/cr800221vPurser, S., Moore, P. R., Swallow, S., & Gouverneur, V. (2008). Fluorine in medicinal chemistry. Chem. Soc. Rev., 37(2), 320-330. doi:10.1039/b610213cMorrill, L. C., Smith, S. M., Slawin, A. M. Z., & Smith, A. D. (2014). Isothiourea-Mediated Asymmetric Functionalization of 3-Alkenoic Acids. 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ChemCatChem, 6(2), 580-591. doi:10.1002/cctc.20130087

Transcriptome Analysis of the Desert Locust Central Nervous System: Production and Annotation of a Schistocerca gregaria EST Database

) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology

High speed functional imaging with source localized multifocal two-photon microscopy

Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals