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04:20 < kanzure> hm.
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05:10 < kanzure> "Genetically targeted all-optical electrophysiology with a transgenic Cre-dependent optopatch mouse" http://www.jneurosci.org/content/36/43/11059.abstract
05:11 < kanzure> "Recent advances in optogenetics have enabled simultaneous optical perturbation and optical readout of membrane potential in diverse cell types. Here, we develop and characterize a Cre-dependent transgenic Optopatch2 mouse line that we call Floxopatch. The animals expressed a blue-shifted channelrhodopsin, CheRiff, and a near infrared Archaerhodopsin-derived voltage indicator, QuasAr2, via targeted knock-in at the rosa26 locus. ... ...
05:11 < kanzure> ... Cell-type-specific expression allowed classification and characterization of neuronal subtypes based on their firing patterns."
05:11 < kanzure> "Calcium imaging has been widely applied in this mode, but is insensitive to the details of action potential waveforms and subthreshold events Simultaneous optical perturbation and optical readout of single-cell electrical activity (“Optopatch”) has been demonstrated in cultured neurons and in organotypic brain slices, but not in acute brain slices or in vivo. Here, we describe a transgenic mouse in which expression of Optopatch ...
05:11 < kanzure> ... constructs is controlled by the Cre-recombinase enzyme. This animal enables fast and robust optical measurements of single-cell electrical excitability in acute brain slices and in somatosensory ganglia in vivo, opening the door to rapid optical mapping of neuronal excitability"
05:13 < kanzure> "voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes"
05:14 < kanzure> "First developed in the Bezanilla lab, hVOS consists of an FP anchored to the plasma membrane with the addition of a small charged molecule, DPA, that binds to the plasma membrane effectively acting as a fluorescent quencher. Since DPA is a lipophilic anion, the quenching agent will move from the outer surface of the plasma membrane to the inner surface upon membrane depolarizations generating a voltage-responsive fluorescent signal. ...
05:14 < kanzure> ... Like the other sensors, hVOS also has drawbacks which are primarily due to the fact that an exogenous chemical must be administered to the sample to be imaged. This is not a trivial process since too much DPA will significantly increase the capacitance of the plasma membrane and alter the neuronal activity of the cell. However, once the appropriate conditions are determined, hVOS gives optical signals for subthreshold potentials as ...
05:14 < kanzure> ... well as action potentials in slice from populations of cells (Wang et al., 2012) or individual cells when expression of the FP is sparser (Ghitani et al., 2015).
05:14 < kanzure> "
05:17 < kanzure> "Azobenzene photocontrol of peptides and proteins" http://pubs.rsc.org/en/content/articlehtml/2016/cc/c6cc04004g
05:21 < kanzure> ... "Although stilbenes, hemithioindigos, thioxopeptides, selenoxopeptides, diarylethenes, acylhydrazones and rhodopsin-like molecules have been used as switches, azobenzenes currently offer the largest range of activation wavelengths and photoactivated state stabilities. In azobenzene-derived photoswitches rotation around the central double bond is transmitted with high fidelity through the rigid rings resulting in a predictable ...
05:21 < kanzure> ... distance changes, particularly between substituents on the para positions."
05:23 < kanzure> "Genetic code expansion can be used to introduce 31 into proteins expressed in E. coli or mammalian cells.65 Azobenzene 32 (Fig. 15) can also be incorporated into expressed proteins; its pentafluoro ring is electrophilic enough to undergo nucleophilic aromatic substitution by a proximal cysteine.66 The fluorine atoms of 32 provide sufficient n–π* band separation for bidirectional switching with 540 nm and 470 nm light to generate ...
05:24 < kanzure> ... ∼70% of the desired isomer in the photostationary state. This switch was incorporated into calmodulin with an orthogonal tRNA synthetase. Whilst a degree of side reaction with glutathione was noted, cyclisation proceeded smoothly to give a visible light responsive calmodulin with distinct binding affinities for neuronal nitric oxide synthase under green and blue light irradiation. Thiol–ene chemistry was also investigated to ...
05:24 < kanzure> ... introduce a bifunctional glycosylated azobenzene photoswitch.67 Using 2,2-dimethoxy-2-phenylacetophenone as a photoinitiator caused 33 to react with glutathione in 70% yield (Fig. 16)."
05:26 < kanzure> "In vitro translation and expanded genetic code techniques have been used to create a library of peptides containing the azobenzyl-lysine 35 (Fig. 18).69 By suppressing the stop codon and conducting the reaction at low temperature, neither the nascent peptide nor the coding mRNA dissociate from the ribosome. Streptavidin-coated beads were placed in the solution in the dark and the beads were washed and exposed to UV light, which resulted ...
05:26 < kanzure> ... in dissociation of the peptide–ribosome–mRNA complex. The complexes were recovered, the mRNA dissociated and subjected to amplification by reverse transcriptase polymerase chain reaction; this identified peptide sequences that bound streptavidin 2.5-fold weaker in their photostationary state than in the dark."
05:26 < kanzure> "Libraries of peptides have also been generated using phage display.70 Peptides containing i,i + 7 spaced cysteines were expressed in E. coli, reduced with resin-bound tris-carboxyethylphosphine and treated with 2. Although the overall yield of modification with 2 was only 50%, peptides were identified with up to 22-fold decreases in affinity for streptavidin in their pure dark and light states. A later study also used i,i + 7 spaced ...
05:26 < kanzure> ... cysteine residues alkylated with 2 and discovered peptides that had a greater affinity in for streptavidin in the light state with the best example found to bind 3-fold more tightly.71"
05:27 < kanzure> ooh ooh this one,
05:27 < kanzure> "The DNA recognition helix from the engrailed homeodomain has been modified to incorporate two i,i + 11 spaced cysteine residues to accommodate 2.72 When the peptide is folded into an α-helix, the intra-cysteine spacing matches the length of E-2 so that the cross-linked peptide is strongly helical and binds to target DNA 25 times more strongly than a control peptide without 2. Irradiation to form Z-2 diminished the DNA binding affinity ...
05:27 < kanzure> ... 18-fold."
05:30 < kanzure> "Introducing azobenzene 42 (Fig. 32) between two helical structural elements resulted in a peptide capable of controlling calcium ion fluxes in pancreatic beta cells and moderating insulin secretion in response to light (Fig. 33).111"
05:32 < kanzure> "Channels modified with 52 returned to their ground state 2200-fold faster than channels modified with 51, allowing much higher temporal resolution control of neuron firing.151 Azobenzene 52 has a much-improved two-photon absorption cross-section that is comparable to that of fluorescent proteins.152"
05:33 < kanzure> "versions of the transient receptor potential pain receptor ion channel TRPV1,159 that respond to compounds including azo-capsaicin 59,160"    right..
05:34 < kanzure> "and endowment of retinal function on blind mice using 63 (Fig. 43).164"
05:35 < kanzure> welp that is all very useful.
05:40 < kanzure> "High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing" http://www.sciencedirect.com/science/article/pii/S0092867416304895 (using crispr of course)
05:40 < kanzure> ah, a more accessible verson of the previous:
05:41 < kanzure> "Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080626/
05:43 < kanzure> ... "This system has been shown to work in cultured cells, embryos, and organisms7,8,11,12,13, but not in postmitotic neurons."
05:45 < kanzure> "Supernova: A versatile vector system for single-cell labeling and gene function studies in vivo" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075795/
05:56 < kanzure> regarding MAPseq and FISSEC-BOINC and dna barcoding,
05:56 < kanzure> "Network cloning using DNA barcodes" https://arxiv.org/abs/1611.00834
05:57 < kanzure> "The ability to measure or manipulate network connectivity is the main challenge in the field of connectomics. Recently, a set of approaches has been developed that takes advantage of next generation DNA sequencing to scan connections between neurons into a set of DNA barcodes. Individual DNA sequences called markers represent single neurons, while pairs of markers, called barcodes contain information about connections. Here we propose a ...
05:57 < kanzure> ... strategy for 'copying' or 'cloning' connectivity contained in barcodes into a clean slate tabula rasa network. We show that a one marker one cell (OMOC) rule, which forces all markers with the same sequence to condense into the same neuron, leads to fast and reliable formation of desired connectivity in a new network. We show that OMOC rule yields convergence in a number of steps given by a power law function of the network size. We ...
05:57 < kanzure> ... thus propose that copying network connectivity from one network to another is theoretically possible."
06:09 < kanzure> "Roadmap on neurophotonics" http://doc.utwente.nl/101196/1/roadmap.pdf
06:13 < kanzure> also mentions photoacoustic microscopy 'Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect'
06:13 < kanzure> https://en.wikipedia.org/wiki/Photoacoustic_imaging
06:14 < kanzure> page 20 has some fancy images of microvasculature resolved by photoacoustic imaging of mouse brain through its skull
06:20 < kanzure> "Engineering prokaryotic channels for control of mammalian tissue excitability" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071848/
06:20 < kanzure> "The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNav) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNav orthologues, an inward-rectifying ...
06:20 < kanzure> ... potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts (‘E-Fibs') retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered ...
06:20 < kanzure> ... BacNav with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K+, decoupling or ischaemia. These studies establish the utility of engineered BacNav channels for induction, control and recovery of mammalian tissue excitability."
06:21 < kanzure> 'single-molecule stochastic localization super-resolution microscopy' 'for the imaging of synaptic proteins'  http://link.springer.com/protocol/10.1007/8623_2016_10
06:26 < kanzure> "Engineering of temperature- and light-switchable Cas9 variants" http://nar.oxfordjournals.org/content/early/2016/10/14/nar.gkw930.abstract
06:31 < kanzure> something about thalamic sensory-modality specific genes http://in.umh.es/datos/noticias/CerebCortex-2016-Gezelius.pdf
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06:41 < kanzure> "Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors" http://www.sciencedirect.com/science/article/pii/S0958166916300684
06:41 < kanzure> "[...] Likewise, genetically labeling desired circuits across mammalian bodies is prohibitively slow and costly via the transgenic route. Emerging breakthroughs in viral vector engineering, genome editing tools, and tissue clearing can render larger opaque organisms genetically tractable and transparent for whole-organ cell phenotyping, tract tracing and imaging at depth."
06:41 < kanzure> oh, the uDISCO paper cited that one. weird.
06:41 < kanzure> i don't remember that.
06:43 < kanzure> short overview of crispr-cas9 and recombinases for editing dna and storing cellular data in vivo http://science.sciencemag.org/content/353/6298/444
06:52 < kanzure> "stop codon suppression" for click chemistry handles and protein photocaged sidechains http://link.springer.com/chapter/10.1007/10_2016_37
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07:18 < kanzure> https://www.coreinfrastructure.org/news/announcements/2016/11/linux-foundations-core-infrastructure-initiative-renews-funding
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08:05 < gnusha> https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=470728c6 Bryan Bishop: update genetic modifications for general cellular changes >> http://diyhpl.us/diyhpluswiki/genetic-modifications/
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13:17 < kanzure> https://deepmind.com/blog/wavenet-generative-model-raw-audio/
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13:28 < kanzure> something something about image recognition tasks on GPUs outperforming human image classification http://www.eetimes.com/document.asp?doc_id=1325712
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13:36 < kanzure> something something nvidia wants to do autonomous cars with vision-only, other folks are saying please use lidar https://news.ycombinator.com/item?id=12938016
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18:08 < kanzure> bloop
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23:25 < vicarion> will the autonomous cars ever rise up and overthrow their human overlords? maybe we shouldn't be arming them with lasers
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--- Log closed Sun Nov 13 00:00:48 2016