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Nature Protocols

Jinglin Fu, Yuhe Renee Yang, Soma Dhakal, Zhao Zhao, Minghui Liu, Ting Zhang, Nils G Walter, Hao Yan
In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths...
November 2016: Nature Protocols
Ido Sagi, Dieter Egli, Nissim Benvenisty
Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency, providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid, their enrichment at an early passage is key for successful derivation. In this protocol, we describe two methodologies, namely metaphase spread analysis and cell sorting, for the identification of haploid human cells within parthenogenetic ESC lines...
November 2016: Nature Protocols
Jibin Song, Peng Huang, Xiaoyuan Chen
Gold nanovesicles contain multiple nanocrystals within a polymeric coating. The strong plasmonic coupling between adjacent nanoparticles in their vesicular shell makes ultrasensitive biosensing and bioimaging possible. In our laboratory, multifunctional plasmonic vesicles are assembled from amphiphilic gold nanocrystals (such as gold nanoparticles and gold nanorods) coated with mixed hydrophilic and hydrophobic polymer brushes or amphiphilic diblock co-polymer brushes. To fulfill the different requirements of biomedical applications, different polymers that are either pH=responsive, photoactive or biodegradable can be used to form the hydrophobic brush, while the hydrophilicity is maintained by polyethylene glycol (PEG)...
November 2016: Nature Protocols
Dong-Wook Park, Sarah K Brodnick, Jared P Ness, Farid Atry, Lisa Krugner-Higby, Amelia Sandberg, Solomon Mikael, Thomas J Richner, Joseph Novello, Hyungsoo Kim, Dong-Hyun Baek, Jihye Bong, Seth T Frye, Sanitta Thongpang, Kyle I Swanson, Wendell Lake, Ramin Pashaie, Justin C Williams, Zhenqiang Ma
Transparent graphene-based neural electrode arrays provide unique opportunities for simultaneous investigation of electrophysiology, various neural imaging modalities, and optogenetics. Graphene electrodes have previously demonstrated greater broad-wavelength transmittance (∼90%) than other transparent materials such as indium tin oxide (∼80%) and ultrathin metals (∼60%). This protocol describes how to fabricate and implant a graphene-based microelectrocorticography (μECoG) electrode array and subsequently use this alongside electrophysiology, fluorescence microscopy, optical coherence tomography (OCT), and optogenetics...
November 2016: Nature Protocols
Alessia Deglincerti, Fred Etoc, M Cecilia Guerra, Iain Martyn, Jakob Metzger, Albert Ruzo, Mijo Simunovic, Anna Yoney, Ali H Brivanlou, Eric Siggia, Aryeh Warmflash
Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results...
November 2016: Nature Protocols
Matthias Schmitz, Maria Cramm, Franc Llorens, Dominik Müller-Cramm, Steven Collins, Ryuichiro Atarashi, Katsuya Satoh, Christina D Orrù, Bradley R Groveman, Saima Zafar, Walter J Schulz-Schaeffer, Byron Caughey, Inga Zerr
The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrP(Sc)) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrP(Sc) seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers...
November 2016: Nature Protocols
Chul-Yong Park, Jin Jea Sung, Sang-Hwi Choi, Dongjin R Lee, In-Hyun Park, Dong-Wook Kim
Genome engineering technology using engineered nucleases has been rapidly developing, enabling the efficient correction of simple mutations. However, the precise correction of structural variations (SVs) such as large inversions remains limited. Here we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol includes the delivery of engineered nucleases with no donor template to hiPSCs, and genotyping and derivation/characterization of gene-manipulated hiPSC clones...
November 2016: Nature Protocols
Marta Czernik, Domenico Iuso, Paola Toschi, Saadi Khochbin, Pasqualino Loi
This protocol describes how to convert the chromatin structure of sheep and mouse somatic cells into spermatid-like nuclei through the heterologous expression of the protamine 1 gene (Prm1). Furthermore, we also provide step-by-step instructions for somatic cell nuclear transfer (SCNT) of Prm1-remodeled somatic nuclei in sheep oocytes. There is evidence that changing the organization of a somatic cell nucleus with that which mirrors the spermatozoon nucleus leads to better nuclear reprogramming. The protocol may have further potential application in determining the protamine and histone footprints of the whole genome; obtaining 'gametes' from somatic cells; and furthering understanding of the molecular mechanisms regulating the maintenance of DNA methylation in imprinted control regions during male gametogenesis...
November 2016: Nature Protocols
Shi Wang, Pingping Liu, Jia Lv, Yangping Li, Taoran Cheng, Lingling Zhang, Yu Xia, Hongzhen Sun, Xiaoli Hu, Zhenmin Bao
Isolength restriction site-associated DNA (isoRAD) sequencing is a very simple but powerful approach that was originally developed for genome-wide genotyping at minimal labor and cost, and it has recently extended its applicability to allow quantification of DNA methylation levels. The isoRAD method is distinct from other genotyping-by-sequencing (GBS) methods because of its use of special restriction enzymes to produce isolength tags (32-36 bp), and sequencing of these uniform tags can bring many benefits...
November 2016: Nature Protocols
Cy M Jeffries, Melissa A Graewert, Clément E Blanchet, David B Langley, Andrew E Whitten, Dmitri I Svergun
Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample...
November 2016: Nature Protocols
Vijay Ramani, Darren A Cusanovich, Ronald J Hause, Wenxiu Ma, Ruolan Qiu, Xinxian Deng, C Anthony Blau, Christine M Disteche, William S Noble, Jay Shendure, Zhijun Duan
With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor...
November 2016: Nature Protocols
Iain C Macaulay, Mabel J Teng, Wilfried Haerty, Parveen Kumar, Chris P Ponting, Thierry Voet
Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products...
November 2016: Nature Protocols
Richard N Day, Wen Tao, Kenneth W Dunn
Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM...
November 2016: Nature Protocols
Tanmay A M Bharat, Sjors H W Scheres
Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis...
November 2016: Nature Protocols
Ramya H Tunuguntla, Artur Escalada, Vadim A Frolov, Aleksandr Noy
Carbon nanotube porins (CNTPs) are 10- to 20-nm-long segments of lipid-stabilized single-walled carbon nanotubes (CNTs) that can be inserted into phospholipid membranes to form nanometer-scale-diameter pores that approximate the geometry and many key transport characteristics of biological membrane channels. We describe protocols for CNTP synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles, and for validation of CNTP incorporation into a lipid membrane using a proton permeability assay...
October 2016: Nature Protocols
Itamar Harel, Dario Riccardo Valenzano, Anne Brunet
A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies. Here we present a comprehensive protocol for efficient genome engineering in the African turquoise killifish (Nothobranchius furzeri), which is the shortest-lived vertebrate in captivity with a median life span of 4-6 months. By taking advantage of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system and the turquoise killifish genome, this platform enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR)...
October 2016: Nature Protocols
Sarita R Shah, Simon Young, Julia L Goldman, John A Jansen, Mark E Wong, Antonios G Mikos
Translational biomaterials targeted toward the regeneration of large bone defects in the mandible require a preclinical model that accurately recapitulates the regenerative challenges present in humans. Computational modeling and in vitro assays do not fully replicate the in vivo environment. Consequently, in vivo models can have specific applications such as those of the mandibular angle defect, which is used to investigate bone regeneration in a nonload-bearing area, and the inferior border mandibular defect, which is a model for composite bone and nerve regeneration, with both models avoiding involvement of soft tissue or teeth...
October 2016: Nature Protocols
Giang K T Nguyen, Yibo Qiu, Yuan Cao, Xinya Hemu, Chuan-Fa Liu, James P Tam
Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His-Val at the P1' and P2' positions...
October 2016: Nature Protocols
Daniel J Hiler, Marie E Barabas, Lyra M Griffiths, Michael A Dyer
Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells...
October 2016: Nature Protocols
Lu Li, Wenbo Liu, Xiaoyue Mu, Zetian Mi, Chao-Jun Li
Aryl iodides are important precursors in synthetic chemistry that form carbon-carbon and carbon-heteroatom bonds. Most methods use transition-metal catalysts, which need to be scrupulously removed before the compounds can be used in the pharmaceutical and electronics industries, where only parts-per-million levels of transition metals are allowed. The aromatic Finkelstein iodination reaction is a powerful method of preparing valuable aryl iodides from cheap but less reactive aryl bromides and chlorides. This protocol describes a transition metal-free method for a photo-induced aromatic Finkelstein iodination reaction that is performed at room temperature (20 °C)...
October 2016: Nature Protocols
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