Hot off the presses! May 01 Biomater

The May 01 issue of the Biomater is now up on Pubget (About Biomater): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial board
  - Biomater 32(13):IFC (2011)
  
• A self-assembling hydrophobically modified chitosan capable of reversible hemostatic action
  - Biomater 32(13):3351-3357 (2011)
  Blood loss at the site of a wound in mammals is curtailed by the rapid formation of a hemostatic plug, i.e., a self-assembled network of the protein, fibrin that locally transforms liquid blood into a gelled clot. Here, we report an amphiphilic biopolymer that exhibits a similar ability to rapidly gel blood; moreover, the self-assembly underlying the gelation readily allows for reversibility back into the liquid state via introduction of a sugar-based supramolecule. The biopolymer is a hydrophobically modified (hm) derivative of the polysaccharide, chitosan. When hm-chitosan is contacted with heparinized human blood, it rapidly transforms the liquid into an elastic gel. In contrast, the native chitosan (without hydrophobes) does not gel blood. Gelation occurs because the hydrophobes on hm-chitosan insert into the membranes of blood cells and thereby connect the cells into a sample-spanning network. Gelation is reversed by the addition of α-cyclodextrin, a supramolecul! e having an inner hydrophobic pocket: polymer hydrophobes unbind from blood cells and embed within the cyclodextrins, thereby disrupting the cell network. We believe that hm-chitosan has the potential to serve as an effective, yet low-cost hemostatic dressing for use by trauma centers and the military. Preliminary tests with small and large animal injury models show its increased efficacy at achieving hemostasis – e.g., a 90% reduction in bleeding time over controls for femoral vein transections in a rat model.
• Transparent, tough collagen laminates prepared by oriented flow casting, multi-cyclic vitrification and chemical cross-linking
  - Biomater 32(13):3358-3366 (2011)
  The lamellar architecture found in many natural fibrous tissues has a significant bearing on their specific functions. However, current engineered tissues have simultaneously no realistic structures and no adequate functions. This study demonstrates a two-step process for obtaining structurally mimicking laminates in natural fibrous tissues with good optical and mechanical characters from purified-clinically-safe collagen molecules. Stacked lamella structures can be created by repeating flow casting, with the controlling parallel/orthogonal directionalities of each thin single-layer (2–5 μm in thickness). The transparency of laminates is successfully improved by a unique multi-cyclic vitrification with chemical cross-linking. The directionalities of optical and mechanical functions in laminates are strongly related with the preferential collagen alignments in the laminates. The tensile strength of laminates is extremely higher than any other engineered materials as ! well as native cornea, which exhibit an orthogonal laminated collagen structure and a good optical transmission.
• Failure of Aβ(1-40) amyloid fibrils under tensile loading
  - Biomater 32(13):3367-3374 (2011)
  Amyloid fibrils and plaques are detected in the brain tissue of patients affected by Alzheimer's disease, but have also been found as part of normal physiological processes such as bacterial adhesion. Due to their highly organized structures, amyloid proteins have also been used for the development of nanomaterials, for a variety of applications including biomaterials for tissue engineering, nanolectronics, or optical devices. Past research on amyloid fibrils resulted in advances in identifying their mechanical properties, revealing a remarkable stiffness. However, the failure mechanism under tensile loading has not been elucidated yet, despite its importance for the understanding of key mechanical properties of amyloid fibrils and plaques as well as the growth and aggregation of amyloids into long fibers and plaques. Here we report a molecular level analysis of failure of amyloids under uniaxial tensile loading. Our molecular modeling results demonstrate that amyloi! d fibrils are extremely stiff with a Young's modulus in the range of 18–30 GPa, in good agreement with previous experimental and computational findings. The most important contribution of our study is our finding that amyloid fibrils fail at relatively small strains of 2.5%–4%, and at stress levels in the range of 1.02 to 0.64 GPa, in good agreement with experimental findings. Notably, we find that the strength properties of amyloid fibrils are extremely length dependent, and that longer amyloid fibrils show drastically smaller failure strains and failure stresses. As a result, longer fibrils in excess of hundreds of nanometers to micrometers have a greatly enhanced propensity towards spontaneous fragmentation and failure. We use a combination of simulation results and simple theoretical models to define critical fibril lengths where distinct failure mechanisms dominate.
• Keratin films for ocular surface reconstruction
  - Biomater 32(13):3375-3386 (2011)
  Human amniotic membrane (AM) is frequently used as a substrate for ocular surface reconstruction. Its disadvantages (e.g., reduced transparency and biomechanical strength, heterogeneity depending on donor) create the need for standardized alternatives. Keratin from hair or wool has been proposed as an appropriate material for producing films or cell cultivation scaffolds. The current study was performed to develop transparent, stable and transferable films based on human hair keratin that support cellular adhesion and proliferation. The films were engineered by a multi-step procedure including keratin extraction, neutral and alkaline dialysis, drying and a curing process. Keratin films were investigated by SDS-PAGE, SEM and X-ray analyses. Furthermore, swelling and water absorption of the films were studied, as were tensile strength and light transmission (UV/VIS). Finally, the growth behavior of corneal epithelial cells on the keratin films and AM was estimated in pro! liferation studies. In addition, we assessed the seeding efficiency and cell detachment behavior during trypsinization. The film-forming process resulted in transparent films composed of nanoparticulate keratin structures. The film characteristics could be varied by changing the protein composition, adding softening agents or varying the curing temperature and duration. Based on these findings, an optimized protocol was developed. The films showed improved light transmission and biomechanical strength in comparison to AM. Furthermore, cell behavior on the films was similar to that found on AM. We conclude that keratin films may represent a new, promising alternative for ocular surface reconstruction.
• Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber–extracellular matrix hydrogel biohybrid scaffold
  - Biomater 32(13):3387-3394 (2011)
  A biohybrid composite consisting of extracellular matrix (ECM) gel from porcine dermal tissue and biodegradable elastomeric fibers was generated and evaluated for soft tissue applications. ECM gel possesses attractive biocompatibility and bioactivity with weak mechanical properties and rapid degradation, while electrospun biodegradable poly(ester urethane)urea (PEUU) has good mechanical properties but limited cellular infiltration and tissue integration. A concurrent gel electrospray/polymer electrospinning method was employed to create ECM gel/PEUU fiber composites with attractive mechanical properties, including high flexibility and strength. Electron microscopy revealed a structure of interconnected fibrous layers embedded in ECM gel. Tensile mechanical properties could be tuned by altering the PEUU/ECM weight ratio. Scaffold tensile strengths for PEUU/ECM ratios of 67/33, 72/28 and 80/20 ranged from 80 to 187 kPa in the longitudinal axis (parallel to the collecting! mandrel axis) and 41–91 kPa in the circumferential axis with 645–938% breaking strains. The 72/28 biohybrid composite and a control scaffold generated from electrospun PEUU alone were implanted into Lewis rats, replacing a full-thickness abdominal wall defect. At 4 wk, no infection or herniation was found at the implant site. Histological staining showed extensive cellular infiltration into the biohybrid scaffold with the newly developed tissue well integrated with the native periphery, while minimal cellular ingress into the electrospun PEUU scaffold was observed. Mechanical testing of explanted constructs showed evidence of substantial remodeling, with composite scaffolds adopting properties more comparable to the native abdominal wall. The described elastic biohybrid material imparts features of ECM gel bioactivity with PEUU strength and handling to provide a promising composite biomaterial for soft tissue repair and replacement.
• The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation
  - Biomater 32(13):3395-3403 (2011)
  Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited simil! ar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.
• Cell affinity for bFGF immobilized heparin-containing poly(lactide-co-glycolide) scaffolds
  - Biomater 32(13):3404-3412 (2011)
  In order to effectively and uniformly immobilize basic fibroblast growth factor (bFGF) to thick PLGA scaffold, the heparin-conjugated PLGA (H-PLGA) was synthesized at the first by reaction between heparin and a low molecular weight PLGA. Then heparin-containing PLGA (H-PLGA/PLGA) scaffold was fabricated by blending the H-PLGA with a high molecular weight PLGA. Finally, bFGF was immobilized on the H-PLGA/PLGA scaffold mainly by static electricity action between them. The effect of H-PLGA content on bFGF binding efficiency of the H-PLGA/PLGA scaffolds was investigated. It was found that bFGF binding efficiency increased with increasing H-PLGA content. The bound bFGF can release in vitro slowly from the H-PLGA/PLGA scaffolds and last over two weeks. The released bFGF has still preserved its bioactivity. The attachment and growth of mouse 3T3 fibroblasts on the H-PLGA/PLGA scaffolds were better than that on the PLGA scaffold, however bFGF immobilized H-PLGA/PLGA scaffolds ! showed much better cell affinity. Therefore, the method to use the H-PLGA/PLGA scaffold for immobilizing bFGF is not only effective for slow delivering bFGF with bioactivity, but also can be used for fabricating thick scaffold where bFGF could be combined and uniformly distributed.
• Engineering spatial control of multiple differentiation fates within a stem cell population
  - Biomater 32(13):3413-3422 (2011)
  The capability to engineer microenvironmental cues to direct a stem cell population toward multiple fates, simultaneously, in spatially defined regions is important for understanding the maintenance and repair of multi-tissue units. We have previously developed an inkjet-based bioprinter to create patterns of solid-phase growth factors (GFs) immobilized to an extracellular matrix (ECM) substrate, and applied this approach to drive muscle-derived stem cells toward osteoblasts 'on-pattern' and myocytes 'off-pattern' simultaneously. Here this technology is extended to spatially control osteoblast, tenocyte and myocyte differentiation simultaneously. Utilizing immunofluorescence staining to identify tendon-promoting GFs, fibroblast growth factor-2 (FGF-2) was shown to upregulate the tendon marker Scleraxis (Scx) in C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells, while downregulating the myofibroblast marker α-smooth muscle! actin (α-SMA). Quantitative PCR studies indicated that FGF-2 may direct stem cells toward a tendon fate via the Ets family members of transcription factors such as pea3 and erm. Neighboring patterns of FGF-2 and bone morphogenetic protein-2 (BMP-2) printed onto a single fibrin-coated coverslip upregulated Scx and the osteoblast marker ALP, respectively, while non-printed regions showed spontaneous myotube differentiation. This work illustrates spatial control of multi-phenotype differentiation and may have potential in the regeneration of multi-tissue units.
• The performance of laminin-containing cryogel scaffolds in neural tissue regeneration
  - Biomater 32(13):3423-3434 (2011)
  Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin – the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds' pore size for neural tissue engineering was 80–100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in "niche"-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be tr! ansplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.
• The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles
  - Biomater 32(13):3435-3446 (2011)
  To systematically elucidate the effect of surface charge on the cellular uptake and in vivo fate of PEG-oligocholic acid based micellar nanoparticles (NPs), the distal PEG termini of monomeric PEG-oligocholic acid dendrimers (telodendrimers) are each derivatized with different number (n = 0, 1, 3 and 6) of anionic aspartic acids (negative charge) or cationic lysines (positive charge). Under aqueous condition, these telodendrimers self-assemble to form a series of micellar NPs with various surface charges, but with similar particle sizes. NPs with high surface charge, either positive or negative, were taken up more efficiently by RAW 264.7 murine macrophages after opsonization in fresh mouse serum. Mechanistic studies of cellular uptake of NPs indicated that several distinct endocytic pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) were involved in the cellular uptake process. After their cellular uptake, the majority ! of NPs were found to localize in the lysosome. Positively charged NPs exhibited dose-dependent hemolytic activities and cytotoxicities against RAW 264.7 cells proportional to the positive surface charge densities; whereas negatively charged NPs did not show obvious hemolytic and cytotoxic properties. In vivo biodistribution studies demonstrated that undesirable liver uptake was very high for highly positively or negatively charged NPs, which is likely due to active phagocytosis by macrophages (Kupffer cells) in the liver. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge of NPs was slightly negative. Based on these studies, we can conclude that slightly negative charge may be introduced to the NPs surface to reduce the undesirable clearance by the reticuloendothelial system (RES) such as liver, improve the blood compatibility, thus deliver the anti-cancer drugs more efficiently to the tumor sites.
• Photosensitizer-conjugated magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging and targeting therapy
  - Biomater 32(13):3447-3458 (2011)
  A major challenge in nanotechnology and nanomedicine is to integrate tumor targeting, imaging, and selective therapy functions into a small single nanoparticle (<50 nm). Herein, photosensitizer-conjugated magnetic nanoparticles with 20 nm in diameter were strategically designed and prepared for gastric cancer imaging and therapy. The second generation photosensitizer chlorin e6 (Ce6) was covalently anchored on the surface of magnetic nanoparticles with silane coupling agent. We found that the covalently incorporated Ce6 molecules retained their spectroscopic and functional properties for near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT), and the core magnetic nanoparticles offered the functions of magnetically guided drug delivery and magnetic resonance imaging (MRI). The as-prepared single particle platform is suitable for simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer or ! other tumors.
• The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes
  - Biomater 32(13):3459-3470 (2011)
  The LPD (liposome-polycation-DNA complex) is an effective nanovector for systemically small interfering RNA (siRNA) delivery which was well characterized previously. However, little effort was spend on the development of targeted LPD conjugated with tumor specific antibody (TLPD) which would be potent in promoting siRNA delivery in tumor. Here, we prepared TLPD through a self-assembling process followed by anti-EGFR antibody conjugation. The effect of antibody type, conjugation strategy and amount on the physicochemical and biological properties of TLPD was investigated. We obtained optimized TLPD conjugated with anti-EGFR Fab' by conventional conjugation (TLPD-FCC), which possessed a small size around 150 nm and superior in vitro stability. Compared with nontargeted LPD (NTLPD), TLPD-FCC showed significantly enhanced binding affinity and luciferase gene silencing activity in EGFR overexpressing MDA-MB-231 breast cancer cells in vitro. Moreover, the in vivo accumulat! ion of TLPD-FCC was obviously higher than that of NTLPD in MDA-MB-231 tumor 24 h post intravenous injection. The promoted uptake of TLPD-FCC in MDA-MB-231 tumor was further confirmed by confocal microscopy. Notably, three intravenous injections of siRNA in TLPD-FCC significantly silenced luciferase expression by 20%, whereas NTLPD showed little effect. All these results suggested that our TLPD-FCC have a great potential in delivering siRNA to EGFR overexpressing breast cancers.
• Gene delivery system based on highly specific recognition of surface-vimentin with N-acetylglucosamine immobilized polyethylenimine
  - Biomater 32(13):3471-3480 (2011)
  Gene and drug-delivery systems that use immobilization of carbohydrates are useful for the specific targeting of lectin-expressing tissues. Here, we report that N-acetylglucosamine (GlcNAc) with polyethylenimine (GlcNAc-PEI) specifically interacted with vimentin-expressing cells such as 293FT and HeLa cells. Recently, the intermediate filaments vimentin and desmin have been reported to have GlcNAc-binding lectin-like properties on the cell surface. Therefore, GlcNAc-conjugated agents can be targeted to vimentin- and desmin-expressing cells and tissues. Vimentin-expressing 293FT and HeLa cells were efficiently transfected with green fluorescent protein and luciferase genes by using GlcNAc-PEI; the expression of these genes in vimentin-knockdown cells were low. Confocal microscopic analysis showed that GlcNAc-PEI complexes interacted with vimentin on the cell surface of HeLa cells. These results demonstrate that GlcNAc-PEI/DNA complexes were specifically taken up by 293F! T and HeLa cells via vimentin. We suggest that this gene-delivery system could be used to target various vimentin-expressing cells such as fibroblasts and tumor cells.
• Co-encapsulation of magnetic nanoparticles and doxorubicin into biodegradable microcarriers for deep tissue targeting by vascular MRI navigation
  - Biomater 32(13):3481-3486 (2011)
  Magnetic tumor targeting with external magnets is a promising method to increase the delivery of cytotoxic agents to tumor cells while reducing side effects. However, this approach suffers from intrinsic limitations, such as the inability to target areas within deep tissues, due mainly to a strong decrease of the magnetic field magnitude away from the magnets. Magnetic resonance navigation (MRN) involving the endovascular steering of therapeutic magnetic microcarriers (TMMC) represents a clinically viable alternative to reach deep tissues. MRN is achieved with an upgraded magnetic resonance imaging (MRI) scanner. In this proof-of-concept preclinical study, the preparation and steering of TMMC which were designed by taking into consideration the constraints of MRN and liver chemoembolization are reported. TMMC were biodegradable microparticles loaded with iron-cobalt nanoparticles and doxorubicin (DOX). These particles displayed high saturation magnetization (Ms = 72 em! u g−1), MRI tracking compatibility (strong contrast on T2*-weighted images), appropriate size for the blood vessel embolization (50 μm), and sustained release of DOX (over several days). The TMMC were successfully steered in vitro and in vivo in the rabbit model. In vivo targeting of the right or left liver lobes was achieved by MRN through the hepatic artery located 4 cm beneath the skin. Parameters such as flow velocity, TMMC release site in the artery, magnetic gradient and TMMC properties, affected the steering efficiency. These data illustrate the potential of MRN to improve drug targeting in deep tissues.
• Tunable physiologic interactions of adhesion molecules for inflamed cell-selective drug delivery
  - Biomater 32(13):3487-3498 (2011)
  Dysregulated inflammation contributes to the pathogenesis of various diseases. Therapeutic efficacy of anti-inflammatory agents, however, falls short against resilient inflammatory responses, whereas long-term and high-dose systemic administration can cause adverse side effects. Site-directed drug delivery systems would thus render more effective and safer treatments by increasing local dosage and minimizing toxicity. Nonetheless, achieving clinically effective targeted delivery to inflammatory sites has been difficult due to diverse cellular players involved in immunity and endogenous targets being expressed at basal levels. Here we exploit a physiological molecular interaction between intercellular adhesion molecule (ICAM)-1 and lymphocyte function associated antigen (LFA)-1 to deliver a potent anti-inflammatory drug, celastrol, specifically and comprehensively to inflamed cells. We found that affinity and avidity adjusted inserted (I) domain, the major binding site ! of LFA-1, on liposome surface enhanced the specificity toward lipopolysaccharides (LPS)-treated or inflamed endothelial cells (HMEC-1) and monocytes (THP-1) via ICAM-1 overexpression, reflecting inherent affinity and avidity modulation of these molecules in physiology. Targeted delivery of celastrol protected cells from recurring LPS challenges, suppressing pro-inflammatory responses and inflammation-induced cell proliferation. Targeted delivery also blocked THP-1 adhesion to inflamed HMEC-1, forming barriers to immune cell accumulation and to aggravating inflammatory signals. Our results demonstrate affinity and avidity of targeting moieties on nanoparticles as important design parameters to ensure specificity and avoid toxicities. We anticipate that such tunable physiologic interactions could be used for designing effective drug carriers for in vivo applications and contribute to treating a range of immune and inflammatory diseases.
• Hierarchical nanoengineered surfaces for enhanced cytoadhesion and drug delivery
  - Biomater 32(13):3499-3506 (2011)
  Delivering therapeutics to mucosal tissues such as the nasal and gastrointestinal tracts is highly desirable due to ease of access and dense vasculature. However, the mucus layer effectively captures and removes most therapeutic macromolecules and devices. In previous work, we have shown that nanoengineered microparticles (NEMPs) adhere through the mucus layer, exhibiting up to 1000 times the pull-off force of an unmodified microsphere, and showing greater adhesion than some chemical targeting means. In this paper, we demonstrate that nanotopography improves device adhesion in vivo, increasing retention time up to ten-fold over unmodified devices. Moreover, we observe considerable adhesion in several cell lines using an in vitro shear flow model, indicating that this approach is promising for numerous tissues. We then demonstrate that nanowire-mediated adhesion is highly robust to variation in nanowire surface charge and cellular structure and function, and we characte! rize particle loading and elution. We present a form of cytoadhesion that utilizes the physical interaction of nanoengineered surfaces with subcellular structures to produce a robust and versatile cytoadhesive for drug delivery. These nanoscale adhesive mechanisms are also relevant to fields such as tissue engineering and wound healing because they likely affect stem cell differentiation, cell remodeling, migration, etc.
• The intracellular plasmid DNA localization of cationic reducible cholesterol-disulfide lipids
  - Biomater 32(13):3507-3519 (2011)
  Stimuli-responsive biomaterials derived from natural products toward efficient drug/gene delivery have been attracting increasing attention in the past decade. In this work, we first designed and prepared a new series of cholesterol-disulfide lipids, namely CHOSS-N, CHOSS-N+, CHOSS-Lys and CHOSS-4N bearing cholesterol and a variety of headgroups via disulfide and carbonate bond linkages, and their molecular structures were characterized by NMR and ESI-MS. Furthermore, plasmid DNA binding affinity for these new CHOSS lipids was separately examined by ethidium bromide displacement and agarose-gel retardant assay. Average diameter sizes and surface potentials of the CHOSS/pDNA lipoplex particles prepared under various N/P charge ratios were analyzed by dynamic laser light scattering (DLS). Under 10 mm dithiothreitol (DTT), stability and disassembly of the CHOSS/pDNA lipoplex nanoparticles were investigated by agarose-gel retardant assay and atomic force microscopy (AFM). ! Employing a COS-7 cell line, cell viability was examined for the prepared CHOSS lipids and their pDNA lipoplexes with branched PEI-25k as the reference. Finally, COS-7 cell gene transfection efficacies with these CHOSS lipids as potential delivery vectors were investigated by luciferase and EGFP transfection assay in the absence and presence of serum, and intracellular uptake capability, trafficking and cellular localization of Cy3-labeled pEGFP-N1 DNA were studied with a flow cytometer and fluorescent microscopy with Lipofectamine™ 2000 as the control. The results demonstrated low cytotoxicity, strong pDNA binding affinity and high transgenetic efficacy for new prepared CHOSS lipids, and particularly high intracellular uptake capability and specific cellular localization of pDNA at the periphery of cell nuclei were for the first time interestingly observed for the CHOSS lipid delivery carriers. In general, these may pave a new way to utilize cholesterol, amino acids and ! other functional natural products to prepare efficient gene/dr! ug delivery carriers with simple structure and low cytotoxicity.
• Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism
  - Biomater 32(13):3520-3537 (2011)
  Functional peptides that transfer biomaterials, such as semiconductor quantum dots (QDs), into cells in biomaterial research have been developed in recent years. Delivery of QDs conjugated with cell-penetrating peptides (CPPs) into cells by the endocytic pathway was problematic in biomedical applications because of lysosomal trapping. Here, we demonstrate that histidine- and arginine-rich CPPs (HR9 peptides) stably and noncovalently combined with QDs are able to enter into cells in an extremely short period (4 min). Interrupting both F-actin polymerization and active transport did not inhibit the entry of HR9/QD complexes into cells, indicating that HR9 penetrates cell membrane directly. Subcellular colocalization studies indicated that QDs delivered by HR9 stay in cytosol without any organelle capture. Dimethyl sulphoxide, ethanol and oleic acid, but not pyrenebutyrate, enhanced HR9-mediated intracellular delivery of QDs by promoting the direct membrane translocation ! pathway. HR9 and HR9/QDs were not cytotoxic. These findings suggest that HR9 could be an efficient carrier to deliver drugs without interfering with their therapeutic activity.
• Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles
  - Biomater 32(13):3538-3546 (2011)
  TRAIL has received considerable attention as a potential anti-cancer agent due to its specific ability to target tumors. However, recombinant TRAIL has several limitations, such as, its short biological half-life, its inherent instability, and its potential hepatotoxicity. In this study, we developed a sustained release nanoparticle formulation of TRAIL and investigated its therapeutic effects in tumor-bearing mice. TRAIL-loaded nanoparticles (NPs) were prepared by mixing PEGylated heparin (PEG-HE), poly-l-lysine (PLL), and TRAIL. NPs prepared by the ionic interaction between polymer and TRAIL showed uniform spherical structures of diameter 213.3 ± 9.7 nm and a surface charge of 5.33 ± 1.2 mV. An in vitro study of the bioactivity of TRAIL in NPs showed that TRAIL-loaded PEG-HE/PLL NPs (TRAIL-PEG-NPs) were slightly less cytotoxic than TRAIL in vitro. To investigate pharmacokinetic parameters, TRAIL and TRAIL-PEG-NPs were intravenously injected into SD rats. The PEG-NP! -based formulation demonstrated a 28.3 fold greater half-life than TRAIL alone. To evaluate the anti-tumor effect, TRAIL, TRAIL-loaded HE/PLL NPs (TRAIL-NPs), and TRAIL-PEG-NPs were intravenously injected into HCT-116 tumor-bearing BALB/c athymic mice. The TRAIL-PEG-NP formulation efficiently suppressed tumor growth (>70%), and histological findings confirmed that NPs induced significant tumor cell apoptosis without inducing liver toxicity. The PEG-exposed NP fabrication method applied in this study could be widely applied to protein and peptide delivery systems.

Hot off the presses! Feb 18 Cell

The Feb 18 issue of the Cell is now up on Pubget (About Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• In This Issue
  - cell 144(4):455, 457 (2011)
  
• Cell Culture: Academy Awards
  - cell 144(4):459, 461 (2011)
  On February 27, 2011, Hollywood's royalty will gather in Kodak Theater to honor the best films of 2010. To add a scientific twist on this year's Academy Awards, Cell Culture dives beneath the skin of the top films' protagonists, identifying a brain structure that impacts our "friend count," genes that make a king stammer, a cellular fragmentation process that saves a solo hiker, and stem cells required for a ballerina to grow feathers. May I have the viral envelope, please?
• The Grand Finale
  - cell 144(4):463-464 (2011)
  
• Targeting Aneuploidy for Cancer Therapy
  - cell 144(4):465-466 (2011)
  Tumor cells frequently display an abnormal number of chromosomes, a phenomenon known as aneuploidy. Tang et al. (2011) now show that aneuploid cells are particularly sensitive to compounds that induce proteotoxic and energy stress. Could this vulnerability lead to new cancer therapies?
• IL-7 Knocks the Socs Off Chronic Viral Infection
  - cell 144(4):467-468 (2011)
  Chronic viral infections represent a major burden to human health, and modulation of the immune system is emerging as a novel approach to fighting such infections. Pellegrini et al. (2011) demonstrate that treatment with the cytokine IL-7 may reinvigorate the immune response to persistent infection by targeting immunosuppressive Socs3 proteins.
• Microbial Communication Superhighways
  - cell 144(4):469-470 (2011)
  Exchange of information is critical for bacterial social behaviors. Now Dubey and Ben-Yehuda (2011) provide evidence for bacterial "nanotube" conduits that allow microbes to directly exchange cytoplasmic factors. Protein and DNA transfer between distantly related species raises the prospect of a new, widely distributed mechanism of bacterial communication.
• Epigenetic Centromere Propagation and the Nature of CENP-A Nucleosomes
  - cell 144(4):471-479 (2011)
  Centromeres direct chromosome inheritance, but in multicellular organisms their positions on chromosomes are primarily specified epigenetically rather than by a DNA sequence. The major candidate for the epigenetic mark is chromatin assembled with the histone H3 variant CENP-A. Recent studies offer conflicting evidence for the structure of CENP-A-containing chromatin, including the histone composition and handedness of the DNA wrapped around the histones. We present a model for the assembly and deposition of centromeric nucleosomes that couples these processes to the cell cycle. This model reconciles divergent data for CENP-A-containing nucleosomes and provides a basis for how centromere identity is stably inherited.
• Revisiting the Central Dogma One Molecule at a Time
  - cell 144(4):480-497 (2011)
  The faithful relay and timely expression of genetic information depend on specialized molecular machines, many of which function as nucleic acid translocases. The emergence over the last decade of single-molecule fluorescence detection and manipulation techniques with nm and Å resolution and their application to the study of nucleic acid translocases are painting an increasingly sharp picture of the inner workings of these machines, the dynamics and coordination of their moving parts, their thermodynamic efficiency, and the nature of their transient intermediates. Here we present an overview of the main results arrived at by the application of single-molecule methods to the study of the main machines of the central dogma.
• Identification of Aneuploidy-Selective Antiproliferation Compounds
  - cell 144(4):499-512 (2011)
  Aneuploidy, an incorrect chromosome number, is a hallmark of cancer. Compounds that cause lethality in aneuploid, but not euploid, cells could therefore provide new cancer therapies. We have identified the energy stress-inducing agent AICAR, the protein folding inhibitor 17-AAG, and the autophagy inhibitor chloroquine as exhibiting this property. AICAR induces p53-mediated apoptosis in primary mouse embryonic fibroblasts (MEFs) trisomic for chromosome 1, 13, 16, or 19. AICAR and 17-AAG, especially when combined, also show efficacy against aneuploid human cancer cell lines. Our results suggest that compounds that interfere with pathways that are essential for the survival of aneuploid cells could serve as a new treatment strategy against a broad spectrum of human tumors.
• Role for Dpy-30 in ES Cell-Fate Specification by Regulation of H3K4 Methylation within Bivalent Domains
  - cell 144(4):513-525 (2011)
  Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental g! enes during the ESC fate transitions.
• ATP Binds to Proteasomal ATPases in Pairs with Distinct Functional Effects, Implying an Ordered Reaction Cycle
  - cell 144(4):526-538 (2011)
  In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.
• Phosphorylation of Nup98 by Multiple Kinases Is Crucial for NPC Disassembly during Mitotic Entry
  - cell 144(4):539-550 (2011)
  Disassembly of nuclear pore complexes (NPCs) is a decisive event during mitotic entry in cells undergoing open mitosis, yet the molecular mechanisms underlying NPC disassembly are unknown. Using chemical inhibition and depletion experiments we show that NPC disassembly is a phosphorylation-driven process, dependent on CDK1 activity and supported by members of the NIMA-related kinase (Nek) family. We identify phosphorylation of the GLFG-repeat nucleoporin Nup98 as an important step in mitotic NPC disassembly. Mitotic hyperphosphorylation of Nup98 is accomplished by multiple kinases, including CDK1 and Neks. Nuclei carrying a phosphodeficient mutant of Nup98 undergo nuclear envelope breakdown slowly, such that both the dissociation of Nup98 from NPCs and the permeabilization of the nuclear envelope are delayed. Together, our data provide evidence for a phosphorylation-dependent mechanism underlying disintegration of NPCs during prophase. Moreover, we identify mitotic pho! sphorylation of Nup98 as a rate-limiting step in mitotic NPC disassembly.
• Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles
  - cell 144(4):551-565 (2011)
  Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo, but their overall composition on axonal vesicles and whether this composition directly modulates transport activity are unknown. Here we characterize the intracellular transport and steady-state motor subunit composition of mammalian prion protein (PrPC) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrPC vesicle motor complexes and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model wherein PrPC vesicles maintain a stable population of associated motors whose activity is ! modulated by regulatory factors instead of by structural changes to motor-cargo associations.
• DNA Damage in Oocytes Induces a Switch of the Quality Control Factor TAp63α from Dimer to Tetramer
  - cell 144(4):566-576 (2011)
  TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with 20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that pro! vide the basis for regulating quality control in oocytes.
• The Basement Membrane of Hair Follicle Stem Cells Is a Muscle Cell Niche
  - cell 144(4):577-589 (2011)
  The hair follicle bulge in the epidermis associates with the arrector pili muscle (APM) that is responsible for piloerection ("goosebumps"). We show that stem cells in the bulge deposit nephronectin into the underlying basement membrane, thus regulating the adhesion of mesenchymal cells expressing the nephronectin receptor, α8β1 integrin, to the bulge. Nephronectin induces α8 integrin-positive mesenchymal cells to upregulate smooth muscle markers. In nephronectin knockout mice, fewer arrector pili muscles form in the skin, and they attach to the follicle above the bulge, where there is compensatory upregulation of the nephronectin family member EGFL6. Deletion of α8 integrin also abolishes selective APM anchorage to the bulge. Nephronectin is a Wnt target; epidermal β-catenin activation upregulates epidermal nephronectin and dermal α8 integrin expression. Thus, bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon ! cells for the APM. Our results reveal a functional role for basement membrane heterogeneity in tissue patterning. PaperClip
• Intercellular Nanotubes Mediate Bacterial Communication
  - cell 144(4):590-600 (2011)
  Bacteria are known to communicate primarily via secreted extracellular factors. Here we identify a previously uncharacterized type of bacterial communication mediated by nanotubes that bridge neighboring cells. Using Bacillus subtilis as a model organism, we visualized transfer of cytoplasmic fluorescent molecules between adjacent cells. Additionally, by coculturing strains harboring different antibiotic resistance genes, we demonstrated that molecular exchange enables cells to transiently acquire nonhereditary resistance. Furthermore, nonconjugative plasmids could be transferred from one cell to another, thereby conferring hereditary features to recipient cells. Electron microscopy revealed the existence of variously sized tubular extensions bridging neighboring cells, serving as a route for exchange of intracellular molecules. These nanotubes also formed in an interspecies manner, between B. subtilis and Staphylococcus aureus, and even between B. subtilis and the evo! lutionary distant bacterium Escherichia coli. We propose that nanotubes represent a major form of bacterial communication in nature, providing a network for exchange of cellular molecules within and between species. PaperFlick
• IL-7 Engages Multiple Mechanisms to Overcome Chronic Viral Infection and Limit Organ Pathology
  - cell 144(4):601-613 (2011)
  Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications f! or its use as a therapeutic in the treatment of chronic viral diseases.
• The Coding of Temperature in the Drosophila Brain
  - cell 144(4):614-624 (2011)
  Thermosensation is an indispensable sensory modality. Here, we study temperature coding in Drosophila, and show that temperature is represented by a spatial map of activity in the brain. First, we identify TRP channels that function in the fly antenna to mediate the detection of cold stimuli. Next, we identify the hot-sensing neurons and show that hot and cold antennal receptors project onto distinct, but adjacent glomeruli in the Proximal-Antennal-Protocerebrum (PAP) forming a thermotopic map in the brain. We use two-photon imaging to reveal the functional segregation of hot and cold responses in the PAP, and show that silencing the hot- or cold-sensing neurons produces animals with distinct and discrete deficits in their behavioral responses to thermal stimuli. Together, these results demonstrate that dedicated populations of cells orchestrate behavioral responses to different temperature stimuli, and reveal a labeled-line logic for the coding of temperature informat! ion in the brain.
• SnapShot: Chromatin Remodeling: CHD
  - cell 144(4):626-626.e1 (2011)
  

Hot off the presses! Mar 01 Nat Meth

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Latest Articles Include:

• Into the fold
  - Nat Meth 8(3):185 (2011)
  Nature Methods | Editorial Into the fold Journal name:Nature MethodsVolume: 8,Page:185Year published:(2011)DOI:doi:10.1038/nmeth0311-185Published online25 February 2011 In spite of its promise, nanotechnology has seen little uptake among biologists. DNA origami may be able to avoid this fate. View full text Additional data
• The author file: Thomas Clandinin
  - Nat Meth 8(3):187 (2011)
  Nature Methods | This Month The author file: Thomas Clandinin * Monya BakerJournal name:Nature MethodsVolume: 8,Page:187Year published:(2011)DOI:doi:10.1038/nmeth0311-187Published online25 February 2011 A new genetic construct enhances enhancer traps. View full text Additional data
• Points of view: Points of review (part 2)
  - Nat Meth 8(3):189 (2011)
  Nature Methods | This Month Points of view: Points of review (part 2) * Bang Wong1Journal name:Nature MethodsVolume: 8,Page:189Year published:(2011)DOI:doi:10.1038/nmeth0311-189Published online25 February 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. I will continue to demonstrate how judicious choice of graphical representations can improve visual communication. Here I will focus on data figures. The power and primary purpose of graphs is to reveal connections in data. As opposed to tables, in which there is little visual association between individual values, graphs and charts depend on readers to form patterns. In reading graphs, we observe individual data points, keep each of them in memory and construct an image from the constituents. The entire process can be exceedingly fast and attest to the power of visual perception. Graphical encoding needs to support the detection and assembly process of reading graphs. We are more accurate at certain types of visual estimation than others (September 2010 column)1. For example, to understand relative differences between categories, a standard bar chart might be easier to read than a pie chart, particularly to appreciate the direction and magnitude of change (Fig. 1). Small differences are more readily apparent when we compare length of bars (Fig. 1c) than sizes of pie slices (Fig. 1a)2. Figure 1: Certain visual encodings are easier to read. (,) Analysis of genetic interactions. Adapted and reprinted from Nature Methods2. () A bar chart showing data from the pie chart in . () A method for ordering slices of a pie chart. () Multiple views to show overlapping data from . Former 'yellow' and 'blue' categories are shown in purple and green, respectively. * Full size image (80 KB) * Figures index * Next figure Pie charts can be useful. Although they are not intended to show complex relationships, pie charts do well to depict parts of a whole. The Wall Street Journal Guide to Information Graphics3 suggests an ordering of slices to aid reading: place the largest wedge to the right of 12 o'clock, the second largest to the left of 12 o'clock and the remainder counter-clockwise descending in size (Fig. 1d). In this way, the largest (and presumably most important) wedges end up at the top. With the two largest slices sharing a vertical edge, we can rely on reading angles to estimate proportion. View full text Figures at a glance * Figure 1: Certain visual encodings are easier to read. (,) Analysis of genetic interactions. Adapted and reprinted from Nature Methods2. () A bar chart showing data from the pie chart in . () A method for ordering slices of a pie chart. () Multiple views to show overlapping data from . Former 'yellow' and 'blue' categories are shown in purple and green, respectively. * Figure 2: Color is not ideal for presenting quantitative data. () Shifts in color scales (circles) are not visually commensurate with change in value. Reprinted from Nature Methods2, 5. () A gradation from 10–90% black produces even transitions. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Bang Wong is the creative director of the Broad Institute of the Massachusetts Institute of Technology and Harvard and an adjunct assistant professor in the Department of Art as Applied to Medicine at The Johns Hopkins University School of Medicine. Author Details * Bang Wong Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Taxonomic metagenome sequence assignment with structured output models
  - Nat Meth 8(3):191-192 (2011)
  Nature Methods | Correspondence Taxonomic metagenome sequence assignment with structured output models * Kaustubh R Patil1 * Peter Haider2 * Phillip B Pope3 * Peter J Turnbaugh4 * Mark Morrison3 * Tobias Scheffer2 * Alice C McHardy1, 5 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:191–192Year published:(2011)DOI:doi:10.1038/nmeth0311-191Published online25 February 2011 To the Editor: Computational inference of the taxonomic origin of sequence fragments is an essential step in metagenome analysis1. Fragments can be assigned to individual populations or corresponding higher-level evolutionary clades using methods based on homology, sequence similarity or sequence composition2. This is a challenging task, as for most uncultured microorganisms reference sequences are unavailable, and large amounts of data have to be processed. With this in mind, we introduce PhyloPythiaS a successor of our previously published method PhyloPythia3. It is a fast and accurate sequence compositional classifier based on the structured output paradigm4. We evaluated PhyloPythiaS on simulated and real metagenome data in comparison to four other methods: PhyloPythia3, metagenome analyzer (MEGAN)5, Phymm and PhymmBL6. PhyloPhythiaS performed particularly well for taxonomic assignment of populations from novel genera, order or higher-level clades, when limited amounts of reference data were available. We observed that accurate assignments were obtained based on 100 kilobases (kb) of training data for a sample population. We observed this for simulated data (Fig. 1a, Supplementary Fig. 1 and Supplementary Table 1) and a predominant population of a novel family of the order of Aeromonadales from the Australian Tammar wallaby gut (Fig. 1b, Supplementary Fig. 2 and Supplementary Tables 2–4). We performed experiments on simulated data for four settings: with genomes of the same species for the dominant strains made available as reference data (known species) or with genomes of the corresponding higher-level clades (genus, order an! d class) removed, while retaining 100 kb of sequence for the dominant strains. In this scenario, alignment-based methods performed poorly. If closely related genomeswere available, the performance of all methods became more similar, with a slight advantage for alignment-based approaches. We observed this for simulated data and the predominant genera of two human gut metagenomes (Supplementary Tables 5–8). back to article Figure 1: Comparison of taxonomic classification methods. () Average performance per contig for the simMC dataset at genus rank. () Scaffold-contig consistency for the WG-1 population (uncultured Succinivibrionaceae bacterium) of the Tammar wallaby gut metagenome. Contig coloring reflects taxonomic assignment consistency with respect to WG-1. Only scaffolds longer than 20 kb are shown. () Empirical execution time evaluated on a Linux machine with 3-gigahertz processor and 4 gigabytes main memory. Results for MEGAN and PhymmBL were determined with a reference database of size 2.1 gigabases. View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Max-Planck Research Group for Computational Genomics and Epidemiology, Max Planck Institute for Informatics, Saarbrücken, Germany. * Kaustubh R Patil & * Alice C McHardy * University of Potsdam, Department of Computer Science, Potsdam, Germany. * Peter Haider & * Tobias Scheffer * Commonwealth Scientific and Industrial Research Organization Livestock Industries, Queensland Bioscience Precinct, St Lucia, Australia. * Phillip B Pope & * Mark Morrison * Harvard Faculty of Arts and Sciences Center for Systems Biology, Cambridge Massacusetts, USA. * Peter J Turnbaugh * Department of Algorithmic Bioinformatics, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany. * Alice C McHardy Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alice C McHardy Author Details * Kaustubh R Patil Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Haider Search for this author in: * NPG journals * PubMed * Google Scholar * Phillip B Pope Search for this author in: * NPG journals * PubMed * Google Scholar * Peter J Turnbaugh Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Morrison Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Scheffer Search for this author in: * NPG journals * PubMed * Google Scholar * Alice C McHardy Contact Alice C McHardy Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–5, Supplementary Tables 1–14, Supplementary Note Additional data * Journal home * Current issue * For authors * Subscribe * E-alert sign up * RSS feed Science jobs from naturejobs * Research Scientist – Gene Therapy * GlaxoSmithKline * Stevenage, Hertfordshire, UK * Junior-Professor W 1 "New Innovative Materials for Photonic Technologies" * Friedrich-Schiller-Universitat Jena * Jena, Germany * Research Professorship "Clinical Epidemiology / Public Health" (W2) * Friedrich-Schiller-Universitat Jena * Jena, Germany * Post a free job * More science jobs Related content Articles * Recovery of intact DNA nanostructures after agarose gel–based separation Nature Methods 25 Feb 2011 * Designer enzymes for glycosphingolipid synthesis by directed evolution Nature Chemical Biology 14 Jun 2009 * Gene-specific RNA polymerase II phosphorylation and the CTD code Nature Structural & Molecular Biology 12 Sep 2010 * The human cytomegalovirus microRNA miR-UL112 acts synergistically with a cellular microRNA to escape immune elimination Nature Immunology 08 Aug 2010 * A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans Nature Medicine 22 Feb 2009 View all Open innovation challenges * Derivation of Trophoblast Stem Cells from Human iPS Cells or Human ES Cells Deadline:Mar 13 2011Reward:$50,000 USD The Seeker wishes to derive trophoblast stem (TS) cells from human induced pluripotent stem (iPS) ce… * RNAi Sequences Targeted to the Asian Citrus Psyllid Genome Deadline:May 03 2011Reward:$100,000 USD The non-profit Citrus Research and Development Foundation, desires proposals for RNA sequences that … * Powered by: * More challenges Top content Emailed * Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns Nature Methods 06 Feb 2011 * Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster Nature Methods 06 Feb 2011 * High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision Nature Methods 18 Apr 2010 * Strategies for protein coexpression in Escherichia coli Nature Methods 01 Jan 2006 * Unrestrained worms bridled by the light Nature Methods 28 Jan 2011 View all Downloaded * Mapping and quantifying mammalian transcriptomes by RNA-Seq Nature Methods 30 May 2008 * Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes Nature Methods 13 Feb 2011 * A microprobe for parallel optical and electrical recordings from single neurons in vivo Nature Methods 13 Feb 2011 * Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster Nature Methods 06 Feb 2011 * Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns Nature Methods 06 Feb 2011 View all Blogged * Limitations of next-generation genome sequence assembly Nature Methods 21 Nov 2010 * Rapid blue-light–mediated induction of protein interactions in living cells Nature Methods 31 Oct 2010 View all * Nature Methods * ISSN: 1548-7091 * EISSN: 1548-7105 * About NPG * Contact NPG * RSS web feeds * Help * Privacy policy * Legal notice * Accessibility statement * Terms * Nature News * Naturejobs * Nature Asia * Nature EducationSearch:Go © 2011 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.partner of AGORA, HINARI, OARE, INASP, CrossRef and COUNTER
• Recovery of intact DNA nanostructures after agarose gel–based separation
  - Nat Meth 8(3):192-194 (2011)
  Nature Methods | Correspondence Recovery of intact DNA nanostructures after agarose gel–based separation * Gaëtan Bellot1, 2, 4 * Mark A McClintock1, 2, 4 * Chenxiang Lin1, 2, 3 * William M Shih1, 2, 3 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:192–194Year published:(2011)DOI:doi:10.1038/nmeth0311-192Published online25 February 2011 To the Editor: Molecular self-assembly using DNA as a structural building block has proven an efficient route for construction of nanoscale objects and arrays of ever-increasing complexity1. An important catalyst for advancing the field in recent years has been the scaffolded DNA origami strategy, in which a long 'scaffold' strand derived from a viral genome (M13) can be folded with hundreds of short synthetic 'staple' strands into a variety of custom two- and three-dimensional shapes2, 3. This technology is being used to develop molecular tools for applications in fields such as structural biology4, single-molecule biophysics and drug delivery. Many of these applications require a homogenous sample of properly folded nanostructures greatly enriched over the misfolded intermediates and large aggregates characteristic of multilayer DNA-origami self-assembly. Agarose-gel electrophoresis is now the most effective method available for high-resolution separation of well-folded objects on this size scale, but extraction of intact DNA nanostructures with high yield from the agarose matrix is problematic. Existing methods rely on thermal, chemical and/or mechanical destruction of the agarose gel or else electroelution of the DNA to a solid support, leading to problems of low yield, damage to structures and/or contamination with residual agarose. We modified a DNA electroelution method for recovery of DNA from a standard horizontal agarose-gel electrophoresis apparatus to optimize it for efficient, high-resolution and scalable recovery of large and complex intact DNA nanostructures5, 6. Our initial attempts to purify DNA nanostructures by electroelution revealed the need for a well-sealed elution bed to eliminate high-conductivity buffer paths that served as escape routes for the nanostructures. To address this problem, we poured a 1–! 2% agarose resolving gel on top of a thinner and more rigid basement layer of 4% agarose previously set in the gel-casting tray (Supplementary Fig. 1 and Supplementary Methods). Once the sample was sufficiently resolved on our dual-layer agarose system, we cut an elution well in the resolving gel directly in front of the band of interest and filled it with a viscous solution of 30–50% sucrose. The elution well is simple to cut down to the interface with the 4% agarose layer because of the difference in rigidity of the layers, and the seal between the layers adjacent to the elution well is not disturbed. To eliminate high-conductivity paths in buffer above the gel, we maintained the running buffer level even with, or below, the surface of the resolving gel. We eluted the band by electrophoresis of the sample into the sucrose bed where movement of the DNA was slowed enough to allow efficient recovery by UV-light detection and micropipetting. The identity of the elution buffer has profound consequences for the efficacy of purification. Using a 400-nm-long 6-helix bundle nanostructure as a model to assess purification performance (Fig. 1a and Supplementary Table 1), we screened three solutes at varying concentrations. Use of glycerol or polyethylene glycol resulted in retarded migration of the DNA band and a slow elution time of 1–3 hours, with inconsistent recovery yields between 20% and 60% (Supplementary Fig. 2). We obtained the greatest yields with solutions of 30–50% sucrose (Supplementary Fig. 3). ImageJ analysis of the gels for purified 6-helix bundles indicated 71 ± 3% of the well-folded structure could be recovered from the agarose matrix versus 15 ± 5% by the pellet-pestle homogenization method7. Our analysis by negative-stain transmission electron microscopy (TEM) also indicated a strong enrichment of the properly folded structures. back to article Figure 1: Agarose-gel and TEM analyses of various DNA origami objects after gel purification. (–) Cylinder models (left; each cylinder represents a DNA double helix) of a 6-helix bundle (), 12-helix bundle (), 6-helix bundle ring () and prestressed tensegrity-structure kite (). In gel images (middle), lanes for each object were cropped from a single gel. Ladder, kilobase ladder. For unpurified DNA nanostructures (unpurified), arrows indicate the region of each lane that was extracted from the gel during purification before TEM imaging. Also shown are 30% sucrose gel–purified nanostructures (sucrose) and pellet-pestle homogenization–recovered gel-purified nanostructures (homogenization), with estimates of yields after purification indicated. TEM micrographs (right) of the nanostructures after 30% sucrose gel purification. Scale bars, 100 nm (,,), 50 nm () and in insets, 70 nm (), 80 nm (), 25 nm () and 50 nm (). View full text Subject terms: * Molecular Engineering * Single Molecule Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Primary authors * These authors contributed equally to this work. * Gaëtan Bellot & * Mark A McClintock Affiliations * Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Gaëtan Bellot, * Mark A McClintock, * Chenxiang Lin & * William M Shih * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Gaëtan Bellot, * Mark A McClintock, * Chenxiang Lin & * William M Shih * Wyss Institute for Biologically Inspired Engineering at Harvard, Cambridge, Massachusetts, USA. * Chenxiang Lin & * William M Shih Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * William M Shih Author Details * Gaëtan Bellot Search for this author in: * NPG journals * PubMed * Google Scholar * Mark A McClintock Search for this author in: * NPG journals * PubMed * Google Scholar * Chenxiang Lin Search for this author in: * NPG journals * PubMed * Google Scholar * William M Shih Contact William M Shih Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–9, Supplementary Table 1, Supplementary Methods Additional data * Journal home * Current issue * For authors * Subscribe * E-alert sign up * RSS feed Science jobs from naturejobs * Research Professorship "Clinical Epidemiology / Public Health" (W2) * Friedrich-Schiller-Universitat Jena * Jena, Germany * Research Scientist – Gene Therapy * GlaxoSmithKline * Stevenage, Hertfordshire, UK * Professor for Radiology (W3) * Friedrich-Schiller-Universitat Jena * Jena, Germany * Post a free job * More science jobs Related content Articles * Inhibition of a viral enzyme by a small-molecule dimer disruptor Nature Chemical Biology 26 Jul 2009 * Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/β-catenin signaling Nature Genetics 01 Aug 2008 * Taxonomic metagenome sequence assignment with structured output models Nature Methods 25 Feb 2011 * Correlated conformational events in EF-G and the ribosome regulate translocation Nature Structural & Molecular Biology 07 Nov 2010 * Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers Nature Structural & Molecular Biology 31 Oct 2010 View all Open innovation challenges * Derivation of Trophoblast Stem Cells from Human iPS Cells or Human ES Cells Deadline:Mar 13 2011Reward:$50,000 USD The Seeker wishes to derive trophoblast stem (TS) cells from human induced pluripotent stem (iPS) ce… * Chordoma Cancer Cell Lines Needed to Save Lives! Deadline:Mar 13 2011Reward:$10,000 USD The Chordoma Foundation requests cell lines or animal models that can be used for research into chor… * Powered by: * More challenges Top content Emailed * Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns Nature Methods 06 Feb 2011 * Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster Nature Methods 06 Feb 2011 * High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision Nature Methods 18 Apr 2010 * Strategies for protein coexpression in Escherichia coli Nature Methods 01 Jan 2006 * Unrestrained worms bridled by the light Nature Methods 28 Jan 2011 View all Downloaded * Mapping and quantifying mammalian transcriptomes by RNA-Seq Nature Methods 30 May 2008 * Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes Nature Methods 13 Feb 2011 * A microprobe for parallel optical and electrical recordings from single neurons in vivo Nature Methods 13 Feb 2011 * Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster Nature Methods 06 Feb 2011 * Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns Nature Methods 06 Feb 2011 View all Blogged * Limitations of next-generation genome sequence assembly Nature Methods 21 Nov 2010 * Rapid blue-light–mediated induction of protein interactions in living cells Nature Methods 31 Oct 2010 View all * Nature Methods * ISSN: 1548-7091 * EISSN: 1548-7105 * About NPG * Contact NPG * RSS web feeds * Help * Privacy policy * Legal notice * Accessibility statement * Terms * Nature News * Naturejobs * Nature Asia * Nature EducationSearch:Go © 2011 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.partner of AGORA, HINARI, OARE, INASP, CrossRef and COUNTER
• TALEs for the masses
  - Nat Meth 8(3):197 (2011)
  Nature Methods | Research Highlights TALEs for the masses * Nicole RuskJournal name:Nature MethodsVolume: 8,Page:197Year published:(2011)DOI:doi:10.1038/nmeth0311-197Published online25 February 2011 An optimized transcription activator–like effector (TALE) and an improved assembly method promise efficient genome editing and transcriptome modulation. View full text Subject terms: * Genomics Additional data Author Details * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google Scholar
• Brains gone wild
  - Nat Meth 8(3):198-199 (2011)
  Nature Methods | Research Highlights Brains gone wild * Erika PastranaJournal name:Nature MethodsVolume: 8,Pages:198–199Year published:(2011)DOI:doi:10.1038/nmeth0311-198aPublished online25 February 2011 Analyzing brain signals from freely moving rodents in the wild is possible using a wireless neural recording system. View full text Subject terms: * Neuroscience Additional data Author Details * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google Scholar
• Bacteria's puppeteers
  - Nat Meth 8(3):198-199 (2011)
  Nature Methods | Research Highlights Bacteria's puppeteers * Erika PastranaJournal name:Nature MethodsVolume: 8,Pages:198–199Year published:(2011)DOI:doi:10.1038/nmeth0311-198bPublished online25 February 2011 Gene expression in bacteria can be modulated in response to unnatural amino acids with engineered transcriptional systems. View full text Subject terms: * Synthetic Biology Additional data Author Details * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google Scholar
• News in brief
  - Nat Meth 8(3):199 (2011)
  Nature Methods | Research Highlights News in brief Journal name:Nature MethodsVolume: 8,Page:199Year published:(2011)DOI:doi:10.1038/nmeth0311-199Published online25 February 2011 Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Advances in label-free chemical imaging Stimulated Raman scattering is a label-free biomedical imaging technique based on vibrational spectroscopy. In its original implementation, narrow-band laser beams had been used to excite a single Raman-active mode, but molecules with overlapping Raman bands could not be distinguished. Freudiger et al. now introduce spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy, which applies collective excitation of selected vibrational frequencies to allow specific molecules to be imaged, even when interfering species are present. Freudiger, C.W.et al. Nat. Photonics5, 103–109 (2011). View full text Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• A day in the half-life of a protein
  - Nat Meth 8(3):201 (2011)
  Nature Methods | Research Highlights A day in the half-life of a protein * Allison DoerrJournal name:Nature MethodsVolume: 8,Page:201Year published:(2011)DOI:doi:10.1038/nmeth0311-201Published online25 February 2011 Researchers describe a method called bleach-chase to quantitatively measure the half-lives of fluorescently tagged proteins in human cancer cells. View full text Subject terms: * Proteomics Additional data Author Details * Allison Doerr Search for this author in: * NPG journals * PubMed * Google Scholar
• Imaging impedance
  - Nat Meth 8(3):202 (2011)
  Nature Methods | Research Highlights Imaging impedance * Allison DoerrJournal name:Nature MethodsVolume: 8,Page:202Year published:(2011)DOI:doi:10.1038/nmeth0311-202Published online25 February 2011 A label-free microscopy technique based on electrochemical impedance offers a new way of studying electrochemical processes in single cells. View full text Subject terms: * Microscopy Additional data Author Details * Allison Doerr Search for this author in: * NPG journals * PubMed * Google Scholar
• What makes flies and worms tick
  - Nat Meth 8(3):204 (2011)
  Nature Methods | Research Highlights What makes flies and worms tick * Nicole RuskJournal name:Nature MethodsVolume: 8,Page:204Year published:(2011)DOI:doi:10.1038/nmeth0311-204Published online25 February 2011 The comprehensive mapping of transcripts, histone modifications and transcription factor binding allows for the functional annotation of fly and worm genomes. View full text Subject terms: * Systems Biology Additional data Author Details * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google Scholar
• qPCR: quicker and easier but don't be sloppy
  - Nat Meth 8(3):207-212 (2011)
  Nature Methods | Technology Feature qPCR: quicker and easier but don't be sloppy * Monya Baker1Journal name:Nature MethodsVolume: 8,Pages:207–212Year published:(2011)DOI:doi:10.1038/nmeth0311-207Published online25 February 2011 Gene profiling using quantitative PCR is becoming higher throughput, but researchers must be careful in gathering their data. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker Author Details * Monya Baker Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• A triple threat to single molecules
  - Nat Meth 8(3):213-215 (2011)
  Nature Methods | News and Views A triple threat to single molecules * Martin Gruebele1Journal name:Nature MethodsVolume: 8,Pages:213–215Year published:(2011)DOI:doi:10.1038/nmeth0311-213Published online25 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Three single-molecule methods promise to increase the time resolution of experiments, to allow better access to sparsely populated molecular states and to permit combinatorial high-throughput analysis. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Martin Gruebele is in the Departments of Chemistry and Physics, and in the Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Martin Gruebele Author Details * Martin Gruebele Contact Martin Gruebele Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Double Brainbow
  - Nat Meth 8(3):217-218 (2011)
  Nature Methods | News and Views Double Brainbow * Sebastian Cachero1 * Gregory S X E Jefferis1 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:217–218Year published:(2011)DOI:doi:10.1038/nmeth0311-217Published online25 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Brainbow is a powerful genetic tool for multicolor labeling in mice with applications in fields including developmental biology and neuroanatomy. Now two groups have ported the approach to the fruit fly where it may have even greater impact. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Sebastian Cachero and Gregory S.X.E. Jefferis are in the Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gregory S X E Jefferis Author Details * Sebastian Cachero Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory S X E Jefferis Contact Gregory S X E Jefferis Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Neutral not a loss: phosphoinositides beyond the head group
  - Nat Meth 8(3):219-220 (2011)
  Nature Methods | News and Views Neutral not a loss: phosphoinositides beyond the head group * Matthias P Wymann1 * Markus R Wenk2 * Affiliations * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:219–220Year published:(2011)DOI:doi:10.1038/nmeth0311-219Published online25 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Detection of cellular PtdIns(3,4,5)P3 by combination of chemical derivatization and tandem mass spectroscopy has been demonstrated. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Matthias P. Wymann is at the Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland. * Markus R. Wenk is in the Department of Biochemistry and Department of Biological Sciences, National University of Singapore, Singapore, and the Swiss Tropical and Public Health Institute, University of Basel, Basel, Switzerland. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Matthias P Wymann or * Markus R Wenk Author Details * Matthias P Wymann Contact Matthias P Wymann Search for this author in: * NPG journals * PubMed * Google Scholar * Markus R Wenk Contact Markus R Wenk Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Methods for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• A primer to scaffolded DNA origami
  - Nat Meth 8(3):221-229 (2011)
  Nature Methods | Perspective A primer to scaffolded DNA origami * Carlos Ernesto Castro1 * Fabian Kilchherr1 * Do-Nyun Kim2 * Enrique Lin Shiao1 * Tobias Wauer1 * Philipp Wortmann1 * Mark Bathe2 * Hendrik Dietz1 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:221–229Year published:(2011)DOI:doi:10.1038/nmeth.1570Published online25 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Molecular self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA origami objects. We also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure. View full text Subject terms: * Biochemistry * Synthetic Biology * Molecular Engineering * Biophysics Figures at a glance * Figure 1: Examples of objects built with scaffolded DNA origami. () Designs of single-layer DNA origami shapes (top) and AFM images of these objects (middle and bottom). The pointed star and the smiley face each have diameters of ~100 nm. Reprinted from ref. 1. () AFM image of crystalline DNA origami arrays formed from several hundred copies of a cross-shaped single-layer DNA origami object. Inset, image of a 100-nm-long cross-shaped origami monomer. Reprinted from ref. 18. () Container-like DNA origami objects (left) imaged with negative-stain TEM (top) and cryogenic TEM (bottom); reprinted from ref. 5 (top) and ref. 6 (bottom). () Design and images of multilayer DNA origami objects7. () Image of a multimeric multilayer DNA origami object with global twist deformation8. () Design and images of space-filling multilayer DNA origami objects such as bent bars () and a gear with square teeth () displaying custom curvature8. () Tensegrity prism created by combining multilayer DNA origami struts and ssDNA strings; reprinted from ref. 16. () Des! ign and image of a single-layer DNA origami shape with site-directed protein attachments; reprinted from ref. 19. Scale bars, 100 nm (), 1,000 nm () and 20 nm (). * Figure 2: The scaffolded DNA origami design concept. () In primitives of scaffolded DNA origami, DNA double helices are represented schematically either as two adjacent lines (left; the white line represents the scaffold strand in white and the staple strand in color) or solid cylinders (middle). A detailed rendering of a B-form double-helical domain is also shown (right). () Individual DNA double-helical domains may be connected to adjacent double-helical domains by interhelix cross-overs (arrows). The interhelix connections are formed by U-turns of the covalent phosphate backbone of either the staple or scaffold strand. Interhelix connections are depicted schematically as lines perpendicular to the lines that represent helices. In the cylinder representation, cross-overs are not drawn. () Examples of single- and multilayer scaffold routing solutions through DNA origami object. () Examples for complete scaffold-staple layouts, with staples colored differently to highlight their individual paths through the structures. () Sing! le- and multilayer DNA origami objects in cylinder representation. * Figure 3: Packing and cross-over spacing rules for multilayer DNA origami. () Cross-sectional view of multilayer DNA origami objects in square lattice (left) and honeycomb lattice (right) packing. () Cross-overs in multilayer objects with honeycomb lattice packing, spaced in constant intervals of 7 bp along the helical axis to link double-helical domains to each of three possible neighbors. The cross-over spacing of 7 bp complies with the natural B-form DNA twist density of 10.5 bp per turn, which corresponds to an average backbone rotation of 240° for a given strand in a DNA double-helical domain. * Figure 4: CanDo. () caDNAno design diagram for multilayer DNA origami objects in honeycomb lattice packing with deviations from the constant 7-bp cross-over spacing rule (left). Base-pair insertions and deletions are depicted as loops and crosses, respectively. CanDo 3D structure and local flexibility prediction shown as a heatmap that indicates local root-mean-square fluctuations (RMSFs) (middle). Representative negative-stain TEM micrographs (right). Scale bars, 20 nm. The objects shown in and form circular gears upon multimerization as described elsewhere8. The object shown in was made for this work; note the asymmetry in RMSF between the two 'shoulders' of the object, which can be mapped to an asymmetric distribution of cross-overs in the object design. () CanDo 3D structure and flexibility prediction for a caDNAno design of a tetrameric 60-helix bundle object in honeycomb lattice packing in which insertions are used to create an effective underwinding to 11 bp per turn for each double-h! elical domain in the object. The caDNAno design file is provided in Supplementary Figure 5. CanDo predicts handedness correctly and reproduces within 15% error the extent of global twist deformation as quantified by direct TEM imaging8. Typical TEM data for the twisted ribbon is shown in Fig. 1e. Rendering of a 50 bp long B-form DNA double helix is included as a length reference (50 bp = 17 nm). * Figure 5: Thermal stability and resistance against nucleases of three multilayer scaffolded DNA origami test structures. () Cylinder models of three multilayer DNA origami objects in honeycomb packing used for the stability screens. The object lengths are 140 nm (18-helix bundle), 100 nm (24-helix bundle) and 70 nm (32-helix bundle). The cross-section of a 400-nm-long six-helix bundle was also subjected to melting experiments. () Representative negative-stain TEM micrographs of the three test origami objects. Scale bars, 20 nm. () Melting profiles for a 6-, 18-, 24- and 32-helix bundles, and for a 20-nucleotide DNA duplex of sequence 5′-ATTCATATGGTTTACCAGCG-3′. () Representative single-particle negative-stain TEM micrographs taken after incubating the objects for 2 h at 37 °C, 55 °C and 65 °C. Scale bars, 20 nm. () Representative single-particle negative-stain TEM micrographs taken after incubating an 18-helix bundle (left), 24-helix bundle (middle) and 32-helix bundle (right) with 10 units (U) of T7 endonuclease I and 1 U of DNase I as indicated. () Photograph of a UV-irradiated ethidi! um bromide–stained 2% agarose gel run after incubating purified 32-helix bundles with exo- and endonucleases (10 U each) at 37 °C for 1 h. () Photograph of a UV-light-irradiated ethidium bromide–stained 2% agarose gel after incubating 2 ng of a 24-helix bundle (left) and 65 ng of a conventional double-stranded DNA plasmid (pET24b, right) with DNase I for indicated amounts of time. * Figure 6: Step-by-step guide through molecular self-assembly with scaffolded DNA origami. Step 1 involves conceiving a target shape for the intended application. Our robot shape was divided into three modules: body (red), arms (blue) and legs (orange). Step 2 covers designing a scaffold-staple layout for the target shape, evaluating the design and determining the set of staple sequences to build the design. Black vertical lines trace the scaffold strand (as in Fig. 2c,d), and colored lines indicate the staple paths. In step 3, scaffold DNA is prepared and staple oligonucleotide synthesis (typically in multiwell plates) is performed. Step 4 involves pooling staple oligonucleotides according to structural modules. In step 5 self-assembly reactions are prepared and subjected to a thermal annealing procedure. Step 6 covers an analysis of the overall folding quality by agarose gel electrophoresis, followed by purification of desired species. Shown is an example of increasing folding quality as evidenced by increasing migration speed of DNA origami folding products obs! erved for longer thermal annealing left to right: annealing from 80 °C to 20 °C in 2 h, 5 h, 10 h, 1 d, 5 d and 7 d as well as scaffold without staples as reference). In step 7 purified objects are subjected to single-particle structural analysis. Scale bar, 70 nm. Author information * Abstract * Author information * Supplementary information Affiliations * Center for Integrated Protein Science Munich, Physics Department and Walter Schottky Institut, Technische Universität München, Garching, Germany. * Carlos Ernesto Castro, * Fabian Kilchherr, * Enrique Lin Shiao, * Tobias Wauer, * Philipp Wortmann & * Hendrik Dietz * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Do-Nyun Kim & * Mark Bathe Competing financial interests A patent has been filed on behalf of the Massachusetts Institute of Technology and Dana Farber Cancer Institute by Ditthavong Mori & Steiner, P.C. listing M.B., D.K. and H.D. as co-inventors of CanDo. Corresponding author Correspondence to: * Hendrik Dietz Author Details * Carlos Ernesto Castro Search for this author in: * NPG journals * PubMed * Google Scholar * Fabian Kilchherr Search for this author in: * NPG journals * PubMed * Google Scholar * Do-Nyun Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Enrique Lin Shiao Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Wauer Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Wortmann Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Bathe Search for this author in: * NPG journals * PubMed * Google Scholar * Hendrik Dietz Contact Hendrik Dietz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–5, Supplementary Protocols 1–5, Supplementary Notes 1–2, Supplementary Methods Additional data
• A versatile in vivo system for directed dissection of gene expression patterns
  - Nat Meth 8(3):231-237 (2011)
  Nature Methods | Resource A versatile in vivo system for directed dissection of gene expression patterns * Daryl M Gohl1 * Marion A Silies1 * Xiaojing J Gao2 * Sheetal Bhalerao1 * Francisco J Luongo1 * Chun-Chieh Lin3 * Christopher J Potter3 * Thomas R Clandinin1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:231–237Year published:(2011)DOI:doi:10.1038/nmeth.1561Received19 November 2010Accepted17 December 2010Published online30 January 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This! system is modular, allowing future genetic tools to be easily incorporated into the existing framework. View full text Subject terms: * Genetics * Model Organisms * Neuroscience Figures at a glance * Figure 1: The InSITE system. () Schematic illustration of the InSITE system, which can be used to convert a GAL4 enhancer trap to another sequence, X, which will then be expressed under the control of local enhancers (En). P/T, P transposase promoter; PB, piggyBac transposon. () Schematic illustration of the procedure for genetically swapping GAL4 with sequence X. (–) Fluorescence images showing the results of an immunohistochemical analysis of InSITE enhancer trap expression in the adult brain: P element line P{IT.GAL4}A110.1 (), P element line P{IT.GAL4}A130.1 () and PiggyBac line PBac{IT.GAL4}5.1 (). Green, anti-mCD8; magenta, anti-Bruchpilot. Scale bars, 100 μm. () Schematic illustrating the insertion of the InSITE-compatible enhancer fusion vector, pBMPGal4LWL into a genomic attP site. * Figure 2: Molecular and genetic validation of the enhancer-trap exchange. (,) Results of PCR analyses () to confirm each step of the genetic conversion of line PBac{IT.GAL4}6.1 to the VP16AD hemi-driver, with amplicons numbered as in schematics in . Locations of PCR primers are shown under each construct. () Images of flies with P{ID.VP16AD}D37.1/+ (left) and y, w, eyFLP2; P{ID.VP16AD}D37.1/+ (right), showing that genetic donor constructs lose mini-white expression when crossed to eyFLP2. (–) Images of heat-shocked adult flies carrying the InSITE donor and recipient transgenes, as well as hs-FLP and vas-ΦC31 integrase transgenes. () y, w (yw), hs-FLP, vas-ΦC31; P{ID.VP16AD}D33.1/CyO; PBac{IT.GAL4.w-}3.1/+. () y, w, hs-FLP, vas-ΦC31; P{ID.VP16AD}D33.1/CyO; PBac{IT.GAL4.w–}6.1/+. () mini-white expression in w; PBac{IS.VP16AD.GAL4}6.1/+ (top right), w; PBac{IS.VP16AD.w-}6.1/+, (bottom) and y, w, eyFLP2; PBac{IS.VP16AD.GAL4}6.1/+ (top left) flies. Scale bars, 100 μm. () Sequence of the PCR product of primer pair 5, including the loxP, FRT and ! attL sites. * Figure 3: Functional validation of the QF and LexA enhancer trap swaps. (–) Expression of the PBac{IT.GAL4.w–}6.1 enhancer trap. (,) Expression of the PBac{IT.GAL4}1.1 enhancer trap. (,) Expression of the PBac{IS.QF.w–}6.1 swap. () Expression of the PBac{IS.LexA.w–}1.1 swap. GFP fluorescence (,) in adult antennae (arrowheads) and maxillary palps (arrows). Adult brains immunostained with anti-mCD8 (green) and anti-Bruchpilot (magenta) () or with anti-GFP (green) and anti-Bruchpilot (magenta) (). mCD8 (green) channel only for images in and is shown in and , and GFP (green) channel only for images in and is shown in and . Asterisks denote a group of cells in which GFP expression was observed in PBac{IT.GAL4}1.1 but not in the PBac{IS.LexA.w–}1.1 swap. Scale bars, 100 μm (,), 50 μm (–,–). * Figure 4: Functional validation of the split-GAL4 and GAL80 enhancer trap swaps. (–) GFP expression in the antennae (arrowheads) of adult flies of the indicated lines. (–) Adult brains immunostained with anti-mCD8 (green) and anti-Bruchpilot (magenta). (–) mCD8 (green) channel only of images in –. (–) Adult brains immunostained with anti-mCD8 (green) and anti-Bruchpilot (magenta). (–) mCD8 (green) channel only of images in –. Asterisks denote a small number of central brain neurons not targeted by the split-GAL4 or GAL80 swaps. Scale bars, 100 μm (–) and 50 μm (–). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * HQ888842 * HQ888843 * HQ888844 * HQ888845 * HQ888846 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Neurobiology, Stanford University, Stanford, California, USA. * Daryl M Gohl, * Marion A Silies, * Sheetal Bhalerao, * Francisco J Luongo & * Thomas R Clandinin * Department of Biological Sciences, Stanford University, Stanford, California, USA. * Xiaojing J Gao * Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Chun-Chieh Lin & * Christopher J Potter Contributions D.M.G. and T.R.C. designed the experiments; D.M.G., M.A.S., X.J.G., F.J.L., C.-C.L., C.J.P. and T.R.C. performed the experiments; S.B. provided critical reagents; and D.M.G. and T.R.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas R Clandinin Author Details * Daryl M Gohl Search for this author in: * NPG journals * PubMed * Google Scholar * Marion A Silies Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaojing J Gao Search for this author in: * NPG journals * PubMed * Google Scholar * Sheetal Bhalerao Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco J Luongo Search for this author in: * NPG journals * PubMed * Google Scholar * Chun-Chieh Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Potter Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas R Clandinin Contact Thomas R Clandinin Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–8 and Supplementary Tables 1–3 Additional data
• Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing
  - Nat Meth 8(3):239-241 (2011)
  Nature Methods | Brief Communication Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing * Yann Gambin1, 3 * Virginia VanDelinder2, 3, 4 * Allan Chris M Ferreon1, 4 * Edward A Lemke1, 3 * Alex Groisman2 * Ashok A Deniz1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:239–241Year published:(2011)DOI:doi:10.1038/nmeth.1568Received09 September 2010Accepted14 January 2010Published online06 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution. View full text Subject terms: * Biophysics * Single Molecule * Lab-on-a-chip * Structural Biology Figures at a glance * Figure 1: Microfluidic setup for kinetic smFRET measurements. () After passing through a mixing region, the protein stream and two buffer streams (fed from three separate inlets) are directed to three outlets, which are connected to separate reservoirs, whose heights are adjusted to tune mixing and dilution. () Diagram of the device showing inlets and outlets. () Micrograph of the functional region with a fluorescent solution fed to the protein inlet. Arrows indicate buffer flow (blue), protein flow before mixing (yellow) and protein flow in mixing and detection regions (red). Scale bar, 25 μm. () Flow velocity as measured with fluorescence correlation spectroscopy and time after mixing or dilution, both as functions of the position along the channel in the deceleration region; dashed line is from three-dimensional flow simulations (Comsol). () Flow velocity (as in ) in the focusing, mixing and deceleration regions. () Flow diagram illustrating how the protein stream is squeezed horizontally by two buffer streams for medium exchange a! nd then directed to the smFRET detection region. () Simulations of flow velocities and streamlines in two device regions highlighted in insets. * Figure 2: Folding and unfolding of α-synuclein. (,) Histograms of EFRET for the folding () and unfolding () reactions, obtained at different time points, are used to generate a three-dimensional histogram (~50,000 events) in coordinates of time and EFRET, with the percentage of total events color-coded as indicated. () Representative EFRET histograms for various states: intrinsically disordered (U state, EFRET≈ 0.42, obtained before mixing), collapsed unfolded (U* state, EFRET≈ 0.6, 490 μs after mixing), intermediate (I state, EFRET≈ 0.8, 1.2 ms after mixing) and extended structures (F state, EFRET≈ 0.1, >10 ms after mixing). () Model of the α-synuclein conformational transitions; the mirror representation emphasizes the asymmetry between the folding and unfolding pathways. Shown are random coil (brown), α-helix (turquoise), as well as donor (green sphere) and acceptor (purple sphere) dye molecules. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Virginia VanDelinder & * Allan Chris M Ferreon Affiliations * Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. * Yann Gambin, * Allan Chris M Ferreon, * Edward A Lemke & * Ashok A Deniz * Department of Physics, University of California San Diego, San Diego, California, USA. * Virginia VanDelinder & * Alex Groisman * Present addresses: Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia (Y.G.) and Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany (V.V. and E.A.L.). * Yann Gambin, * Virginia VanDelinder & * Edward A Lemke Contributions Y.G., A.G. and A.A.D. designed research; Y.G. performed smFRET experiments; Y.G. and V.V. characterized the device; A.C.M.F. provided α-synuclein expertise and samples; E.A.L. provided instrumentation support; and all authors contributed to writing the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Yann Gambin or * Alex Groisman or * Ashok A Deniz Author Details * Yann Gambin Contact Yann Gambin Search for this author in: * NPG journals * PubMed * Google Scholar * Virginia VanDelinder Search for this author in: * NPG journals * PubMed * Google Scholar * Allan Chris M Ferreon Search for this author in: * NPG journals * PubMed * Google Scholar * Edward A Lemke Search for this author in: * NPG journals * PubMed * Google Scholar * Alex Groisman Contact Alex Groisman Search for this author in: * NPG journals * PubMed * Google Scholar * Ashok A Deniz Contact Ashok A Deniz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–23 and Supplementary Note 1 Additional data
• High-throughput single-molecule optofluidic analysis
  - Nat Meth 8(3):242-245 (2011)
  Nature Methods | Brief Communication High-throughput single-molecule optofluidic analysis * Soohong Kim1, 8 * Aaron M Streets2, 8 * Ron R Lin1 * Stephen R Quake2, 3, 4 * Shimon Weiss1, 5, 6 * Devdoot S Majumdar1, 7 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:242–245Year published:(2011)DOI:doi:10.1038/nmeth.1569Received09 July 2010Accepted12 January 2011Published online06 February 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment. View full text Subject terms: * Single Molecule * Lab-on-a-chip * Structural Biology * Biophysics Figures at a glance * Figure 1: A microfluidic formulator for high-throughput single-molecule FRET measurements. () Device image (left) with the mixing ring highlighted (arrow). Scale bar, 5 mm. The schematic (right) depicts critical features of the control and flow layer (control and flow channels). () A schematic plot representing smFRET measurements as a two-dimensional histogram of FRET versus stoichiometry. The subpopulations of interest are hybridized poly(dT) (low FRET population; dsDNA) and unhybridized poly(dT) (high FRET population; ssDNA). () Heat map of hybridization efficiency (ratio of dsDNA to total DNA) for various concentrations of NaCl and complementary strand. () Matrix of FRET-stoichiometry scatter plots with contour overlay of fits used to generate heat map shown in . * Figure 2: RNAP activity measured with smFRET. () A schematic of the assay depicts RNAP transcribing the template, thus producing complementary transcript that hybridizes to the poly(dT) probe. () A matrix of FRET-stoichiometry scatter plots (as in Fig. 1) depicting hybridization of the poly(dT) probe to newly produced transcript upon titrating RNAP and glutamate. () Heat map showing quantification of the data in . Amount of transcript is plotted for various concentrations of RNAP and glutamate. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Soohong Kim & * Aaron M Streets Affiliations * Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA. * Soohong Kim, * Ron R Lin, * Shimon Weiss & * Devdoot S Majumdar * Department of Applied Physics, Stanford University, Stanford, California, USA. * Aaron M Streets & * Stephen R Quake * Department of Bioengineering, Stanford University, Stanford, California, USA. * Stephen R Quake * Howard Hughes Medical Institute, Stanford, California, USA. * Stephen R Quake * Department of Physiology, University of California, Los Angeles, Los Angeles, California, USA. * Shimon Weiss * California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California, USA. * Shimon Weiss * Present address: Division of Biology, California Institute of Technology, Pasadena, California, USA. * Devdoot S Majumdar Contributions S.K., A.M.S. and D.S.M. designed experiments, conducted experiments, wrote and implemented data acquisition and analysis software, and analyzed data. R.R.L. analyzed data. S.K., A.M.S., S.R.Q., S.W. and D.S.M. assisted in writing and editing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Devdoot S Majumdar or * Shimon Weiss Author Details * Soohong Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron M Streets Search for this author in: * NPG journals * PubMed * Google Scholar * Ron R Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen R Quake Search for this author in: * NPG journals * PubMed * Google Scholar * Shimon Weiss Contact Shimon Weiss Search for this author in: * NPG journals * PubMed * Google Scholar * Devdoot S Majumdar Contact Devdoot S Majumdar Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Zip files * Supplementary Software (116M) Software used in this study to control and coordinate microfluidics and optical components. PDF files * Supplementary Text and Figures (942K) Supplementary Figures 1–10, Supplementary Note 1, Supplementary Table 1 Additional data
• Micropilot: automation of fluorescence microscopy–based imaging for systems biology
  - Nat Meth 8(3):246-249 (2011)
  Nature Methods | Brief Communication Micropilot: automation of fluorescence microscopy–based imaging for systems biology * Christian Conrad1 * Annelie Wünsche2 * Tze Heng Tan2 * Jutta Bulkescher1 * Frank Sieckmann3 * Fatima Verissimo2 * Arthur Edelstein4 * Thomas Walter2 * Urban Liebel2, 5 * Rainer Pepperkok1, 2 * Jan Ellenberg2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:246–249Year published:(2011)DOI:doi:10.1038/nmeth.1558Received26 May 2010Accepted06 December 2010Published online23 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology. View full text Subject terms: * Cell Biology * Microscopy * Imaging * Systems Biology Figures at a glance * Figure 1: Schematic workflow of Micropilot. () After autofocussing different positions to find the best focal plane (yellow frame), low-resolution prescan images (optionally maximum z-dimension projections, gray frames) are presented to the automatic classification. If a cell is selected, a complex imaging protocol is executed; otherwise the system continues to prescan. After completion of the complex imaging protocol, the system loops back to prescan mode, continuing at the sample position where it stopped for the complex imaging mode. () Communication steps executed by the different microscope systems (red outlines) and the Micropilot software (blue outlines). The microscope sends the image path either via windows registry or socket interface to Micropilot. In the synchronous modes, each positive classification launches the complex imaging mode. In the asynchronous mode, microscope and Micropilot send and receive messages via transmission control protocol or internet protocol (TCP/IP), allowing classification of sev! eral different positions before launching the complex imaging protocol for a list of positions. () After reading the low-resolution image, Micropilot segments, extracts the feature set per object and classifies the cells during scanning to return eventually the positions of interest. After the criteria are met (time or number of positions) Micropilot deploys the complex imaging and the microscope switches back to prescan mode (). * Figure 2: Assays of SEC31 and H2B-tubulin HeLa cells. () Examples for Hoechst-labeled (blue; DNA label) and SEC31-labeled (green) cells representing null or artifact and anaphase or telophase cells (insets, close-up images). Scale bars, 10 μm. () Confusion matrix of the prediction shows true positives (TP) horizontally against the predicted class vertically for cells. At edges the total numbers of the cells are given (overall total, 10,793 cells) corresponding to PPV = TP / (TP + false positives) and sensitivity = TP / (TP + false negatives). () Examples of null or artifact (left) and anaphase or telophase (right) cells stained with Hoechst (blue) and ERES spot (green) (50 slices of 0.2 μm). Scale bar, 10 μm. () Number of ERES spots of 91 anaphase cells to late-telophase cells plotted versus volume of nuclei, with exponential fit plotted. Red and blue data points correspond to the nuclei in the left and right images in , respectively. () Example of negative control experiment (time resolution, 3 min; 30 slices of 1 μm; maxi! mum projections) started after prophase recognition. Times indicated are after prophase recognition. Scale bar, 10 μm.() Spindle lengths after treatment with scrambled siRNA. () Example images after treatment with siRNA to CENPE, showing centrosome poles (arrows; left) for the first recognizable metaphase (acquisition as in ). Scale bar, 10 μm. () Normal mixture modeling of pole-pole distances in metaphase from 71 movies after treatment with siRNA to CENPE resulted in three distributions, which are shown as colored curves. * Figure 3: Examples and measurements of automatic FRAP on CBX1-EGFP cells. () After the automatic selection of an interphase or prophase cell with a trained prophase SVM classifier, a prebleached image was taken, followed by bleaching of the predefined upper half of the nucleus and subsequent time-lapse imaging with 2-s time resolution for 60 s (values in the lower images indicate time relative to bleaching). Scale bar, 5 μm. () Normalized intensities for CBX1-EGFP measured during fluorescence relaxation after photobleaching in interphase and prophase cells. We measured, normalized, averaged and plotted over time fluorescence intensities in the bleached region of the nucleus. Error bars, s.d. () Recovery rates as box plots for interphase and prophase cell populations. Author information * Author information * Supplementary information Affiliations * Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Heidelberg, Germany. * Christian Conrad, * Jutta Bulkescher & * Rainer Pepperkok * Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. * Annelie Wünsche, * Tze Heng Tan, * Fatima Verissimo, * Thomas Walter, * Urban Liebel, * Rainer Pepperkok & * Jan Ellenberg * Leica Microsystems GmbH, Mannheim, Germany. * Frank Sieckmann * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. * Arthur Edelstein * Present address: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany. * Urban Liebel Contributions C.C. developed the 'Micropilot' software and drafted the manuscript. A.W. developed the Visual Basic for Applications macro and performed and analyzed the automatic FRAP experiments. T.H.T. developed the feature selection, extended classification to multiple channels and acquired and analyzed the ERES images. F.V. performed the ERES experiments. J.B. performed and analyzed the spindle length experiments. F.S. and U.L. developed the computer-aided microscopy interface and set up software prototypes. A.E. developed the communication of μManager with Micropilot. T.W. helped with image processing and object feature design. R.P. supervised the project. J.E. supervised the project and revised the manuscript. Competing financial interests F.S and U.L. filed a patent application covering the CAM approach (Patent Cooperation Treaty/European Patent 2007/059351/US patent application 20100103253). F.S. is employed by Leica Microsystems. Corresponding authors Correspondence to: * Rainer Pepperkok or * Jan Ellenberg Author Details * Christian Conrad Search for this author in: * NPG journals * PubMed * Google Scholar * Annelie Wünsche Search for this author in: * NPG journals * PubMed * Google Scholar * Tze Heng Tan Search for this author in: * NPG journals * PubMed * Google Scholar * Jutta Bulkescher Search for this author in: * NPG journals * PubMed * Google Scholar * Frank Sieckmann Search for this author in: * NPG journals * PubMed * Google Scholar * Fatima Verissimo Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur Edelstein Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Walter Search for this author in: * NPG journals * PubMed * Google Scholar * Urban Liebel Search for this author in: * NPG journals * PubMed * Google Scholar * Rainer Pepperkok Contact Rainer Pepperkok Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Ellenberg Contact Jan Ellenberg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (1M) Example video of H2B-tubulin HeLa cells negative control from prophase recognition on. * Supplementary Video 2 (3M) Example video of FRAP on CBX1-EGFP interphase. * Supplementary Video 3 (4M) Example video of FRAP on CBX1-EGFP early prophase (slow recovery). * Supplementary Video 4 (5M) Example video of FRAP on CBX1-EGFP late prophase (fast recovery). Zip files * Supplementary Software (38M) Micropilot software source code, documentation, microscope scripts and demonstration images. PDF files * Supplementary Text and Figures (328K) Supplementary Figures 1–3 and Supplementary Table 1 Additional data
• Codon adaptation–based control of protein expression in C. elegans
  - Nat Meth 8(3):250-252 (2011)
  Nature Methods | Brief Communication Codon adaptation–based control of protein expression in C. elegans * Stefanie Redemann1 * Siegfried Schloissnig2 * Susanne Ernst1 * Andrey Pozniakowsky1 * Swathi Ayloo1 * Antony A Hyman1 * Henrik Bringmann3 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:250–252Year published:(2011)DOI:doi:10.1038/nmeth.1565Received28 September 2010Accepted17 December 2010Published online30 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter. View full text Subject terms: * Cell Biology * Gene Expression * Synthetic Biology Figures at a glance * Figure 1: Codon-adaptation of the gpr-1 gene determines the amount of YFP–GPR-1 protein. () Fluorescence images showing localization of YFP–GPR-1 expressed from the indicated constructs in embryos at early anaphase. Images were acquired using different imaging conditions, and different contrast was used to display localization patterns. Scale bar, 10 μm. () Mean YFP fluorescence intensity over the surface of the embryo expressing the indicated constructs. yfp::gpr-1(endogenous, CAI 0.3), 28 ± 13 arbitrary units (mean ± s.d.; n = 6 embryos); yfp::gpr-1(synthetic, CAI 0.3), 12 ± 3 arbitrary units (n = 6 embryos); yfp::gpr-1(synthetic, CAI 0.6), 75 ± 22 arbitrary units (n = 6 embryos); and yfp::gpr-1(synthetic, CAI 1.0), 202 ± 34 arbitrary units (n = 6 embryos). N2, wild type expressing no transgene, 0 ± 5 arbitrary units (n = 6 embryos). * Figure 2: Increasing GPR-1 amounts causes an increase in force acting on the mitotic spindle. () Time-lapse images of an embryo expressing yfp::gpr-1(synthetic, CAI 1.0) throughout the first cell division. Scale bar, 10 μm. () Histogram of centrosomal velocity after spindle cut by a UV light laser and spindle break (n = 5 embryos for all three strains; error bars, s.d.). Author information * Author information * Supplementary information Affiliations * Max Planck Institute of Molecular Cell Biology and Genetics, Dresden Germany. * Stefanie Redemann, * Susanne Ernst, * Andrey Pozniakowsky, * Swathi Ayloo & * Antony A Hyman * European Molecular Biology Laboratory Heidelberg, Heidelberg, Germany. * Siegfried Schloissnig * Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Henrik Bringmann Contributions S.R. characterized all strains. S.S. wrote the web tool algorithm and analyzed genome-wide CAI. S.E. bombarded all constructs. A.P. cloned constructs. S.A. filmed embryos. A.A.H. mentored and financed the project. S.R., A.A.H. and H.B. wrote the paper. H.B. conceived the general synthetic gene design, cloned gpr-1 constructs and preliminarily characterized gpr-1 strains. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Henrik Bringmann Author Details * Stefanie Redemann Search for this author in: * NPG journals * PubMed * Google Scholar * Siegfried Schloissnig Search for this author in: * NPG journals * PubMed * Google Scholar * Susanne Ernst Search for this author in: * NPG journals * PubMed * Google Scholar * Andrey Pozniakowsky Search for this author in: * NPG journals * PubMed * Google Scholar * Swathi Ayloo Search for this author in: * NPG journals * PubMed * Google Scholar * Antony A Hyman Search for this author in: * NPG journals * PubMed * Google Scholar * Henrik Bringmann Contact Henrik Bringmann Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–8 and Supplementary Table 1 Additional data
• Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns
  - Nat Meth 8(3):253-259 (2011)
  Nature Methods | Article Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns * Stefanie Hampel1, 2 * Phuong Chung1, 2 * Claire E McKellar1 * Donald Hall1 * Loren L Looger1 * Julie H Simpson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:253–259Year published:(2011)DOI:doi:10.1038/nmeth.1566Received19 August 2010Accepted20 December 2010Published online06 February 2011Corrected online16 February 2011 Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We developed a multicolor neuron labeling technique in Drosophila melanogaster that combines the power to specifically target different neural populations with the label diversity provided by stochastic color choice. This adaptation of vertebrate Brainbow uses recombination to select one of three epitope-tagged proteins detectable by immunofluorescence. Two copies of this construct yield six bright, separable colors. We used Drosophila Brainbow to study the innervation patterns of multiple antennal lobe projection neuron lineages in the same preparation and to observe the relative trajectories of individual aminergic neurons. Nerve bundles, and even individual neurites hundreds of micrometers long, can be followed with definitive color labeling. We traced motor neurons in the subesophageal ganglion and correlated them to neuromuscular junctions to identify their specific proboscis muscle targets. The ability to independently visualize multiple lineage or neuron projections i! n the same preparation greatly advances the goal of mapping how neurons connect into circuits. View full text Subject terms: * Neuroscience * Imaging * Genetics Figures at a glance * Figure 1: Schematic of the dBrainbow construct. UAS-dBrainbow contains a UAS that allows its expression to be cell-specifically controlled by the presence of GAL4. Cre recombination occurs only between matched lox sites. The selection of lox site recombination in a given cell is stochastic. In the absence of recombinase, no fluorescent proteins are made because of a stop cassette with three-frame translation terminators and an SV40 polyadenylation signal. Recombination between lox2272 sites removes this stop cassette and permits expression of EGFP-V5; recombination between the lox5171 sites results in expression of EBFP2-HA; and recombination between loxP sites produces mKO2-Myc. Cre-mediated recombination is irreversible. Colors from one or two copies of UAS-dBrainbow are shown on the right. All fluorescent proteins are cytoplasmic and epitope-tagged as indicated. * Figure 2: Comparison of endogenous and antibody-based fluorescence of UAS-dBrainbow flies. (–) Projections of two 1-μm slices through the antennal lobes from adult brains of hs-Cre; GH146-GAL4; UAS-dBrainbow flies, imaged without fixation. Merged image () reveals endogenous fluorescence of EGFP (green), mKO2 (red) and EBFP2 (blue; arrow); there is some bleed-through into the GFP channel. Raw grayscale images show mKO2 (), EGFP () and EBFP2 () fluorescence; laser wavelengths are indicated in the images. (–) Flies of the same genotype were fixed and stained with primary antibodies to GFP (α-GFP), Myc (α-Myc) and HA (α-HA), and secondary antibodies coupled to Alexa Fluors 488, 568 and 633, respectively. Merged image () shows all three colors and individual images (–) show spectral separation of Alexa Fluor dyes and lack of antibody cross-reactivity. (–) Maximum intensity projections of 20× confocal stacks for whole brains labeled with the nc82 antibody as a neuropil marker (gray), with antibodies to GFP (green) and HA (blue), and showing mKO2 endogenous ! fluorescence (red). Merged image (), EGFP, mKO2 and EBFP signals alone (), and nc82 signal alone () are shown. Scale bars, 50 μm. * Figure 3: Expression of UAS-dBrainbow in three projection neuron lineages. (–) Single (–) and double (–) copies of UAS-dBrainbow were used along with hs-Cre; GH146-GAL4 to label the three projection neuron lineages that express GH146-GAL4 (adPN, lPN and vPN) as well as the axon tracts (iACT and mACT) that connect the projection neuron cell bodies to the lateral horn (LH) () and calyx (ca) (,–). Shown are maximum intensity projections of several 1-μm confocal sections (–) with each projection neuron lineage expressing a different fluorescent protein–epitope cassette and thus pseudocolored differently (arrowheads; ). The number and depth of confocal slices used to produce the merged images are indicated. In the right lPN lineage (arrowhead; ), recombination in the neuroblast occurred to select blue in one copy of UAS-dBrainbow but the recombinase did not act on the second copy until later to select green, so a subset of later-born neurons is labeled in cyan. In , the antennal lobe (AL) and efferent neurons projecting to the lateral horn ! via the mACT and iACT; individual neurites can also be traced from the antennal lobe to the lateral horn via an alternative pathway (arrows). Higher-magnification views of the lateral horn from different orientations (,–). Scale bars, 50 μm (–,) and 20 μm (,–). * Figure 4: dBrainbow labeling of lineages or individual neurons in different colors. (–) Maximum intensity projections of confocal stacks of OK107-GAL4 (–) and Tdc2-GAL4 (–) with UAS-dBrainbow and hs-Cre shown as merged images (,) and grayscale red (,) green (,) and blue (,) channels, with the imaged wavelengths indicated (blue was used to represent the Alexa Fluor 633). Arrows and arrowheads trace individual ventral unpaired medial (VUM) neurons (–). ASM, anterior superior medial protocerebrum and AL2, anterior lateral cluster 2. Arrowhead in indicates gamma lobe of the mushroom body. (–) Three 35-μm substacks of hs-Cre; UAS-dBrainbow; UAS-dBrainbow, FruM-GAL4 divide the ~2,000 neurons labeled by FruM-GAL4 into many subpopulations, demarcated by different colors. Arrowhead in indicates the pars intercerebrales. Scale bars, 50 μm. * Figure 5: Expression of UAS-dBrainbow in motor neurons that connect the subesophageal ganglion to the proboscis muscles in sections of a single example of an hs-cre; UAS-dBrainbow; R12D05-GAL4 fly. () Maximum projection confocal stack showing a cluster of several cell types in the subesophageal ganglion. () Frontal and lateral schematics of a fly head with proboscis extended, showing brain in gray and subesophageal ganglion (SOG) in darker gray. Two motor neuron types (D and V-L pairs) project to neuromuscular junctions on proboscis muscles. Proboscis was dissected separately (dashed lines) to allow antibody penetration. () Confocal substack showing dorsal motor neurons in red and green (arrowheads) that send axons out the pharyngeal nerve (arrow). Neurites in the middle do not belong to these neurons as they are labeled in blue and green. () Confocal substack showing a pair of ventrolateral neurons with C-shaped arbors in the SOG in blue and red (arrowheads). Axons exit via labial nerves (arrows). () Confocal substack of the rostrum, lateral view, as shown in the inset. The red and green axons travel through the proboscis together (arrows), to terminate at neuromuscul! ar junctions near the distal end of the rostrum (arrowheads). Background was caused by autofluorescence of intact cuticle. (,) Blue and red axons terminate in neuromuscular junctions on the transverse muscles in the distal end of the haustellum. Substacks at different levels show faint blue () and bright red () terminals (arrowheads). At higher gain (), autofluorescence in the blue channel also shows the muscle fibers of the haustellum: longitudinal muscles (vertical fibers in the image) and transverse muscles (horizontal). Scale bars, 50 μm. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Entrez Nucleotide * JF267350 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 16 February 2011In the version of this article initially published online, accession codes were not included. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Stefanie Hampel & * Phuong Chung Affiliations * Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. * Stefanie Hampel, * Phuong Chung, * Claire E McKellar, * Donald Hall, * Loren L Looger & * Julie H Simpson Contributions S.H. designed and performed cloning, tested constructs in S2 cells and made the figures. P.C. performed the fly genetics, immunohistochemistry and confocal imaging. C.E.M. generated and analyzed the subesophageal ganglion and proboscis data. D.H. generated the recombinant fly stocks. L.L.L. advised on selection of fluorescent proteins, construct design and the conversion from endogenous fluorescence to antibody. J.H.S. conceived the project, cloned initial test constructs and wrote the paper with help from S.H., P.C., C.E.M. and L.L.L. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Julie H Simpson Author Details * Stefanie Hampel Search for this author in: * NPG journals * PubMed * Google Scholar * Phuong Chung Search for this author in: * NPG journals * PubMed * Google Scholar * Claire E McKellar Search for this author in: * NPG journals * PubMed * Google Scholar * Donald Hall Search for this author in: * NPG journals * PubMed * Google Scholar * Loren L Looger Search for this author in: * NPG journals * PubMed * Google Scholar * Julie H Simpson Contact Julie H Simpson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–4 and Supplementary Tables 1–4 Additional data
• Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster
  - Nat Meth 8(3):260-266 (2011)
  Nature Methods | Article Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster * Dafni Hadjieconomou1 * Shay Rotkopf2, 5 * Cyrille Alexandre3 * Donald M Bell4 * Barry J Dickson2 * Iris Salecker1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:260–266Year published:(2011)DOI:doi:10.1038/nmeth.1567Received28 October 2010Accepted28 December 2010Published online06 February 2011Corrected online16 February 2011 Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marke! r; this could further support the genetic analysis of neural circuit assembly and function. View full text Subject terms: * Neuroscience * Genetics * Model Organisms * Imaging Figures at a glance * Figure 1: Schematic of Flybow variants. (–) Pairs of fluorescent protein–encoding cDNAs are arranged in opposing orientations and flanked by mFRT71 sites (black arrowheads). Fluorescent proteins are membrane-tethered either by a Cd8a (cd8) or the palm-myr (pm) sequence of Lyn kinase. Fluorescent protein sequences are followed by SV40 and hsp70Ab polyadenylation (pA) signals. Constructs were subcloned into a modified pKC26 UAS vector, which contains ten UAS (10xUAS) sites and a short attB recognition sequence. mFlp5 under the control of the heat-shock promoter (hs-mFlp5) induces inversions of DNA cassettes by recombining mFRT71 sites in opposing orientations, or excisions (Flp-out) by recombining mFRT71 sites placed in the same orientation. FB1.0 () consists of one invertible cassette encoding two fluorescent proteins (mCherry and Cerulean-V5). FB1.1 () contains two invertible cassettes, each encoding two fluorescent proteins (EGFP and mCitrine; mCherry and Cerulean-V5). FB2.0 () contains an additional stop cas! sette, flanked by canonical FRT sites (white arrowheads) facing in the same orientation, which can be excised by wild-type Flp. The stop cassette consists of lamin cDNA, followed by two HA tag sequences and hsp70Aa and hsp27 polyadenylation signals. * Figure 2: Activity of FB1.0 and FB1.1 transgenes. (,) Schematic of the third instar larval (3L) () and adult visual system (). Shown are R1–R8 photoreceptor axons, lamina neurons (ln) L1–L5, and the medulla neuron (mn) and lobula neuron and lobula plate neuron subtypes Tm, TmY, Dm and T, which are main target neuron subtype classes. Glial subtypes include epithelial glia (eg), marginal glia (mg), medulla glia (meg) and medulla neuropil glia (mng)15. GMC, ganglion mother cell; LPC, lamina precursor cells; MF, morphogenetic furrow; Nb, neuroblasts; and OPC, outer proliferation center. () Schematic showing that FB1.0 enables expression of Cerulean-V5 instead of mCherry in a Gal4-expressing cell population upon heat-shock induction of mFlp5. (,) Confocal images of a larval eye disc () and optic lobe () with some R cells and lamina neurons expressing Cerulean-V5. la, lamina; me, medulla. () Schematic showing that FB1.1 leads to expression of EGFP, mCitrine, mCherry and Cerulean-V5. (,) Confocal images of larval R cells () an! d of lineages of younger (y, asterisks) and individual older (o, arrowheads) medulla neurons () expressing different fluorescent proteins. (–) Differentially labeled adult neuron subtypes14: lamina neurons L5 (,), lineage-related ascending T2–T5 neurons (), an amacrine Dm neuron and the transmedullary neurons Tm18 (arrows) and TmY5a (arrowheads) (). lo, lobula; lop, lobula plate. (,) Sagittal view of a live stage 16 embryo with clusters and single neurons expressing mCitrine or mCherry (arrowheads). Arrow, cluster expressing EGFP and mCherry owing to perdurance; asterisk, unlabeled cluster of Cerulean-V5–expressing neurons. Insets, live growth cone extending from the ventral nerve cord (VNC) into the peripheral nervous system (PNS). (,) Flat preparation of a fixed VNC with large (arrows) or smaller (arrowheads) growth cones exploring lateral (l) tracts and anterior (ac) or posterior (pc) commissures. () PNS neurons, including the lateral chordotonal organ (lch). elav-! Gal4c155 was used as pan-neuronal driver. EGFP, mCitrine and m! Cherry were detected using endogenous fluorescence signals and Cerulean using immunolabeling with antibody to V5. Scale bars, 50 μm (,,–) and 20 μm (insets, ). * Figure 3: Expression of FB1.1 transgenes in distinct cell populations. (–) Confocal images showing third instar larval (3L) and adult R1–R8 photoreceptor axons labeled using GMR-Gal4. R1–R6 growth cones (arrowheads) in the lamina (la), and young (double arrowheads) and mature (arrows). R8 growth cones in the medulla (me) are highlighted in ,. Adult R8 and R7 terminals in a column express the same fluorescent protein (arrowhead) or combinations of two fluorescent proteins (asterisks) (–). (–) Single optical sections (,) and a 10 μm z-stack projection of the mCitrine channel () of adult optic lobes in which MzVum-Gal4 drives FB1.1 expression in medulla neurons. One neuron (arrow; ,) traced through a series of consecutive sections had TmY5a neuron-like features; asterisks indicate additional or absent branches compared to reported morphology14. (,) Epithelial (eg) and marginal (mg) glia in the lamina, and medulla neuropil glia (mng) at the distal medulla neuropil border were labeled with different fluorescent proteins in third instar la! rval () and adult () optic lobes using repo-Gal4 and FB1.1. In , fluorescence signals in the lamina above the white line were reduced relative to those in the medulla. (–) Higher magnification of the image in , showing elaborate shapes of epithelial and medulla neuropil glia. () Area with overlapping medulla neuropil glial cell branches (arrowhead) in a medulla cross-section. (,) en-Gal4–driven expression of different fluorescent proteins in epithelial cell clones in the posterior (p) compartment of wing discs. d, dorsal. Scale bars, 50 μm (,,–,–) and 10 μm (,,,–). * Figure 4: FB2.0 facilitates sparse labeling of cells within a Gal4-expressing cell population. () Schematic showing that upon heat induction, Flp excises the upstream FRT stop cassette to enable reporter expression in a subset of Gal4-expressing cells. Induction of hs-mFlp5 randomizes the fluorescent protein selection as in FB1.1. Expression of four fluorescent proteins is restricted to cells with overlapping Gal4, Flp and mFlp5 activities. (–) Confocal images showing that elav-Gal4c155 in conjunction with FB2.0 led to fluorescent protein expression in only a small subset of R cells in eye discs posterior to the morphogenic furrow (), and of lamina and medulla neurons (mn) in larval () and adult (,) optic lobes. Labeling of fewer cells facilitated the identification of neuron subtypes in the dense neuropils of the lamina (la), medulla (me), lobula (lo) and lobula plate (lop). Surrounded by mCherry-expressing medulla neurons, lamina neurons subtype L3 can be identified by mCitrine expression in the adult medulla. Scale bars, 50 μm. * Figure 5: Combining Flybow and MARCM for functional mosaic analysis. () Schematic of Flp-FRT–mediated mitotic recombination in trans during the G2-M phase of the cell cycle and subsequent chromosome segregation, which leads to the loss of the Gal80 repressor in one of the daughter cells, enabling reporter gene expression. This Gal80-free cell is homozygous for any mutation located on the homologous chromosome arm (vertical bar on black chromosome). mFlp5-mFRT71–mediated recombination of the FB1.1 transgene in cis leads to the stochastic expression of one of four fluorescent proteins in progeny not expressing Gal80. (–) Confocal images of adult optic lobes (,) and higher magnifications of medulla neuropil (–) showing that elav-Gal4c155 in conjunction with FB1.1 drives expression of EGFP, mCitrine and mCherry in wild-type control and CadNM19 homozygous mutant neurons in the adult lamina (la), medulla (me), lobula (lo) and lobula plate (lop). ln, lamina neurons. R-cell axons were labeled with the photoreceptor-specific antibody mAb24B10 ! (blue). Shown are mCitrine-expressing lamina neuron L1 innervating layers M1 and M5 () and mCherry-expressing lamina neurons L5 terminating in the M1, M2 and M5 layers () in controls. In the latter, axonal arbors can be distinguished from overlapping branches of neighboring neurons expressing EGFP or mCitrine (arrowhead). Also shown are mCherry-expressing L1 and L5 neurons with aberrant projections in the absence of CadNM19 (). () Schematic illustrating the axonal arborizations of control and CadNM19 homozygous mutant L1 and L5 neurons. Scale bars, 50 μm (,) and 10 μm (–). Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Entrez Nucleotide * HQ998853 * HQ998854 * HQ998855 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 16 February 2011In the version of this article initially published online, accession codes were not included. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * Medical Research Council (MRC) National Institute for Medical Research, Division of Molecular Neurobiology, London, UK. * Dafni Hadjieconomou & * Iris Salecker * Research Institute of Molecular Pathology, Vienna, Austria. * Shay Rotkopf & * Barry J Dickson * MRC National Institute for Medical Research, Division of Developmental Neurobiology, London, UK. * Cyrille Alexandre * MRC National Institute for Medical Research, Confocal Image Analysis Laboratory, London, UK. * Donald M Bell * Present address: Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel. * Shay Rotkopf Contributions I.S., B.J.D., D.H. and C.A. designed the Flybow strategy. D.H. cloned the Flybow constructs, D.H. and I.S. generated the transgenic fly stocks, and D.H. conducted the experimental analysis. S.R. and B.J.D. developed the modified Flp-FRT system, and provided the original pKC26 UAS vector and the wild-type Flp-out cassette. C.A. provided expert advice for cloning, and D.M.B. provided expert advice for image acquisition and analysis. I.S. and D.H. wrote the manuscript in interaction with all contributing authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Iris Salecker Author Details * Dafni Hadjieconomou Search for this author in: * NPG journals * PubMed * Google Scholar * Shay Rotkopf Search for this author in: * NPG journals * PubMed * Google Scholar * Cyrille Alexandre Search for this author in: * NPG journals * PubMed * Google Scholar * Donald M Bell Search for this author in: * NPG journals * PubMed * Google Scholar * Barry J Dickson Search for this author in: * NPG journals * PubMed * Google Scholar * Iris Salecker Contact Iris Salecker Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (647K) Supplementary Figures 1–5 and Supplementary Table 1 Additional data
• Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry
  - Nat Meth 8(3):267-272 (2011)
  Nature Methods | Article Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry * Jonathan Clark1, 2, 5 * Karen E Anderson1, 5 * Veronique Juvin1 * Trevor S Smith1 * Fredrik Karpe3, 4 * Michael J O Wakelam1 * Len R Stephens1, 5 * Phillip T Hawkins1, 5 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:267–272Year published:(2011)DOI:doi:10.1038/nmeth.1564Received13 October 2010Accepted20 December 2010Published online30 January 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P3 have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography–mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P3 and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP2 are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P3 in un! stimulated mouse and human cells (≥105) or tissues (≥0.1 mg) and their increase upon appropriate stimulation. View full text Subject terms: * Mass Spectrometry * Chemistry * Cell Biology * Biochemistry Figures at a glance * Figure 1: Analysis of phosphoinositides in control and fMLP-stimulated human neutrophils. (–) Neutral loss scans of a derivatized phosphoinositide extract, from unstimulated control (,) or fMLP-stimulated (,) neutrophils. The two most abundant species of endogenous PtdIns(3,4,5)P3 and PtdInsP2 are labeled with full masses and corresponding fatty acid species of diacylglycerol unit. Cps, counts per second. () Overlay of m/z chromatograms for parent ions with masses similar to those of derivatized C18:0/C20:4-Ptdins(3,4,5)P3) from extracts from 105 human neutrophils, highlighting elution at 10.75 min (ions that increase with fMLP stimulation in a wortmannin-sensitive manner). () Overlay of MRM chromatograms (m/z 1,225 to m/z 627 + 598) of samples from . Data were collected using Quattro Ultima (Waters) (–) and QTRAP 4000 (AB Sciex) (–) mass spectrometers. Representative traces from several independent experiments are shown. * Figure 2: Validation of the robustness, signal-to-noise ratio and linearity of the assay. () C17:0/C16:0-PtdIns(3,4,5)P3 internal standard spiked into unstimulated human neutrophil extract. () C18:0/C20:4-PtdIns(3,4,5)P3 standard spiked into unstimulated human neutrophil extract. () Data for 0.25 ng C18:0/C20:4-PtdIns(3,4,5)P3 standard spiked into unstimulated neutrophil extract. Signal to noise, root mean square = 114. () Response to increasing amounts of C18:0/C20:4-PtdIns(3,4,5)P3 added to extracts of unstimulated neutrophils (2.25 × 106). () Relationship between cell number (fMLP-stimulated neutrophils) and estimated endogenous C18:0/C20:4-PtdIns(3,4,5)P3 by using the internal standard to correct for recovery. The term 'response ratio' in (,,) is the integrated ion current response to the defined phosphoinositide divided by that to the internal standard. () MRM chromatograms for 1 μg each of synthetic C18:0/C20:4-PtdIns(4,5)P2 (left), synthetic C18:0/C20:4-PtdIns(3,4,5)P3 (middle) and internal standard (C17:0/C16:0-PtdIns(3,4,5)P3) (right) in water. * Figure 3: fMLP-stimulated changes in C18:0/C20:4 and C18:0/C18:1 PtdInsP2 and PtdIns(3,4,5)P3 species in human neutrophils. (,) Amounts of the C18:0/C20:4 and C18:0/C18:1 molecular species of PtdInsP2 () and PtdIns(3,4,5)P3 () determined at indicated times after addition of fMLP. The data are expressed as either response ratios (as defined in Fig. 2), or through use of the calibration curve presented in Figure 2d and protein assays, picomoles of C18:0/C20:4 PtdIns(3,4,5)P3 per milligram protein. Error bars, s.e.m. (n = 4). () The ratio of the quantity of each molecular species of PtdIns(3,4,5)P3 divided by that of its respective PtdInsP2 species. * Figure 4: Identification and quantification of the molecular species of PtdIns(3,4,5)P3 in wild-type and PTEN−/− MCF10a cells. (,) Neutral loss scans of the common families of molecular species of PtdInsP2 () and PtdIns(3,4,5)P3 () in EGF-stimulated, wild-type MCF10a cells. Further fragmentation and analysis of the relevant daughter ions indicated they possessed the fatty acids indicated in . Data were collected using a QTRAP 4000 mass spectrometer. (,) The levels of these species of PtdInsP2 () and PtdIns(3,4,5)P3 () in the indicated cell lines and conditions are presented as mean response ratios normalized for cell input via the recovered C18:0/C18:1-PtdSer (error bars, s.e.m.; n = 3). () The ratio of the quantity of each molecular species of PtdIns(3,4,5)P3 divided by that of their respective PtdInsP2 species. * Figure 5: Detection and quantification of insulin-stimulated PtdIns(3,4,5)P3 responses in mouse liver and human adipose tissue. () Neutral loss scan, collected using a QTRAP 4000 mass spectrometer, of PtdIns(3,4,5)P3 species in wild-type, insulin-stimulated mouse liver. () Levels of C18:0/C20:4-PtdIns(3,4,5)P3 in the livers of wild-type (WT) or Gnasxlm+/p− mice after injection of insulin or saline (error bars, s.e.m.; n = 4). () Phosphorylation status of S473 in PKB for parallel samples to those analyzed in ; data were normalized for input material via immunoblotting for β-COP. () Neutral loss scan of PtdIns(3,4,5)P3 species in human adipose tissue after oral ingestion of glucose. () C18:0/C20:4-PtdIns(3,4,5)P3 amounts in healthy human adipose tissue after overnight starvation either before (fasting) or 90 min after (glucose) oral ingestion of glucose, for three individuals (error bars, s.e.m.; technical replicates n = 4). () Phosphorylation status of S473 in PKB for parallel samples to those analyzed in ; data normalized for input material via immunoblotting for actin. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jonathan Clark, * Karen E Anderson, * Len R Stephens & * Phillip T Hawkins Affiliations * Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, UK. * Jonathan Clark, * Karen E Anderson, * Veronique Juvin, * Trevor S Smith, * Michael J O Wakelam, * Len R Stephens & * Phillip T Hawkins * Babraham Bioscience Technologies Ltd., Babraham Research Campus, Babraham, Cambridge, UK. * Jonathan Clark * Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK. * Fredrik Karpe * National Institute for Health Research, Oxford Biomedical Research Centre, Oxford Radcliffe Hospitals Trust, Churchill Hospital, UK. * Fredrik Karpe Contributions J.C. and K.E.A. designed and performed experiments, developed methods, analyzed data and contributed to writing the manuscript; V.J. designed and performed PKB experiments; T.S.S. performed experiments; F.K. designed experiments (Fig. 5 and Supplementary Figs. 9 and 10) to generate samples for analysis; M.J.O.W. developed methods and provided reagents; and L.R.S. and P.T.H. designed the study/experiments, developed methods, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Phillip T Hawkins or * Len R Stephens Author Details * Jonathan Clark Search for this author in: * NPG journals * PubMed * Google Scholar * Karen E Anderson Search for this author in: * NPG journals * PubMed * Google Scholar * Veronique Juvin Search for this author in: * NPG journals * PubMed * Google Scholar * Trevor S Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Fredrik Karpe Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J O Wakelam Search for this author in: * NPG journals * PubMed * Google Scholar * Len R Stephens Contact Len R Stephens Search for this author in: * NPG journals * PubMed * Google Scholar * Phillip T Hawkins Contact Phillip T Hawkins Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (904K) Supplementary Figures 1–10, Supplementary Tables 1–2, Supplementary Data 1–2 Additional data