SmartEM: machine-learning guided electron microscopy.

Connectomics provides essential nanometer-resolution, synapse-level maps of neural circuits to understand brain activity and behavior. However, few researchers have access to the high-throughput electron microscopes necessary to generate enough data for whole circuit or brain reconstruction. To date, machine-learning methods have been used after the collection of images by electron microscopy (EM) to accelerate and improve neuronal segmentation, synapse reconstruction and other data analysis.

Sparse Scanning Electron Microscopy Data Acquisition and Deep Neural Networks for Automated Segmentation in Connectomics.

With the growing importance of three-dimensional and very large field of view imaging, acquisition time becomes a serious bottleneck. Additionally, dose reduction is of importance when imaging material like biological tissue that is sensitive to electron radiation. Random sparse scanning can be used in the combination with image reconstruction techniques to reduce the acquisition time or electron dose in scanning electron microscopy.

A bimodal tomographic reconstruction technique combining EDS-STEM and HAADF-STEM.

A three-dimensional (3D) chemical characterization of nanomaterials can be obtained using tomography based on high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) or energy dispersive X-ray spectroscopy (EDS) STEM. These two complementary techniques have both advantages and disadvantages. The Z-contrast images have good image quality but lack robustness in the compositional analysis, while the elemental maps give more element-specific information, but at a low signal-to-noise ratio and a longer exposure time.

Foil-hole and data image quality assessment in 3DEM: Towards high-throughput image acquisition in the electron microscope.

Automatic or semiautomatic data collection approaches on a transmission electron microscope (TEM) for Single Particle Analysis, capable of acquiring large datasets composed of only high quality images, are of great importance to obtain 3D density maps with the highest resolution possible. Typically, this task is performed by an experienced microscopist, who manually decides to keep or discard images according to subjective criteria. Therefore, this methodology is slow, intensive in human work and subjective.

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography.

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages.

Evaluation of bicinchoninic acid as a ligand for copper(I)-catalyzed azide-alkyne bioconjugations.

The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid).

A fast algorithm for computing and correcting the CTF for tilted, thick specimens in TEM.

Today, the resolution in phase-contrast cryo-electron tomography is for a significant part limited by the contrast transfer function (CTF) of the microscope. The CTF is a function of defocus and thus varies spatially as a result of the tilting of the specimen and the finite specimen thickness. Models that include spatial dependencies have not been adopted in daily practice because of their high computational complexity.

Enzyme mediated site-specific surface modification.

Stable tethering of bioactive peptides like RGD to surfaces can be achieved via chemical bonding, biotin streptavidin interaction, or photocross-linking. More challenging is the immobilization of proteins, since methods applied to immobilize peptides are either not specific or versatile enough or might even compromise the protein's bioactivity. To overcome this limitation, we have employed a scheme that by enzymatic (transglutaminase) reaction allows the site-directed and site-specific coupling of growth factors and other molecules to nonfouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coated surfaces under physiological conditions.

Drug-sensing hydrogels for the inducible release of biopharmaceuticals.

Drug-dependent dissociation or association of cellular receptors represents a potent pharmacologic mode of action for regulating cell fate and function. Transferring the knowledge of pharmacologically triggered protein-protein interactions to materials science will enable novel design concepts for stimuli-sensing smart hydrogels. Here, we show the design and validation of an antibiotic-sensing hydrogel for the trigger-inducible release of human vascular endothelial growth factor.

A synthetic mammalian gene circuit reveals antituberculosis compounds.

Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In Mycobacterium tuberculosis EthR binds to a specific operator (O(ethR)) thereby repressing ethA and preventing EthA-catalyzed conversion of the prodrug ethionamide, which increases the resistance of the pathogen to this last-line-of-defense treatment. We have designed a synthetic mammalian gene circuit that senses the EthR-O(ethR) interaction in human cells and produces a quantitative reporter gene expression readout.

Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: systematic modulation of a synthetic cell-responsive PEG-hydrogel.

We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices (E=322+/-64.

Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions.

We present polymeric hydrogel biomaterials that are biomimetic both in their synthesis and degradation. The design of oligopeptide building blocks with dual enzymatic responsiveness allows us to create polymer networks that are formed and functionalized via enzymatic reactions and are degradable via other enzymatic reactions, both occurring under physiological conditions. The activated transglutaminase enzyme factor XIIIa was utilized for site-specific coupling of prototypical cell adhesion ligands and for simultaneous cross-linking of hydrogel networks from factor XIIIa substrate-modified multiarm poly(ethylene glycol) macromers.

Recent advances in electron tomography: TEM and HAADF-STEM tomography for materials science and semiconductor applications.

Electron tomography is a well-established technique for three-dimensional structure determination of (almost) amorphous specimens in life sciences applications. With the recent advances in nanotechnology and the semiconductor industry, there is also an increasing need for high-resolution three-dimensional (3D) structural information in physical sciences. In this article, we evaluate the capabilities and limitations of transmission electron microscopy (TEM) and high-angle-annular-dark-field scanning transmission electron microscopy (HAADF-STEM) tomography for the 3D structural characterization of partially crystalline to highly crystalline materials.

6-hydroxy-nicotine-inducible multilevel transgene control in mammalian cells.

The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O(NIC)), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O(NIC) operators 3' of a constitutive promoter.

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.

Quorum-sensing-based toolbox for regulatable transgene and siRNA expression in mammalian cells.

Technologies for regulated expression of multiple transgenes in mammalian cells have gathered momentum for bioengineering, gene therapy, drug discovery, and gene-function analyses. Capitalizing on recently developed mammalian transgene modalities (QuoRex) derived from Streptomyces coelicolor, we have designed a flexible and highly compatible expression vector set that enables desired transgene/siRNA control in response to the nontoxic butyrolactone SCB1. The construction-kit-like expression portfolio includes (i) multicistronic (pTRIDENT), (ii) autoregulated, (iii) bidirectional (pBiRex), (iv) oncoretro- and lentiviral transduction, and (v) RNA polymerase II-based siRNA transcription-fine-tuning vectors for straightforward implementation of QuoRex-controlled (trans)gene modulation in mammalian cells.

Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins.

Hydrogels were formed by conjugate addition of polyethylene glycol (PEG) multiacrylates and dithiothreitol (DTT) for encapsulation and sustained release of protein drugs; human growth hormone (hGH) was considered as an example. Prior to encapsulation, the hGH was precipitated either by Zn2+ ions or by linear PEG, to protect the hGH from reaction with the gel precursors during gelation. Precipitation by Zn2+ ions yielded precipitates that dissolved slowly and delayed release from even highly permeable gels, whereas linear PEG yielded rapidly dissolving precipitates.

Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice.

Background: Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes.

Methods: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1. Fusion of the S.

The effect of the linker on the hydrolysis rate of drug-linked ester bonds.

Tailoring the length of a sulfide containing linker adjusts the hydrolysis of a drug-linked ester bond to values appropriate for once-a-week administrations. A model drug of paclitaxel was coupled using a hydrolyzable linker to a poly(ethylene glycol) macromonomer, via a conjugate addition reaction between a thiol and an acrylamide. The macromonomers were synthesized in three steps with an average overall yield of 70%.

Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice.

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene-function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum-sensing to coordinate population-wide responses to physiological and/or physicochemical signals. A generic bacterial quorum-sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon.

Structure-transport relationship for the intestinal small-peptide carrier: is the carbonyl group of the peptide bond relevant for transport?

Purpose: The objective of this study was to determine the influence of the peptide bond with emphasis on the carbonyl group on the interaction with and transport by the intestinal small-peptide carrier. Therefore enalapril, a known substrate for the small-peptide carrier, has been modified to an analogue with a reduced peptide bond, enamipril. The transport characteristics of both compounds have been determined.

Monoclonal antibodies against two electron reduced riboflavin and a quantification of affinity constants for this oxygen-sensitive molecule.

In order to create a protein environment that binds preferentially to the two-electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two-electron-reduced flavin using standard techniques. Because of its sensitivity, time-resolved fluorescence can be used to overcome this problem.