Integration of 3D-printed cerebral cortical tissue into an ex vivo lesioned brain slice.

Engineering human tissue with diverse cell types and architectures remains challenging. The cerebral cortex, which has a layered cellular architecture composed of layer-specific neurons organised into vertical columns, delivers higher cognition through intricately wired neural circuits. However, current tissue engineering approaches cannot produce such structures. Here, we use a droplet printing technique to fabricate tissues comprising simplified cerebral cortical columns. Human induced pluripotent stem cells are differentiated into upper- and deep-layer neural progenitors, which are then printed to form cerebral cortical tissues with a two-layer organization. The tissues show layer-specific biomarker expression and develop a structurally integrated network of processes. Implantation of the printed cortical tissues into ex vivo mouse brain explants results in substantial structural implant-host integration across the tissue boundaries as demonstrated by the projection of processes and the migration of neurons, and leads to the appearance of correlated Ca2+ oscillations across the interface. The presented approach might be used for the evaluation of drugs and nutrients that promote tissue integration. Importantly, our methodology offers a technical reservoir for future personalized implantation treatments that use 3D tissues derived from a patient's own induced pluripotent stem cells.

A microscale soft ionic power source modulates neuronal network activity.

Bio-integrated devices need power sources to operate1,2. Despite widely used technologies that can provide power to large-scale targets, such as wired energy supplies from batteries or wireless energy transduction3, a need to efficiently stimulate cells and tissues on the microscale is still pressing. The ideal miniaturized power source should be biocompatible, mechanically flexible and able to generate an ionic current for biological stimulation, instead of using electron flow as in conventional electronic devices4-6. One approach is to use soft power sources inspired by the electrical eel7,8; however, power sources that combine the required capabilities have not yet been produced, because it is challenging to obtain miniaturized units that both conserve contained energy before usage and are easily triggered to produce an energy output. Here we develop a miniaturized soft power source by depositing lipid-supported networks of nanolitre hydrogel droplets that use internal ion gradients to generate energy. Compared to the original eel-inspired design7, our approach can shrink the volume of a power unit by more than 105-fold and it can store energy for longer than 24 h, enabling operation on-demand with a 680-fold greater power density of about 1,300 W m-3. Our droplet device can serve as a biocompatible and biological ionic current source to modulate neuronal network activity in three-dimensional neural microtissues and in ex vivo mouse brain slices. Ultimately, our soft microscale ionotronic device might be integrated into living organisms.

Enzyme-less nanopore detection of post-translational modifications within long polypeptides.

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through the nanopore, with directional co-translocational unfolding occurring unit by unit from either the C or N terminus. Chaotropic reagents at non-denaturing concentrations accelerate the analysis. By monitoring the ionic current flowing through the nanopore, we locate post-translational modifications deep within the polypeptide chains, laying the groundwork for compiling inventories of the proteoforms in cells and tissues.

Mobile Molecules: Reactivity Profiling Guides Faster Movement on a Cysteine Track.

We previously reported a molecular hopper, which makes sub-nanometer steps by thiol-disulfide interchange along a track with cysteine footholds within a protein nanopore. Here we optimize the hopping rate (ca. 0.1 s-1 in the previous work) with a view towards rapid enzymeless biopolymer characterization during translocation within nanopores. We first took a single-molecule approach to obtain the reactivity profiles of individual footholds. The pKa values of cysteine thiols within a pore ranged from 9.17 to 9.85, and the pH-independent rate constants of the thiolates with a small-molecule disulfide varied by up to 20-fold. Through site-specific mutagenesis and a pH increase from 8.5 to 9.5, the overall hopping rate of a DNA cargo along a five-cysteine track was accelerated 4-fold, and the rate-limiting step 21-fold.

Mobile Molecules: Reactivity Profiling Guides Faster Movement on a Cysteine Track.

We previously reported a molecular hopper, which makes sub-nanometer steps by thiol-disulfide interchange along a track with cysteine footholds within a protein nanopore. Here we optimize the hopping rate (ca. 0.1 s-1 in the previous work) with a view towards rapid enzymeless biopolymer characterization during translocation within nanopores. We first took a single-molecule approach to obtain the reactivity profiles of individual footholds. The pK a values of cysteine thiols within a pore ranged from 9.17 to 9.85, and the pH-independent rate constants of the thiolates with a small-molecule disulfide varied by up to 20-fold. Through site-specific mutagenesis and a pH increase from 8.5 to 9.5, the overall hopping rate of a DNA cargo along a five-cysteine track was accelerated 4-fold, and the rate-limiting step 21-fold.

A rapid high throughput bioprinted colorectal cancer spheroid platform forin vitrodrug- and radiation-response.

We describe the development of a high-throughput bioprinted colorectal cancer (CRC) spheroid platform with high levels of automation, information content, and low cell number requirement. This is achieved via the formulation of a hydrogel bioink with a compressive Young's modulus that is commensurate with that of colonic tissue (1-3 kPa), which supports exponential growth of spheroids from a wide range of CRC cell lines. The resulting spheroids display tight cell-cell junctions, bioink matrix-cell interactions and necrotic hypoxic cores. By combining high content light microscopy imaging and processing with rapid multiwell plate bioprinting, dose-response profiles are generated from CRC spheroids challenged with oxaliplatin (OX) and fluorouracil (5FU), as well as radiotherapy. Bioprinted CRC spheroids are shown to exhibit high levels of chemoresistance relative to cell monolayers, and OX was found to be significantly less effective against tumour spheroids than in monolayer culture, when compared to 5FU.

Nanopore-based technologies beyond DNA sequencing.

Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.

Parallel transmission in a synthetic nerve.

Bioelectronic devices that are tetherless and soft are promising developments in medicine, robotics and chemical computing. Here, we describe bioinspired synthetic neurons, composed entirely of soft, flexible biomaterials, capable of rapid electrochemical signal transmission over centimetre distances. Like natural cells, our synthetic neurons release neurotransmitters from their terminals, which initiate downstream reactions. The components of the neurons are nanolitre aqueous droplets and hydrogel fibres, connected through lipid bilayers. Transmission is powered at these interfaces by light-driven proton pumps and mediated by ion-conducting protein pores. By bundling multiple neurons into a synthetic nerve, we have shown that distinct signals can propagate simultaneously along parallel axons, thereby transmitting spatiotemporal information. Synthetic nerves might play roles in next-generation implants, soft machines and computing devices.

Reconstruction of the Gram-Negative Bacterial Outer-Membrane Bilayer.

The outer membrane (OM) of gram-negative bacteria is highly asymmetric. The outer leaflet comprises lipopolysaccharides (LPS) and the inner leaflet phospholipids. Here, it is shown that the outer membrane lipid bilayer (OMLB) of Escherichia coli can be reconstructed as a droplet interface bilayer (DIB), which separates two aqueous droplets in oil. The trimeric porin OmpF is inserted into the model OMLB and the translocation of the bacteriocin colicin E9 (colE9) through it is monitored. By contrast with LPS-free bilayers, it is found that colE9 made multiple failed attempts to engage with OmpF in an OMLB before successful translocation occurred. In addition, the observed rate for the second step of colE9 translocation is 3-times smaller than that in LPS-free bilayers, and further, the colE9 dissociates when the membrane potential is reversed. The findings demonstrate the utility of the DIB approach for constructing model OMLBs from physiologically realistic lipids and that the properties of the model OMLBs differ from those of a simple lipid bilayer. The model OMLB offers a credible platform for screening the properties of antibiotics.

A Lipid-Based Droplet Processor for Parallel Chemical Signals.

A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life is the ability to process several external chemical signals, often in parallel. Until now, cell- and tissue-like structures have been constructed with no more than one signaling pathway. Many pathways rely on signal transport across membranes using protein nanopores. However, such systems currently suffer from the slow transport of molecules. We have optimized the application of these nanopores to permit fast molecular transport, which has allowed us to construct a processor for parallel chemical signals from the bottom up in a modular fashion. The processor comprises three aqueous droplet compartments connected by lipid bilayers and operates in an aqueous environment. It can receive two chemical signals from the external environment, process them orthogonally, and then produce a distinct output for each signal. It is suitable for both sensing and enzymatic processing of environmental signals, with fluorescence and molecular outputs. In the future, such processors could serve as smart drug delivery vehicles or as modules within synthetic tissues to control their behavior in response to external chemical signals.

Enzymeless DNA Base Identification by Chemical Stepping in a Nanopore.

The stepwise movement of a single biopolymer strand through a nanoscopic detector for the sequential identification of its building blocks offers a universal means for single-molecule sequencing. This principle has been implemented in portable sequencers that use enzymes to move DNA or RNA through hundreds of individual nanopore detectors positioned in an array. Nevertheless, its application to the sequencing of other biopolymers, including polypeptides and polysaccharides, has not progressed because suitable enzymes are lacking. Recently, we devised a purely chemical means to move molecules processively in steps comparable to the repeat distances in biopolymers. Here, with this chemical approach, we demonstrate sequential nucleobase identification during DNA translocation through a nanopore. Further, the relative location of a guanine modification with a chemotherapeutic platinum derivative is pinpointed with single-base resolution. After further development, chemical translocation might replace stepping by enzymes for highly parallel single-molecule biopolymer sequencing.

Constructing ion channels from water-soluble α-helical barrels.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.

Determining the Orientation of Porins in Planar Lipid Bilayers.

Single-channel planar lipid bilayer (PLB) recording of bacterial porins has revealed molecular details of transport across the outer membrane of Gram-negative bacteria, including antibiotic permeation and protein translocation. To explore directional transport processes across cellular membranes, the orientation of porins or other pore-forming proteins must be established in a lipid bilayer prior to experimentation. Here, we describe a direct method for determining the orientation of porins in a PLB-with a focus on E. coli OmpF-by using targeted covalent modification of cysteine mutants. Each of the two possible orientations can be correlated with the porin conductance asymmetry, such that thereafter an I-V curve taken at the start of an experiment will suffice to establish orientation.

Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules.

Nanopore enzymology is a powerful single-molecule technique for the label-free study of enzymes using engineered protein nanopore sensors. The technique has been applied to protein kinases, where it has enabled the full repertoire of kinase function to be observed, including: kinetics of substrate binding and dissociation, product binding and dissociation, nucleotide binding, and reversible phosphorylation. Further, minor modifications enable the screening of type I kinase inhibitors and the determination of inhibition constants in a facile and label-free manner. Here, we describe the design and production of suitably engineered protein nanopores and their use for the determination of key mechanistic parameters of kinases. We also provide procedures for the determination of inhibition constants of protein kinase inhibitors.

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer.

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

Single-Molecule Observation of Intermediates in Bioorthogonal 2-Cyanobenzothiazole Chemistry.

We report a single-molecule mechanistic investigation into 2-cyanobenzothiazole (CBT) chemistry within a protein nanoreactor. When simple thiols reacted reversibly with CBT, the thioimidate monoadduct was approximately 80-fold longer-lived than the tetrahedral bisadduct, with important implications for the design of molecular walkers. Irreversible condensation between CBT derivatives and N-terminal cysteine residues has been established as a biocompatible reaction for site-selective biomolecular labeling and imaging. During the reaction between CBT and aminothiols, we resolved two transient intermediates, the thioimidate and the cyclic precursor of the thiazoline product, and determined the rate constants associated with the stepwise condensation, thereby providing critical information for a variety of applications, including the covalent inhibition of protein targets and dynamic combinatorial chemistry.

Direct detection of molecular intermediates from first-passage times.

All natural phenomena are governed by energy landscapes. However, the direct measurement of this fundamental quantity remains challenging, particularly in complex systems involving intermediate states. Here, we uncover key details of the energy landscapes that underpin a range of experimental systems through quantitative analysis of first-passage time distributions. By combined study of colloidal dynamics in confinement, transport through a biological pore, and the folding kinetics of DNA hairpins, we demonstrate conclusively how a short-time, power-law regime of the first-passage time distribution reflects the number of intermediate states associated with each of these processes, despite their differing length scales, time scales, and interactions. We thereby establish a powerful method for investigating the underlying mechanisms of complex molecular processes.

Lipid-Bilayer-Supported 3D Printing of Human Cerebral Cortex Cells Reveals Developmental Interactions.

Current understanding of human brain development is rudimentary due to suboptimal in vitro and animal models. In particular, how initial cell positions impact subsequent human cortical development is unclear because experimental spatial control of cortical cell arrangement is technically challenging. 3D cell printing provides a rapid customized approach for patterning. However, it has relied on materials that do not represent the extracellular matrix (ECM) of brain tissue. Therefore, in the present work, a lipid-bilayer-supported printing technique is developed to 3D print human cortical cells in the soft, biocompatible ECM, Matrigel. Printed human neural stem cells (hNSCs) show high viability, neural differentiation, and the formation of functional, stimulus-responsive neural networks. By using prepatterned arrangements of neurons and astrocytes, it is found that hNSC process outgrowth and migration into cell-free matrix and into astrocyte-containing matrix are similar in extent. However, astrocytes enhance the later developmental event of axon bundling. Both young and mature neurons migrate into compartments containing astrocytes; in contrast, astrocytes do not migrate into neuronal domains signifying nonreciprocal chemorepulsion. Therefore, precise prepatterning by 3D printing allows the construction of natural and unnatural patterns that yield important insights into human cerebral cortex development.

Bifurcated binding of the OmpF receptor underpins import of the bacteriocin colicin N into Escherichia coli.

Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.