Hagan Bayley

Glycosaminoglycan (GAG) polysaccharides play vital roles in animal physiology and disease ¹ . Their diverse and intricate patterns of sulfation and epimerization endow them with an extensive potential to encode functional information ^2,3 . GAG characterization, however, remains a formidable challenge for state-of-the-art ensemble-based techniques ^4,5 . Single-molecule techniques are uniquely suited for analysing complex mixtures ^6,7 . Here, we report the single-molecule resolution and counting of diverse GAG di- and oligosaccharides derived from longer heterogeneous chains as part of a deconvolutive nanopore-based workflow that requires no fractionation and is operationally simple. Modular chemical deacylation and amino-selective ring-contractive formation of electrophilic aldehydes enable the parsing of libraries of GAG structures into simplified sets of reactive anhydrosugars for nanopore readout via reversible covalent adduct formation. Discrete clustering of event amplitudes enables direct sugar sizing (∼10 % step change per residue), which can be coupled to precisely resolved amplitude differences that further reveal sugar fine structure—including the number and position of sulfate groups (∼2 % step change per sulfate) alongside single- atom stereochemistry (∼0.5 % step change between epimers). Guided by chemical logic, the reverse mapping of resolved anhydrosugars to their precursors covers ∼84–100 % of all disaccharides and their eliminative digestion variants in natural heparan sulfate (HS). We demonstrate the practical utility and scope of our approach through the compositional analysis of a panel of HS polysaccharides that together encompass natural GAG structural diversity. Moreover, we detect contaminants in heparin, including oversulfated chondroitin sulfate found in an authentic pharmaceutical heparin sample previously implicated in a global healthcare crisis. Together, our results suggest a general chemo-biophysical framework for the precise and sensitive characterization of GAGs that extends to other aminosugar biopolymers. When adapted for portable, widely used nanopore sequencing devices, our approach may offer a path towards the long-sought ‘democratization’ of glycan analysis.

Molecular systems can store data, and chemical reaction networks can form reservoirs for computation, but architectures that integrate both functions remain elusive. Here we show that a single nanopore can both store and process information by using its own chemical dynamics. Confined in an individual protein nanopore acting as a nanoreactor, reactive cysteine residues on a single β strand encode ‘1’ bits, and unreactive residues encode ‘0’ bits – together constituting configurable memory tapes. Reversible thiol–selenosulfide exchange reactions convert cysteine thiolates into transient selenosulfide adducts, producing complex reaction dynamics that enable data retrieval. The chemical reaction network inside the nanoreactor acts as an autonomous physical reservoir. The data-reading process generates the temporal dynamics, which are decoded with a simple linear readout for memory tape classification and future reaction-trend prediction. All 15 reactive 4-bit memory tapes are distinguishable and identified with >90% overall accuracy in 15-way classification. The timescale over which the reservoir retains predictive capacity is chemically tunable by reactant concentration, network complexity or memory tape composition. Our design principles generalize to (bio)chemical reaction networks and information processing systems with a single-molecule readout and reversible transitions.

Regenerative medicine offers a promising approach to treat brain injuries, yet challenges persist in promoting neuronal survival and integration. Recent studies demonstrate that human cells implanted into rodent brains can exhibit plasticity, integrate into neural circuits and alleviate functional deficits. However, integration is often poor, with inadequate vascularization, and insufficient support cells such as astrocytes. Astrocytes play a crucial role in neuronal development and recovery by releasing growth factors, facilitating synaptogenesis, and promoting blood vessel formation. This study investigated human neuronal progenitor cells cultured alone or cultured with mouse astrocytes and formed into 3D constructs using microfluidics. Co‐cultures exhibited enhanced neuronal maturation, viability, and density. Following implantation into mouse brains, co‐cultures reduced lesion size, increased axonal growth, and improved astrocyte coupling to blood vessels within the graft. Additionally, we show that NPCs and co‐cultures increased astrocyte size in implants. Deconvolved high‐resolution microscopy identified synapses and optogenetics showed functional connections between the host and implants. These findings underscore the essential role of astrocytes in enhancing neuronal tissue integration and advancing brain injury treatments.

Volatile organic compounds are choice analytes in a variety of contexts. For example, humans release over 4000 volatile organic compounds, many of which are diagnostic of life-threatening medical conditions. The analysis of combinations of a large number of potential analytes requires the application of costly, cumbersome technology. In this work, we show that covalent nanopore sensing can be used for the targeted detection of a reduced set of analytes in a mixture. In this case aldehydes, which constitute ~5% of human volatiles, can be selectively detected by using reversible thiol-aldehyde chemistry. Further, nanopore engineering permits high-resolution detection, which allows closely related aldehydes, including isomers, to be distinguished. Differential sensing of members of other chemical classes, such as mono alcohols, is also demonstrated by leveraging their enzymatic conversion into aldehydes. Our approach is compatible with the use of low-cost, portable, user-friendly diagnostic devices applicable to a wide variety of objectives, including pollutant monitoring, food and beverage testing and the quality control of pharmaceuticals, as well as disease diagnostics.

Synthetic cells, such as giant unilamellar vesicles, can be engineered to detect and release chemical signals to control target cell behavior. However, control over the targeting of cell populations is limited due to poor spatial or temporal resolution and the inability of synthetic cells to deliver patterned signals. Here, 3D‐printed picoliter droplet networks are described that direct gene expression in underlying bacterial populations by patterned release of a chemical signal with temporal control. Shrinkage of the droplet networks prior to use achieves spatial control over gene expression with ≈50 µm resolution. Ways to store chemical signals in the droplet networks and to activate release at controlled points in time are also demonstrated. Finally, it is shown that the spatially‐controlled delivery system can regulate competition between bacteria by inducing the patterned expression of toxic bacteriocins. This system provides the groundwork for the use of picoliter droplet networks in fundamental biology and in medicine in applications that require the controlled formation of chemical gradients (i.e., for the purpose of local control of gene expression) within a target group of cells.

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane α-helical barrels with conductive channels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) low-conductance states that correlate with variations in the designed amino-acid sequences and modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single channels change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic channels, as observed in natural barrel-stave peptide channels. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

The poor survival of ovarian cancer patients is linked to their high likelihood of relapse. In spite of full apparent macroscopic clearance, tumor recurrences arise from cells that are resistant to primary chemotherapy in the form of minimal residual disease (MRD). MRD exhibits distinct molecular drivers from bulk cancer and therefore necessitates alternative therapeutic strategies. However, there is a lack of 3D models that faithfully recapitulate MRD ex vivo for therapy development. This study constructs microfluidics‐based 3D microtumors to generate a clinically‐relevant model for ovarian cancer MRD. The microtumors recapitulate the non‐genetic heterogeneity of ovarian cancer, capturing the “Oxford Classic” five molecular signatures. Gene expression in the 3D microtumors aligns closely with MRD from ovarian cancer patients and features the upregulation of fatty acid metabolism genes. Finally, the MRD 3D microtumors respond to the approved fatty acid oxidation inhibitor, perhexiline, demonstrating their utility in drug discovery. This system might be used as a drug‐testing platform for the discovery of novel MRD‐specific therapies in ovarian cancer.

Nanoscale photoswitchable proteins could facilitate precise spatiotemporal control of transmembrane communication and support studies in synthetic biology, neuroscience and bioelectronics. Here, through covalent modification of the α-haemolysin protein pore with arylazopyrazole photoswitches, we produced ‘photopores’ that transition between iontronic resistor and diode modes in response to irradiation at orthogonal wavelengths. In the diode mode, a low-leak OFF-state nanopore exhibits a reversible increase in unitary conductance of more than 20-fold upon irradiation at 365 nm. A rectification ratio of >5 was achieved with photopores in the diode state by either direct or alternating voltage input. Unlike conventional electronic phototransistors with intensity-dependent photoelectric responses, the photopores regulated current output solely based on the wavelength(s) of monochromatic or dual-wavelength irradiation. Dual-wavelength irradiation at various relative intensities allowed graded adjustment of the photopore conductance. By using these properties, photonic signals encoding text or graphic messages were converted into ionic signals, highlighting the potential applications of photopores as components of smart devices in synthetic biology.