Toward single-molecule protein sequencing using nanopores
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Rapid brain tumor classification from sparse epigenomic data
Although the intraoperative molecular diagnosis of the approximately 100 known brain tumor entities described to date has been a goal of neuropathology for the past decade, achieving this within a clinically relevant timeframe of under 1 h after biopsy collection remains elusive. Advances in third-generation sequencing have brought this goal closer, but established machine learning techniques rely on computationally intensive methods, making them impractical for live diagnostic workflows in clinical applications. Here we present MethyLYZR, a naive Bayesian framework enabling fully tractable, live classification of cancer epigenomes. For evaluation, we used nanopore sequencing to classify over 200 brain tumor samples, including 10 sequenced in a clinical setting next to the operating room, achieving highly accurate results within 15 min of sequencing. MethyLYZR can be run in parallel with an ongoing nanopore experiment with negligible computational overhead. Therefore, the only limiting factors for even faster time to results are DNA extraction time and the nanopore sequencer’s maximum parallel throughput. Although more evidence from prospective studies is needed, our study suggests the potential applicability of MethyLYZR for live molecular classification of nervous system malignancies using nanopore sequencing not only for the neurosurgical intraoperative use case but also for other oncologic indications and the classification of tumors from cell-free DNA in liquid biopsies.
Geochemistry of lithospheric aqueous fluids modified by nanoconfinement
Water is a principal component of Earth’s fluids, and its interaction with rocks governs lithospheric geochemical and geodynamic processes. Water–rock interactions are crucial in societally relevant resource management, including subsurface extraction and storage of energy, the deep carbon cycle and generating critical metal deposits. The prevailing view is that fluids navigate through the lithosphere without being influenced by the distinct properties that arise from matter confined at the nanoscale. Here we use electron microscopy and neutron scattering data to show that a diverse range of lithospheric rocks, including sandstones, peridotites and serpentinites, consistently show nanoporosity, predominantly with pore sizes < 100 nanometres. Using molecular dynamics simulations, we demonstrate that water’s dielectric permittivity—a fundamental property that governs its geochemical behaviour—diverges in nanoconfinement from its bulk counterpart under conditions ranging from ambient to extremes of 700 °C and 5 GPa. Our geochemical simulations suggest that changes in water permittivity due to confinement will decrease mineral solubility, a process that is not currently considered in models of fluid–rock interactions. Given that permittivity is also intimately linked to ion speciation, pore-size-dependent properties should be expected to exert a primary influence on rock reactivity and the geochemical evolution of fluids during fluid–rock interactions.
Analysis of microbial composition and sharing in low-biomass human milk samples: a comparison of DNA isolation and sequencing techniques
Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.
ON–OFF nanopores for optical control of transmembrane ionic communication
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.
Advance in peptide-based drug development: delivery platforms, therapeutics and vaccines
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