Rational design of a 'two-in-one' immunogen DAM drives potent immune response against mpox virus.

The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a 'two-in-one' immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.

Toehold switches: de-novo-designed regulators of gene expression.

Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. Here, we report a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. We show that switches can be integrated into the genome to regulate endogenous genes and use them as sensors that respond to endogenous RNAs. We exploit the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology.

Paper-based synthetic gene networks.

Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

Single-shot 20-fold expansion microscopy.

Expansion microscopy (ExM) is in increasingly widespread use throughout biology because its isotropic physical magnification enables nanoimaging on conventional microscopes. To date, ExM methods either expand specimens to a limited range (~4-10× linearly) or achieve larger expansion factors through iterating the expansion process a second time (~15-20× linearly). Here, we present an ExM protocol that achieves ~20× expansion (yielding <20-nm resolution on a conventional microscope) in a single expansion step, achieving the performance of iterative expansion with the simplicity of a single-shot protocol. This protocol, which we call 20ExM, supports postexpansion staining for brain tissue, which can facilitate biomolecular labeling. 20ExM may find utility in many areas of biological investigation requiring high-resolution imaging.

Large-Area Epitaxial Growth of Transition Metal Dichalcogenides.

Over the past decade, research on atomically thin two-dimensional (2D) transition metal dichalcogenides (TMDs) has expanded rapidly due to their unique properties such as high carrier mobility, significant excitonic effects, and strong spin-orbit couplings. Considerable attention from both scientific and industrial communities has fully fueled the exploration of TMDs toward practical applications. Proposed scenarios, such as ultrascaled transistors, on-chip photonics, flexible optoelectronics, and efficient electrocatalysis, critically depend on the scalable production of large-area TMD films. Correspondingly, substantial efforts have been devoted to refining the synthesizing methodology of 2D TMDs, which brought the field to a stage that necessitates a comprehensive summary. In this Review, we give a systematic overview of the basic designs and significant advancements in large-area epitaxial growth of TMDs. We first sketch out their fundamental structures and diverse properties. Subsequent discussion encompasses the state-of-the-art wafer-scale production designs, single-crystal epitaxial strategies, and techniques for structure modification and postprocessing. Additionally, we highlight the future directions for application-driven material fabrication and persistent challenges, aiming to inspire ongoing exploration along a revolution in the modern semiconductor industry.

Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.

Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly.

High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

Diverse and robust molecular algorithms using reprogrammable DNA self-assembly.

Molecular biology provides an inspiring proof-of-principle that chemical systems can store and process information to direct molecular activities such as the fabrication of complex structures from molecular components. To develop information-based chemistry as a technology for programming matter to function in ways not seen in biological systems, it is necessary to understand how molecular interactions can encode and execute algorithms. The self-assembly of relatively simple units into complex products1 is particularly well suited for such investigations. Theory that combines mathematical tiling and statistical-mechanical models of molecular crystallization has shown that algorithmic behaviour can be embedded within molecular self-assembly processes2,3, and this has been experimentally demonstrated using DNA nanotechnology4 with up to 22 tile types5-11. However, many information technologies exhibit a complexity threshold-such as the minimum transistor count needed for a general-purpose computer-beyond which the power of a reprogrammable system increases qualitatively, and it has been unclear whether the biophysics of DNA self-assembly allows that threshold to be exceeded. Here we report the design and experimental validation of a DNA tile set that contains 355 single-stranded tiles and can, through simple tile selection, be reprogrammed to implement a wide variety of 6-bit algorithms. We use this set to construct 21 circuits that execute algorithms including copying, sorting, recognizing palindromes and multiples of 3, random walking, obtaining an unbiased choice from a biased random source, electing a leader, simulating cellular automata, generating deterministic and randomized patterns, and counting to 63, with an overall per-tile error rate of less than 1 in 3,000. These findings suggest that molecular self-assembly could be a reliable algorithmic component within programmable chemical systems. The development of molecular machines that are reprogrammable-at a high level of abstraction and thus without requiring knowledge of the underlying physics-will establish a creative space in which molecular programmers can flourish.

Author Correction: Diverse and robust molecular algorithms using reprogrammable DNA self-assembly.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rotation tracking of genome-processing enzymes using DNA origami rotors.

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking1-3, angular optical trapping4 and magnetic tweezers5, have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP)6, gyrase2, a viral DNA packaging motor7 and DNA recombination enzymes8. Despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient for tracking the rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce origami-rotor-based imaging and tracking (ORBIT), a method that uses fluorescently labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair9, as well as from transcription by RNAP. We characterized a series of events that occur during RecBCD-induced DNA unwinding-including initiation, processive translocation, pausing and backtracking-and revealed an initiation mechanism that involves reversible ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA.

Loss of TDP-43 mediates severe neurotoxicity by suppressing PJA1 gene transcription in the monkey brain.

The nuclear loss and cytoplasmic accumulation of TDP-43 (TAR DNA/RNA binding protein 43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previously, we reported that the primate-specific cleavage of TDP-43 accounts for its cytoplasmic mislocalization in patients' brains. This prompted us to investigate further whether and how the loss of nuclear TDP-43 mediates neuropathology in primate brain. In this study, we report that TDP-43 knockdown at the similar effectiveness, induces more damage to neuronal cells in the monkey brain than rodent mouse. Importantly, the loss of TDP-43 suppresses the E3 ubiquitin ligase PJA1 expression in the monkey brain at transcriptional level, but yields an opposite upregulation of PJA1 in the mouse brain. This distinct effect is due to the species-dependent binding of nuclear TDP-43 to the unique promoter sequences of the PJA1 genes. Further analyses reveal that the reduction of PJA1 accelerates neurotoxicity, whereas overexpressing PJA1 diminishes neuronal cell death by the TDP-43 knockdown in vivo. Our findings not only uncover a novel primate-specific neurotoxic contribution to the loss of function theory of TDP-43 proteinopathy, but also underscore a potential therapeutic approach of PJA1 to the loss of nuclear TDP-43.

Fluorescent Probe for Investigating the Mitochondrial Viscosity and Hydrogen Peroxide Changes in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI), a cause of cerebral dysfunction during cerebral infarction treatment, is closely associated with mitochondrial viscosity and hydrogen peroxide (H2O2). However, the accurate measurement of mitochondrial viscosity and H2O2 levels in CIRI is challenging because of the lack of sufficient selectivity and blood-brain barrier (BBB) penetration of existing monitoring tools related to CIRI, hampering the exploration of the role of mitochondrial viscosity and H2O2 in CIRI. To address this issue, we designed an activatable fluorescent probe, mitochondria-targeting styryl-quinolin-ium (Mito-IQS), with excellent properties including high selectivity, mitochondrial targeting, and BBB penetration, for the visualization of mitochondrial viscosity and H2O2 in the brain. Based on the real-time monitoring capabilities of the probe, bursts of mitochondrial viscosity and H2O2 levels were visualized during CIRI. This probe can be used to monitor the therapeutic effects of butylphthalein treatment. More importantly, in vivo experiments further confirmed that CIRI was closely associated with the mitochondrial viscosity and H2O2 levels. This discovery provides new insights and tools for the study of CIRI and is expected to accelerate the process of CIRI diagnosis, treatment, and drug design.

Entropy-Driven Ostwald Ripening Reversal Promotes the Formation of Low-Platinum Intermetallic Fuel Cell Catalysts.

Strain engineering has been widely used to optimize platinum-based oxygen reduction reaction (ORR) catalysts for proton exchange membrane fuel cells (PEMFCs). PtM3 (M is base metals), a well-known high-compressive-strain intermetallic alloy, shows promise as a low platinum ORR catalyst due to high intrinsic activity. However, during the alloying of Pt with a threefold amount of M, a notable phase separation between Pt and M may occur, with M particles rapidly sintering while Pt particles grow slowly, posing a challenge in achieving a well-defined PtM3 intermetallic alloy. Here, an entropy-driven Ostwald ripening reversal phenomenon is discovered that enables the synthesis of small-sized Pt(FeCoNiCu)3 intermetallic ORR catalysts. High entropy promotes the thermodynamic driving force for the alloying Pt with M, which triggers the Ostwald ripening reversal of sintered FeCoNiCu particles and facilitates the formation of uniform Pt(FeCoNiCu)3 intermetallic catalysts. The prepared Pt(FeCoNiCu)3 catalysts exhibit a high specific activity of 3.82 mA cm-2, along with a power density of ≈1.3 W cm-2 at 0.67 V and 94 °C with a cathode Pt loading of 0.1 mg cm-2 in H2-air fuel cell.

Operando Spectroscopy Observation of Mo Clusters-Ti3 C2 TX Catalyst/Support Interface's Dynamic Evolution in Hydrogen Evolution Reaction.

The interaction between catalyst and support plays an important role in electrocatalytic hydrogen evolution (HER), which may explain the improvement in performance by phase transition or structural remodeling. However, the intrinsic behavior of these catalysts (dynamic evolution of the interface under bias, structural/morphological transformation, stability) has not been clearly monitored, while the operando technology does well in capturing the dynamic changes in the reaction process in real time to determine the actual active site. In this paper, nitrogen-doped molybdenum atom-clusters on Ti3 C2 TX (MoACs /N-Ti3 C2 TX ) is used as a model catalyst to reveal the dynamic evolution of MoAcs on Ti3 C2 TX during the HER process. Operando X-ray absorption structure (XAS) theoretical calculation and in situ Raman spectroscopy showed that the Mo cluster structure evolves to a 6-coordinated monatomic Mo structure under working conditions, exposing more active sites and thus improving the catalytic performance. It shows excellent HER performance comparable to that of commercial Pt/C, including an overpotential of 60 mV at 10 mA cm-2 , a small Tafel slope (56 mV dec-1 ), and high activity and durability. This study provides a unique perspective for investigating the evolution of species, interfacial migration mechanisms, and sources of activity-enhancing compounds in the process of electroreduction.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Accelerated differentiation of neo-W nuclear-encoded mitochondrial genes between two climate-associated bird lineages signals potential co-evolution with mitogenomes.

There is considerable evidence for mitochondrial-nuclear co-adaptation as a key evolutionary driver. Hypotheses regarding the roles of sex-linkage have emphasized Z-linked nuclear genes with mitochondrial function (N-mt genes), whereas it remains contentious whether the perfect co-inheritance of W genes with mitogenomes could hinder or facilitate co-adaptation. Young (neo-) sex chromosomes that possess relatively many N-mt genes compared to older chromosomes provide unprecedented hypothesis-testing opportunities. Eastern Yellow Robin (EYR) lineages in coastal and inland habitats with different climates are diverged in mitogenomes, and in a ~ 15.4 Mb nuclear region enriched with N-mt genes, in contrast with otherwise-similar nuclear genomes. This nuclear region maps to passerine chromosome 1A, previously found to be neo-sex in the inland EYR genome. To compare sex-linked Chr1A-derived genes between lineages, we assembled and annotated the coastal EYR genome. We found that: (i) the coastal lineage shares a similar neo-sex system with the inland lineage, (ii) neo-W and neo-Z N-mt genes are not more diverged between lineages than are comparable non-N-mt genes, and showed little evidence for broad positive selection, (iii) however, W-linked N-mt genes are more diverged between lineages than are their Z-linked gametologs. The latter effect was ~7 times stronger for N-mt than non-N-mt genes, suggesting that W-linked N-mt genes might have diverged between lineages under environmental selection through co-evolution with mitogenomes. Finally, we identify a candidate gene driver for divergent selection, NDUFA12. Our data represent a rare example suggesting a possible role for W-associated mitochondrial-nuclear interactions in climate-associated adaptation and lineage differentiation.

Ambient fine particulate matter and Life's essential 8 and mortality in adults in China: A Nationwide retrospective cohort study.

BACKGROUND: Enhanced cardiovascular health (CVH) is linked to reduced mortality risks, whereas long-term exposure to fine particulate matter (PM2.5), elevates these risks. Whether long-term exposure to PM2.5 counteracts the health benefits of high CVH is unknown. The study aims to evaluate whether the association of CVH assessed by Life's Essential 8 (LE8) with death was consistent between participants with different PM2.5 exposures. METHODS: We included 134,727 participants in the field survey of China Chronic Disease and Risk Factor Surveillance which was conducted from August 2013 to June 2014. The deaths of participants were obtained by linking to the National Mortality Surveillance System (2013-2018). The environmental data is obtained by satellite inversion. The participants' CVH scores were calculated using the LE8 method. Hazard ratio (HR) and 95% confidence intervals (95%CI) for mortality were calculated using Cox regression models. RESULTS: A total of 2,936 all-cause deaths and 1,158 cardiovascular disease (CVD) deaths were recorded. Compared to those with low CVH, adults with high CVH demonstrated a reduced risk of all-cause mortality, irrespective of their PM2.5 exposure levels (P < 0.05, all P for interaction >0.05). Furthermore, in comparison to those with low CVH and highest PM2.5 exposure, adults with high CVH and lowest PM2.5 exposure exhibited HR of 0.18 (95%CI, 0.12-0.25) for all-cause mortality and 0.13 (95%CI, 0.08-0.22) for CVD mortality. CONCLUSIONS: High CVH is associated with reduced all-cause mortality risk, regardless of PM2.5 exposure levels. For Chinese adults, sustaining high CVH is advisable, irrespective of their residential location.