Adaptive multi-epitope targeting and avidity-enhanced nanobody platform for ultrapotent, durable antiviral therapy.

Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA's modularity enables rapid, cost-effective production and adaptation to evolving pathogens.

Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.

Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 ß-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.

Neutral sphingomyelinase 2 is required for HIV-1 maturation.

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

A single amino acid transporter controls the uptake of priming-inducing beta-amino acids and the associated tradeoff between induced resistance and plant growth.

Selected ß-amino acids, such as ß-aminobutyric acid (BABA) and R-ß-homoserine (RBH), can prime plants for resistance against a broad spectrum of diseases. Here, we describe a genome-wide screen of fully annotated Arabidopsis thaliana T-DNA insertion lines for impaired in RBH-induced immunity (iri) mutants against the downy mildew pathogen Hyaloperonospora arabidopsidis, yielding 104 lines that were partially affected and four lines that were completely impaired in RBH-induced resistance (IR). We confirmed the iri1-1 mutant phenotype with an independent T-DNA insertion line in the same gene, encoding the high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER 1 (LHT1). Uptake experiments with yeast cells expressing LHT1 and mass spectrometry-based quantification of RBH and BABA in leaves of lht1 mutant and LHT1 overexpression lines revealed that LHT1 acts as the main transporter for cellular uptake and systemic distribution of RBH and BABA. Subsequent characterization of lht1 mutant and LHT1 overexpression lines for IR and growth responses revealed that the levels of LHT1-mediated uptake determine the tradeoff between IR and plant growth by RBH and BABA.

Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function.

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

DYNAMIC VIRAL LOAD MONITORING AND METAGENOMIC SEQUENCING IN ACUTE RETINAL NECROSIS CAUSED BY VARICELLA-ZOSTER VIRUS.

PURPOSE: To analyze the trend of intraocular viral load after antiviral treatment in patients with varicella-zoster virus-induced acute retinal necrosis and to explore the effect of viral genotypes on clinical manifestations. METHODS: In this case series, viral load was detected using polymerase chain reaction from aqueous humor during treatment; viral load curves were fitted, and the time required to reach the inflection point between plateau phase and logarithmic reduction phase was estimated. Variations in viral genomes were detected by metagenomic sequencing. RESULTS: Twenty eyes of 20 patients were included. The median (interquartile range) initial viral load was 5.9 × 10 7 (1.1 × 10 7 -1.1 × 10 8 ) copies/mL. The average duration of retinitis was 5 ± 3 weeks. The average time required to reach the inflection point was 4.2 ± 1.6 days. Time required to reach the inflection point was correlated with the duration of retinitis ( P = 0.025). Patients with varicella-zoster virus carrying the p.S715* variation in ribonucleotide reductase ( RNR ) subunit 1 gene had lower initial viral loads (median 1.3 × 10 7 copies/mL) than those without (median 1.1 × 10 8 copies/mL; adjusted P = 0.030). CONCLUSIONS: The inflection of viral load curve is helpful to estimate the length of plateau phase and the duration of retinitis during antiviral treatment in patients with acute retinal necrosis. Loss-of-function variation in RNR gene might be correlated with lower virulence of varicella-zoster virus.

Exosomal MicroRNAs modulate the cognitive function in fasudil treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by cognitive decline stemming from the accumulation of beta-amyloid (Aß) plaques and the propagation of tau pathology through synapses. Exosomes, crucial mediators in neuronal development, maintenance, and intercellular communication, have gained attention in AD research. Yet, the molecular mechanisms involving exosomal miRNAs in AD remain elusive. In this study, we treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice, a model for AD, with either vehicle (ADNS) or fasudil (ADF), while C57BL/6 (control) mice received vehicle (WT). Cognitive function was evaluated using the Y-maze test, and AD pathology was confirmed through immunostaining and western blot analysis of Aß plaques and phosphorylated tau. Exosomal RNAs were extracted, sequenced, and analyzed from each mouse group. Our findings revealed that fasudil treatment improved cognitive function in AD mice, as evidenced by increased spontaneous alternation in the Y-maze test and reduced Aß plaque load and phosphorylated tau protein expression in the hippocampus. Analysis of exosomal miRNAs identified three miRNAs (mmu-let-7i-5p, mmu-miR-19a-3p, mmu-miR-451a) common to both ADNS vs ADF and WT vs ADNS groups. Utilizing miRTarBase software, we predicted and analyzed target genes associated with these miRNAs. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNA target genes indicated that mmu-miR-19a-3p and mmu-miR-451a are implicated in signal transduction, immune response, cellular communication, and nervous system pathways. Specifically, mmu-miR-19a-3p targeted genes involved in the sphingolipid signaling pathway, such as Pten and Tnf, while mmu-miR-451a targeted Nsmaf, Gnai3, and Akt3. Moreover, mmu-miR-451a targeted Myc in signaling pathways regulating the pluripotency of stem cells. In conclusion, fasudil treatment enhanced cognitive function by modulating exosomal MicroRNAs, particularly mmu-miR-451a and mmu-miR-19a-3p. These miRNAs hold promise as potential biomarkers and therapeutic targets for novel AD treatments.

Comparative genomics provides insights into the aquatic adaptations of mammals.

The ancestors of marine mammals once roamed the land and independently committed to an aquatic lifestyle. These macroevolutionary transitions have intrigued scientists for centuries. Here, we generated high-quality genome assemblies of 17 marine mammals (11 cetaceans and six pinnipeds), including eight assemblies at the chromosome level. Incorporating previously published data, we reconstructed the marine mammal phylogeny and population histories and identified numerous idiosyncratic and convergent genomic variations that possibly contributed to the transition from land to water in marine mammal lineages. Genes associated with the formation of blubber (NFIA), vascular development (SEMA3E), and heat production by brown adipose tissue (UCP1) had unique changes that may contribute to marine mammal thermoregulation. We also observed many lineage-specific changes in the marine mammals, including genes associated with deep diving and navigation. Our study advances understanding of the timing, pattern, and molecular changes associated with the evolution of mammalian lineages adapting to aquatic life.

Survival improvements of marine mammals in zoological institutions mirror historical advances in human longevity.

An intense public debate has fuelled governmental bans on marine mammals held in zoological institutions. The debate rests on the assumption that survival in zoological institutions has been and remains lower than in the wild, albeit the scientific evidence in support of this notion is equivocal. Here, we used statistical methods previously applied to assess historical improvements in human lifespan and data on 8864 individuals of four marine mammal species (harbour seal, Phoca vitulina; California sea lion, Zalophus californianus; polar bear, Ursus maritimus; common bottlenose dolphin, Tursiops truncatus) held in zoos from 1829 to 2020. We found that life expectancy increased up to 3.40 times, and first-year mortality declined up to 31%, during the last century in zoos. Moreover, the life expectancy of animals in zoos is currently 1.65-3.55 times longer than their wild counterparts. Like humans, these improvements have occurred concurrently with advances in management practices, crucial for population welfare. Science-based decisions will help effective legislative changes and ensure better implementation of animal care.

Cryo-electron tomography to study viral infection.

Developments in cryo-electron microscopy (cryo-EM) have been interwoven with the study of viruses ever since its first applications to biological systems. Following the success of single particle cryo-EM in the last decade, cryo-electron tomography (cryo-ET) is now rapidly maturing as a technology and catalysing great advancement in structural virology as its application broadens. In this review, we provide an overview of the use of cryo-ET to study viral infection biology, discussing the key workflows and strategies used in the field. We highlight the vast body of studies performed on purified viruses and virus-like particles (VLPs), as well as discussing how cryo-ET can characterise host-virus interactions and membrane fusion events. We further discuss the importance of in situ cellular imaging in revealing previously unattainable details of infection and highlight the need for validation of high-resolution findings from purified ex situ systems. We give perspectives for future developments to achieve the full potential of cryo-ET to characterise the molecular processes of viral infection.

Making small molecules in plants: A chassis for synthetic biology-based production of plant natural products.

Plant natural products have been extensively exploited in food, medicine, flavor, cosmetic, renewable fuel, and other industrial sectors. Synthetic biology has recently emerged as a promising means for the cost-effective and sustainable production of natural products. Compared with engineering microbes for the production of plant natural products, the potential of plants as chassis for producing these compounds is underestimated, largely due to challenges encountered in engineering plants. Knowledge in plant engineering is instrumental for enabling the effective and efficient production of valuable phytochemicals in plants, and also paves the way for a more sustainable future agriculture. In this manuscript, we briefly recap the biosynthesis of plant natural products, focusing primarily on industrially important terpenoids, alkaloids, and phenylpropanoids. We further summarize the plant hosts and strategies that have been used to engineer the production of natural products. The challenges and opportunities of using plant synthetic biology to achieve rapid and scalable production of high-value plant natural products are also discussed.

Mechanisms of E. coli chemotaxis signaling pathways visualized using cryoET and computational approaches.

Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.

Comparison of gut microbial communities, free amino acids or fatty acids contents in the muscle of wild Aristichthys nobilis from Xinlicheng reservoir and Chagan lake.

BACKGROUND: Fish is favored by consumers, while amino acids and fatty acids are the main nutrients of muscle. At present, it has been found that the gut microbial community may be involved in the regulation of host material anabolism. Juvenile and adult bighead carp (A. nobilis) from Chagan lake and Xinlicheng reservoir were selected, and divided into four groups to compare the differences of gut microbial communities, free amino acid and fatty acids in muscle. RESULTS: The results showed that fish in different lakes or ages contained specific microbiota, the gut microbial structure was similar, but the microbial content was significantly different. Gut microbial abundance of juvenile fish in Chagan lake was significantly higher than that of other groups. Phylum level analysis Proteobacteria was the dominant gut bacteria of fish in both adult and juvenile fish from two separate lakes. Actinobacteria was another dominant bacterial phylum in juvenile fish in both lakes. Contents of free amino acids and fatty acids in muscle were detected, and the relationships between them and gut microbial communities were analyzed. Bighead carp grew from juvenile to adult, Actinobacteria abundance decreased (P < 0.05) and Proteobacteria increased (P < 0.05). Proteobacteria was positively correlated with the contents of Thr, Lys, Pro, Asp, Gly and Glu, Actinobacteria was negatively correlated with Met and His. Meanwhile, EPA and DHA were positively correlated with Proteobacteria, EPA and DHA were not significantly associated with Actinobacteria. CONCLUSION: It was speculated that the contents of free amino acids and fatty acids in muscle might be affected by the difference of gut microbiota, thus affecting the taste and nutritional quality.

Genome-enabled discovery of candidate virulence loci in Striga hermonthica, a devastating parasite of African cereal crops.

Parasites have evolved proteins, virulence factors (VFs), that facilitate plant colonisation, however VFs mediating parasitic plant-host interactions are poorly understood. Striga hermonthica is an obligate, root-parasitic plant of cereal hosts in sub-Saharan Africa, causing devastating yield losses. Understanding the molecular nature and allelic variation of VFs in S. hermonthica is essential for breeding resistance and delaying the evolution of parasite virulence. We assembled the S. hermonthica genome and identified secreted proteins using in silico prediction. Pooled sequencing of parasites growing on a susceptible and a strongly resistant rice host allowed us to scan for loci where selection imposed by the resistant host had elevated the frequency of alleles contributing to successful colonisation. Thirty-eight putatively secreted VFs had very different allele frequencies with functions including host cell wall modification, protease or protease inhibitor and kinase activities. These candidate loci had significantly higher Tajima's D than the genomic background, consistent with balancing selection. Our results reveal diverse strategies used by S. hermonthica to overcome different layers of host resistance. Understanding the maintenance of variation at virulence loci by balancing selection will be critical to managing the evolution of virulence as part of a sustainable control strategy.

Application of super-resolution and correlative double sampling in cryo-electron microscopy.

Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that collecting cryo-ET STA data using the same conditions as SPA, with both correlated double sampling (CDS) and the super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in the super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in the super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising the data size on disk and the area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

Peptoid-Directed Formation of Five-Fold Twinned Au Nanostars through Particle Attachment and Facet Stabilization.

While bio-inspired synthesis offers great potential for controlling nucleation and growth of inorganic particles, precisely tuning biomolecule-particle interactions is a long-standing challenge. Herein, we used variations in peptoid sequence to manipulate peptoid-Au interactions, leading to the synthesis of concave five-fold twinned, five-pointed Au nanostars via a process of repeated particle attachment and facet stabilization. Ex situ and liquid-phase TEM observations show that a balance between particle attachment biased to occur near the star points, preferential growth along the [100] direction, and stabilization of (111) facets is critical to forming star-shaped particles. Molecular simulations predict that interaction strengths between peptoids and distinct Au facets differ significantly and thus can alter attachment kinetics and surface energies to form the stars. This work provides new insights into how sequence-defined ligands affect particle growth to regulate crystal morphology.

emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging.

Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software.

Studying bacterial chemosensory array with CryoEM.

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

Synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model.

Purpose: To explore synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model. Methods: Sprague-Dawley rats were euthanized 1 h, 1 day, 3 days, 7 days, and 14 days after being exposed to intense blue light for 24 h. Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to evaluate changes in the outer nuclear layer (ONL). Transmission electron microscopy (TEM) was applied to observe the ultrastructural changes in the synapses between the photoreceptors and second-order neurons. Western blotting was conducted to evaluate specific proteins, including postsynaptic density-95 (PSD-95), metabotropic glutamate receptor 6 (mGluR6), synapsin I, and synaptophysin. Immunofluorescence of CD11b and PKC-α or mGluR6 was used to explore the spatial relationships between microglial processes and synaptic elements. Immunoelectron microscopy of PSD-95 was performed to further confirm its engulfment of synaptic materials. Results: H&E and TUNEL staining showed that the thickness of the ONL decreased markedly, and the number of apoptotic photoreceptors peaked at day 1. TEM revealed darkened photoreceptor terminals and that ribbons of them were floating in the cytoplasm, coinciding with the downregulation of PSD-95 and mGluR6. Downstream synaptic protein synapsin I and synaptophysin exhibited upregulation in the inner plexiform layer. Activated microglia migrated to the outer retina, and their processes were found in close proximity to synapses in the outer plexiform layer under light and electron microscopy levels. Double immunostaining of CD11b and mGluR6 showed colocalization. PSD-95-immunoreactive electron-dense materials were observed inside the microglia suggesting engulfment of synaptic components. Conclusions: The study showed that there are early synaptic impairment and late compensatory changes in downstream synapses in this photic injury model. Activated microglia touched and directly engulfed synaptic materials. Microglia may play a role or a partial role in synaptic changes.

Machining protein microcrystals for structure determination by electron diffraction.

We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.