D-peptide-magnetic nanoparticles fragment tau fibrils and rescue behavioral deficits in a mouse model of Alzheimer's disease.

Amyloid fibrils of tau are increasingly accepted as a cause of neuronal death and brain atrophy in Alzheimer's disease (AD). Diminishing tau aggregation is a promising strategy in the search for efficacious AD therapeutics. Previously, our laboratory designed a six-residue, nonnatural amino acid inhibitor D-TLKIVW peptide (6-DP), which can prevent tau aggregation in vitro. However, it cannot block cell-to-cell transmission of tau aggregation. Here, we find D-TLKIVWC (7-DP), a d-cysteine extension of 6-DP, not only prevents tau aggregation but also fragments tau fibrils extracted from AD brains to neutralize their seeding ability and protect neuronal cells from tau-induced toxicity. To facilitate the transport of 7-DP across the blood-brain barrier, we conjugated it to magnetic nanoparticles (MNPs). The MNPs-DP complex retains the inhibition and fragmentation properties of 7-DP alone. Ten weeks of MNPs-DP treatment appear to reverse neurological deficits in the PS19 mouse model of AD. This work offers a direction for development of therapies to target tau fibrils.

Oolonghomobisflavans from Camellia sinensis disaggregate tau fibrils across Alzheimer's disease models.

Alzheimer's disease (AD) is a common debilitating neurodegenerative disease with limited treatment options. Amyloid-ß (Aß) and tau fibrils are well-established hallmarks of AD, which can induce oxidative stress, neuronal cell death, and are linked to disease pathology. Here, we describe the effects of Oolonghomobisflavan A (OFA) and Oolonghomobisflavan B (OFB) on tau fibril disaggregation and prionogenic seeding. Transcriptomic analysis of OF-treated animals reveals the induction of a proteostasis-enhancing and health-promoting signature. OFA treatment reduced the burden of Tau protein aggregation in a C. elegans model expressing pathogenic human tau ("hTau-expressing") and promoted Tau disaggregation and inhibited seeding in assays using ex vivo brain-derived paired helical filament tau protein fibrils from Alzheimer's disease brain donors. Correspondingly, treatment with OF improved multiple fitness and aging-related health parameters in the hTau-expressing C. elegans model, including reproductive output, muscle function, and importantly, reversed the shortened lifespan stemming from pathogenic Tau expression. Collectively, this study provides new evidence supporting the neuroprotective effects of OFs and reveal a new therapeutic strategy for targeting AD and other neurodegenerative diseases characterized by tauopathy.

A heterologous expression platform in Aspergillus nidulans for the elucidation of cryptic secondary metabolism biosynthetic gene clusters: discovery of the Aspergillus fumigatus sartorypyrone biosynthetic pathway.

Aspergillus fumigatus is a serious human pathogen causing life-threatening Aspergillosis in immunocompromised patients. Secondary metabolites (SMs) play an important role in pathogenesis, but the products of many SM biosynthetic gene clusters (BGCs) remain unknown. In this study, we have developed a heterologous expression platform in Aspergillus nidulans, using a newly created genetic dereplication strain, to express a previously unknown BGC from A. fumigatus and determine its products. The BGC produces sartorypyrones, and we have named it the spy BGC. Analysis of targeted gene deletions by HRESIMS, NMR, and microcrystal electron diffraction (MicroED) enabled us to identify 12 products from the spy BGC. Seven of the compounds have not been isolated previously. We also individually expressed the polyketide synthase (PKS) gene spyA and demonstrated that it produces the polyketide triacetic acid lactone (TAL), a potentially important biorenewable platform chemical. Our data have allowed us to propose a biosynthetic pathway for sartorypyrones and related natural products. This work highlights the potential of using the A. nidulans heterologous expression platform to uncover cryptic BGCs from A. fumigatus and other species, despite the complexity of their secondary metabolomes.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Selective inhibition of hsp90 paralogs: Structure and binding studies uncover the role of helix 1 in Grp94-selective ligand binding.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the structural elements that mediate each paralog's response to inhibitor binding. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, the 8-aryl moiety of PU-H36 is inserted into Site 2, a conditionally available side pocket, but in Hsp90 it occupies Site 1, a non-selective side pocket that is accessible in all hsp90 paralogs. The structure of Grp94 in complex with the non-selective PU-H71 shows only Site 1 binding. Large conformational shifts involving helices 1, 4 and 5 of the N-terminal domain of Grp94 are associated with the engagement of the Site 2 pocket for ligand binding. To understand the origins of Site 2 pocket engagement, we tested the binding of Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 of the Grp94 N-terminal domain is the discriminating element that allows for remodeling of the ATP binding pocket and exposure of the Site 2 selective pocket.

A heterogeneously integrated lithium niobate-on-silicon nitride photonic platform.

The availability of thin-film lithium niobate on insulator (LNOI) and advances in processing have led to the emergence of fully integrated LiNbO3 electro-optic devices. Yet to date, LiNbO3 photonic integrated circuits have mostly been fabricated using non-standard etching techniques and partially etched waveguides, that lack the reproducibility achieved in silicon photonics. Widespread application of thin-film LiNbO3 requires a reliable solution with precise lithographic control. Here we demonstrate a heterogeneously integrated LiNbO3 photonic platform employing wafer-scale bonding of thin-film LiNbO3 to silicon nitride (Si3N4) photonic integrated circuits. The platform maintains the low propagation loss (<0.1 dB/cm) and efficient fiber-to-chip coupling (<2.5 dB per facet) of the Si3N4 waveguides and provides a link between passive Si3N4 circuits and electro-optic components with adiabatic mode converters experiencing insertion losses below 0.1 dB. Using this approach we demonstrate several key applications, thus providing a scalable, foundry-ready solution to complex LiNbO3 integrated photonic circuits.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Ultrafast tunable lasers using lithium niobate integrated photonics.

Early works1 and recent advances in thin-film lithium niobate (LiNbO3) on insulator have enabled low-loss photonic integrated circuits2,3, modulators with improved half-wave voltage4,5, electro-optic frequency combs6 and on-chip electro-optic devices, with applications ranging from microwave photonics to microwave-to-optical quantum interfaces7. Although recent advances have demonstrated tunable integrated lasers based on LiNbO3 (refs. 8,9), the full potential of this platform to demonstrate frequency-agile, narrow-linewidth integrated lasers has not been achieved. Here we report such a laser with a fast tuning rate based on a hybrid silicon nitride (Si3N4)-LiNbO3 photonic platform and demonstrate its use for coherent laser ranging. Our platform is based on heterogeneous integration of ultralow-loss Si3N4 photonic integrated circuits with thin-film LiNbO3 through direct bonding at the wafer level, in contrast to previously demonstrated chiplet-level integration10, featuring low propagation loss of 8.5 decibels per metre, enabling narrow-linewidth lasing (intrinsic linewidth of 3 kilohertz) by self-injection locking to a laser diode. The hybrid mode of the resonator allows electro-optic laser frequency tuning at a speed of 12 × 1015 hertz per second with high linearity and low hysteresis while retaining the narrow linewidth. Using a hybrid integrated laser, we perform a proof-of-concept coherent optical ranging (FMCW LiDAR) experiment. Endowing Si3N4 photonic integrated circuits with LiNbO3 creates a platform that combines the individual advantages of thin-film LiNbO3 with those of Si3N4, which show precise lithographic control, mature manufacturing and ultralow loss11,12.

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

Structure-based discovery of small molecules that disaggregate Alzheimer's disease tissue derived tau fibrils in vitro.

Alzheimer's disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

De novo designed protein inhibitors of amyloid aggregation and seeding.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-ß (Aß) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aß, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies.

Proteins including FUS, hnRNPA2, and TDP-43 reversibly aggregate into amyloid-like fibrils through interactions of their low-complexity domains (LCDs). Mutations in LCDs can promote irreversible amyloid aggregation and disease. We introduce a computational approach to identify mutations in LCDs of disease-associated proteins predicted to increase propensity for amyloid aggregation. We identify several disease-related mutations in the intermediate filament protein keratin-8 (KRT8). Atomic structures of wild-type and mutant KRT8 segments confirm the transition to a pleated strand capable of amyloid formation. Biochemical analysis reveals KRT8 forms amyloid aggregates, and the identified mutations promote aggregation. Aggregated KRT8 is found in Mallory-Denk bodies, observed in hepatocytes of livers with alcoholic steatohepatitis (ASH). We demonstrate that ethanol promotes KRT8 aggregation, and KRT8 amyloids co-crystallize with alcohol. Lastly, KRT8 aggregation can be seeded by liver extract from people with ASH, consistent with the amyloid nature of KRT8 aggregates and the classification of ASH as an amyloid-related condition.

Microwave-to-optical conversion with a gallium phosphide photonic crystal cavity.

Electrically actuated optomechanical resonators provide a route to quantum-coherent, bidirectional conversion of microwave and optical photons. Such devices could enable optical interconnection of quantum computers based on qubits operating at microwave frequencies. Here we present a platform for microwave-to-optical conversion comprising a photonic crystal cavity made of single-crystal, piezoelectric gallium phosphide integrated on pre-fabricated niobium circuits on an intrinsic silicon substrate. The devices exploit spatially extended, sideband-resolved mechanical breathing modes at ~3.2 GHz, with vacuum optomechanical coupling rates of up to g0/2π ≈ 300 kHz. The mechanical modes are driven by integrated microwave electrodes via the inverse piezoelectric effect. We estimate that the system could achieve an electromechanical coupling rate to a superconducting transmon qubit of ~200 kHz. Our work represents a decisive step towards integration of piezoelectro-optomechanical interfaces with superconducting quantum processors.

Three-repeat and four-repeat tau isoforms form different oligomers.

Different tauopathies are characterized by the isoform-specific composition of the aggregates found in the brain and by structurally distinct tau strains. Although tau oligomers have been implicated as important neurotoxic species, little is known about how the primary structures of the six human tau isoforms affect tau oligomerization because the oligomers are metastable and difficult to analyze. To address this knowledge gap, here, we analyzed the initial oligomers formed by the six tau isoforms in the absence of posttranslational modifications or other manipulations using dot blots probed by an oligomer-specific antibody, native-PAGE/western blots, photo-induced cross-linking of unmodified proteins, mass-spectrometry, and ion-mobility spectroscopy. We found that under these conditions, three-repeat (3R) isoforms are more prone than four-repeat (4R) isoforms to form oligomers. We also tested whether known inhibitors of tau aggregation affect its oligomerization using three small molecules representing different classes of tau aggregation inhibitors, Methylene Blue (MB), the molecular tweezer CLR01, and the all-D peptide TLKIVW, for their ability to inhibit or modulate the oligomerization of the six tau isoforms. Unlike their reported inhibitory effect on tau fibrillation, the inhibitors had little or no effect on the initial oligomerization. Our study provides novel insight into the primary-quaternary structure relationship of human tau and suggests that 3R-tau oligomers may be an important target for future development of compounds targeting pathological tau assemblies.

Maximizing Completion of the Two-Dose COVID-19 Vaccine Series with Aid from Infographics.

Two of the three COVID-19 vaccines approved in the United States require two doses to reach full efficacy, as do others available elsewhere in the world. The complete series of multidose COVID-19 vaccines offers stronger protection against infection by SARS-CoV-2 compared to single-dose injections with the same vaccines. Achieving perfect community-level adherence is a challenge in any public health campaign, even in non-pandemic times. Vaccines requiring multiple doses combined with a surge of vaccine hesitancy and misinformation that has been witnessed by the public during the COVID-19 pandemic are exacerbating the challenge of ensuring the world's population achieves a sufficient level of immunity against COVID-19. Here, we describe the results of our study in which we sought to determine whether completion of a two-dose COVID-19 vaccine regimen could be improved by disseminating infographics that explain what the vaccine is and why returning for the second dose is beneficial. Our results show that the proportion of COVID-19 vaccine recipients returning for a second inoculation grew after COVID-19 vaccine infographics were distributed to first-time vaccine recipients. We suggest that extending communication and outreach initiatives into the clinic positively influences the rate of follow-up visits, and that infographics are useful tools to aid and bolster the deployment of COVID-19 vaccines.

Nanoscopic Charge Fluctuations in a Gallium Phosphide Waveguide Measured by Single Molecules.

We present efficient evanescent coupling of single organic molecules to a gallium phosphide (GaP) subwavelength waveguide (nanoguide) decorated with microelectrodes. By monitoring their Stark shifts, we reveal that the coupled molecules experience fluctuating electric fields. We analyze the spectral dynamics of different molecules over a large range of optical powers in the nanoguide to show that these fluctuations are light-induced and local. A simple model is developed to explain our observations based on the optical activation of charges at an estimated mean density of 2.5×10^{22} m^{-3} in the GaP nanostructure. Our work showcases the potential of organic molecules as nanoscopic sensors of the electric charge as well as the use of GaP nanostructures for integrated quantum photonics.

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2.

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. ONE SENTENCE SUMMARY: Atomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease.

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.

Laser Cooling of a Nanomechanical Oscillator to Its Zero-Point Energy.

Optomechanical systems in the well-resolved-sideband regime are ideal for studying a myriad of quantum phenomena with mechanical systems, including backaction-evading measurements, mechanical squeezing, and nonclassical states generation. For these experiments, the mechanical oscillator should be prepared in its ground state, i.e., exhibit negligible residual excess motion compared to its zero-point motion. This can be achieved using the radiation pressure of laser light in the cavity by selectively driving the lower motional sideband, leading to sideband cooling. To date, the preparation of sideband-resolved optical systems to their zero-point energy has eluded laser cooling because of strong optical absorption heating. The alternative method of passive cooling suffers from the same problem, as the requisite milliKelvin environment is incompatible with the strong optical driving needed by many quantum protocols. Here, we employ a highly sideband-resolved silicon optomechanical crystal in a ^{3}He buffer-gas environment at ∼2 K to demonstrate laser sideband cooling to a mean thermal phonon occupancy of 0.09_{-0.01}^{+0.02} quantum (self-calibrated using motional sideband asymmetry), which is -7.4 dB of the oscillator's zero-point energy and corresponds to 92% ground state probability. Achieving such low occupancy by laser cooling opens the door to a wide range of quantum-optomechanical experiments in the optical domain.