Designed endocytosis-inducing proteins degrade targets and amplify signals.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.

Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.

New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.

De novo design of miniprotein antagonists of cytokine storm inducers.

Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.

Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Computational design of non-porous pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.

Effect of multifunctional cationic polymer coatings on mitigation of broad microbial pathogens.

Infection control measures to prevent viral and bacterial infection spread are critical to maintaining a healthy environment. Pathogens such as viruses and pyogenic bacteria can cause infectious complications. Viruses such as SARS-CoV-2 are known to spread through the aerosol route and on fomite surfaces, lasting for a prolonged time in the environment. Developing technologies to mitigate the spread of pathogens through airborne routes and on surfaces is critical, especially for patients at high risk for infectious complications. Multifunctional coatings with a broad capacity to bind pathogens that result in inactivation can disrupt infectious spread through aerosol and inanimate surface spread. This study uses C-POLAR, a proprietary cationic, polyamine, organic polymer with a charged, dielectric property coated onto air filtration material and textiles. Using both SARS-CoV-2 live viral particles and bovine coronavirus models, C-POLAR-treated material shows a dramatic 2-log reduction in circulating viral inoculum. This reduction is consistent in a static room model, indicating simple airflow through a static C-POLAR hanging can capture significant airborne particles. Finally, Gram-positive and Gram-negative bacteria are applied to C-POLAR textiles using a viability indicator to demonstrate eradication on fomite surfaces. These data suggest that a cationic polymer surface can capture and eradicate human pathogens, potentially interrupting the infectious spread for a more resilient environment. IMPORTANCE: Infection control is critical for maintaining a healthy home, work, and hospital environment. We test a cationic polymer capable of capturing and eradicating viral and bacterial pathogens by applying the polymer to the air filtration material and textiles. The data suggest that the simple addition of cationic material can result in the improvement of an infectious resilient environment against viral and bacterial pathogens.

Computationally designed mRNA-launched protein nanoparticle vaccines.

Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1-6. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7-10, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.

Quantitative analysis and risk assessment to full-size microplastics pollution in the coastal marine waters of Hong Kong.

Given the potential risk to the ecosystem, attention has increased in recent decades to the contamination of the aquatic environment by microplastics (MPs). Due to the limitations of conventional analysis methods of MPs, little is known about the size distribution and abundance of a full-size MPs from 1 µm to 5 mm. The present study quantified MPs with size ranges of 50 µm - 5 mm and 1-50 µm in the coastal marine waters from twelve locations in Hong Kong using fluorescence microscopy and flow cytometry respectively, during the end of wet (September 2021) and dry (March 2022) seasons. The average abundance of MPs with size ranges of 50 µm - 5 mm and 1-50 µm from twelve sampling locations marine surface waters were found ranging from 27 to 104 particles L-1 and 43,675-387,901 particles L-1 in the wet season respectively, and 13-36 particles L-1 and 23,178-338,604 particles L-1 in the dry season respectively. Significant temporal and spatial variations of small MPs abundance might be observed at the sampling locations, which were contributed by the influences of the estuary of Pearl River, sewage discharge points, land structure, and other anthropogenic activities. Based on the MPs abundance information, ecological risk assessment was conducted and revealed that the small MPs (< 10 µm) in coastal marine surface waters may pose potential health risks to aquatic organisms. Additional risk assessments are needed in order to determine whether or not the MPs exposure would cause health risks to the public.

Assessment of novel antiviral filter using pseudo-type SARS-CoV-2 virus in fast air velocity vertical-type wind tunnel.

Current evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can remain suspended spread in aerosols for longer period of time under poorly ventilated indoor setting. To minimize spreading, application of antiviral filter to capture infectious aerosols and to inactivate SARS-CoV-2 can be a promising solution. This study aimed to develop a method to assess simultaneously the filtration and removal efficiency of aerosolized pseudo-type SARS-CoV-2 using a vertical-type wind tunnel with relatively high face velocity (1.3 m/s). Comparing with the untreated spunlace non-woven filter, the C-POLAR™ treated filter increased the filtration efficiency from 74.2 ± 11.5% to 97.2 ± 1.7%, with the removal efficiency of 99.4 ± 0.051%. The results provided not only solid evidence to support the effectiveness of the cationic polymeric coated filter in fighting against the SARS-CoV-2 pandemic, but also a method to test viral filtration and removal efficiency under relative fast air velocity and with a safer environment to the operators.

Antigen spacing on protein nanoparticles influences antibody responses to vaccination.

Immunogen design approaches aim to control the specificity and quality of antibody responses elicited by next-generation vaccines. Here, we use computational protein design to generate a nanoparticle vaccine platform based on the receptor-binding domain (RBD) of influenza hemagglutinin (HA) that enables precise control of antigen conformation and spacing. HA RBDs are presented as either monomers or native-like closed trimers that are connected to the underlying nanoparticle by a rigid linker that is modularly extended to precisely control antigen spacing. Nanoparticle immunogens with decreased spacing between trimeric RBDs elicit antibodies with improved hemagglutination inhibition and neutralization potency as well as binding breadth across diverse H1 HAs. Our "trihead" nanoparticle immunogen platform provides insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used in next-generation vaccines against influenza and other viruses.

Antigen spacing on protein nanoparticles influences antibody responses to vaccination.

Immunogen design approaches aim to control the specificity and quality of antibody responses to enable the creation of next-generation vaccines with improved potency and breadth. However, our understanding of the relationship between immunogen structure and immunogenicity is limited. Here we use computational protein design to generate a self-assembling nanoparticle vaccine platform based on the head domain of influenza hemagglutinin (HA) that enables precise control of antigen conformation, flexibility, and spacing on the nanoparticle exterior. Domain-based HA head antigens were presented either as monomers or in a native-like closed trimeric conformation that prevents exposure of trimer interface epitopes. These antigens were connected to the underlying nanoparticle by a rigid linker that was modularly extended to precisely control antigen spacing. We found that nanoparticle immunogens with decreased spacing between closed trimeric head antigens elicited antibodies with improved hemagglutination inhibition (HAI) and neutralization potency as well as binding breadth across diverse HAs within a subtype. Our "trihead" nanoparticle immunogen platform thus enables new insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used to generate next-generation vaccines against influenza and other viruses.

Computational design of non-porous, pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

Quantitative Assessment of Full Size Microplastics in Bottled and Tap Water Samples in Hong Kong.

Human exposure to microplastics (MPs) through drinking water has drawn serious concern recently because of the potential adverse health effects. Although there are reports on the occurrence of MPs in bottled water, little is known about the abundance of a whole spectrum of MPs with sizes ranging from 1 µm to 5 mm due to the restrictions of conventional MPs detection methods. Some studies using micro-Raman spectroscopy can achieve MPs with a size of <10 µm, however, quantitation of all MPs was extremely time consuming and only a small portion (<10%) of MPs would be analyzed. The present study quantified MPs from nine brands of bottled water using fluorescence microscopy and flow cytometry for MPs with a size of ≥50 µm and a size of <50 µm, respectively. The average abundance of MPs with a size of ≥50 µm in bottled water samples was found ranging from 8-50 particles L-1, while MPs with a size of <50 µm were found to be 1570-17,817 particles L-1, where the MPs abundance from mineral water samples were significantly more than distilled and spring water samples. The modal size and shape of MPs were found at 1 µm and fragments, respectively. Besides, three tap water samples obtained locally were analyzed and compared with the bottled water samples, where less MPs were found in tap water samples. In addition, contamination of MPs from bottle and cap and interference by addition of mineral salts were studied, where no significant difference from all these processes to the control sample was found, suggesting the major contamination of MPs was from other manufacturing processes. Estimated daily intake (EDI) of MPs increased substantially when data of small MPs are included, suggesting that previously reports on exposure of MPs from drinking water might be underestimated, as only large MPs were considered.

Astaxanthin Influence on Health Outcomes of Adults at Risk of Metabolic Syndrome: A Systematic Review and Meta-Analysis.

The use of medication is effective in managing metabolic syndrome (MetS), but side effects have led to increased attention on using nutraceuticals and supplements. Astaxanthin shows positive effects in reducing the risk of MetS, but results from individual studies are inconclusive. This systematic review summarizes the latest evidence of astaxanthin in adults with risk factors of MetS. A systematic search of English and Chinese randomized controlled trials in 14 electronic databases from inception to 30 June 2021 was performed. Two reviewers independently screened the titles and abstracts, and conducted full-text review, quality appraisal, and extraction of data. Risk of bias was assessed by PEDro. A total of 7 studies met the inclusion criteria with 321 participants. Six studies were rated to have excellent methodological quality, while the remaining one was rated at good. Results show marginal effects of astaxanthin on reduction in total cholesterol and systolic blood pressure, and a significant attenuating effect on low-density lipoprotein cholesterol. Further robust evidence is needed to examine the effects of astaxanthin in adults at risk of MetS.

Macroalgal morphology mediates microplastic accumulation on thallus and in sediments.

The accumulation process of microplastics (MPs) is a key to understanding their fate in the environment. However, there is limited information about the short-term accumulation of MPs on macrophytes. The ability of macrophyte to attenuate wave and reduce current velocity is potentially facilitating MPs deposition. We hypothesize that the macroalgae retain MPs with their morphologies (filamentous and non-filamentous) being one of the factors to govern retention. Our hypothesis was tested by field observation during the dry season in Hong Kong when the macroalgae communities were the most diverse. MPs per biomass, surface area, or interstitial volume were used to represent the abundances on macroalgae. We found that filamentous algae retained a 2.35 times higher number of MPs when compared with non-filamentous algae if unit per biomass was considered. Other units, however, showed insignificant differences in MPs abundances between algal morphologies. Fibre was the most dominant shape of MPs with no significant difference in their abundances between filamentous and non-filamentous algae, suggesting fibres were retained regardless of the algal morphologies. To further evaluate the potential accumulation in the environment, sediment samples were also collected under the algal mat and immediate vicinity (~50 cm) of the algal mat. We found that sediment collected under the vegetated area contained significantly higher MPs. This was 3.39 times higher than the unvegetated area. Sediment collected under/near filamentous algae retained much higher abundances of MPs than those of non-filamentous algae. Provided that the observed retention of MPs on macroalgae, we speculate macrophyte system is one of the short-term MPs accumulation hotspots where the temporal increase of MPs depends on the seasonality of macrophyte in a given region.

Structure-based design of stabilized recombinant influenza neuraminidase tetramers.

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.

Determination of hydroxy metabolites of polycyclic aromatic hydrocarbons by fully automated solid-phase microextraction derivatization and gas chromatography-mass spectrometry.

A fully automated sample pretreatment method was developed for the detection of mono and dihydroxy metabolites of polycyclic aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry in the selected ion monitoring mode. Direct immersion solid-phase microextraction for the extraction of target compounds and the headspace on-fiber silylation with N,O-bis(trimethylsilyl)trifluoroacetamide were performed automatically by a multipurpose autosampler (MPS2). The operating conditions including extraction time, derivatization time, ionic strength, pH, and incubation temperature were optimized. Calibration responses of nine metabolites of PAHs over a concentration range of 0.1-100 microg L(-1) with a correlation coefficient of 0.999 were obtained. The detection limits of the nine metabolites in mini pore water, minimal salts medium and soil extract culture medium were in the range of 0.001-0.013, 0.002-0.024 and 0.002-0.134 microg L(-1), respectively, while the respective quantification limits were 0.003-0.044, 0.005-0.081 and 0.008-0.447 microg L(-1). The reliability was confirmed by the traditional solid-phase extraction method. The proposed method could be used to analyze the metabolites of PAHs degraded by microorganisms such as algae and to determine the biodegradation pathways of PAHs.

Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum.

The removal and degradation of a mixture of polycyclic aromatic hydrocarbons (PAHs), namely phenanthrene (PHE), fluoranthene (FLA), and pyrene (PYR), by a green microalgal species, Selenastrum capricornutum, at different initial cell densities were studied. The PAH removal efficiency increased with the initial cell density, and 96% of PHE, 100% of FLA, and 100% of PYR in the medium were removed by live S. capricornutum at the density of 1 x 10(7) cells/ml in 4 d, whereas less than 50% of PAHs were removed at the lowest cell density (5 x 10(4) cells/ml) in 7 d. The removal mechanisms included initial adsorption onto the cell walls of both live and dead cells, and the adsorbed PAHs were then absorbed and degraded in live cells only. Among different PAHs in a mixture, irrespective of whether they were added to medium at the same or different concentrations, the removal preference by live S. capricornutum was in the descending order of PYR > FLA > PHE, whereas the biodegradation rates followed the descending order of FLA > PYR > PHE. Initial findings regarding PAH metabolites revealed that PHE was converted into four different monohydroxyphenanthrenes and two dihydroxyphenanthrenes, whereas FLA and PYR were converted into three hydroxylated derivatives through the monooxygenase pathway. The presence of dihydroxylated PAHs suggested that the dioxygenase pathway also might have taken place at the same time.