Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

De novo design of buttressed loops for sculpting protein functions.

In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9-14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.

Design of amyloidogenic peptide traps.

Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.

De novo design of small beta barrel proteins.

Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy-based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.

IQGAP1 overexpression attenuates chemosensitivity through YAP-mediated ferroptosis inhibition in esophageal squamous cell cancer cells.

Chemoresistance is one of the major hindrances to many cancer therapies, including esophageal squamous cell carcinoma (ESCC). Ferroptosis, a new programmed cell death, plays an essential role in chemoresistance. IQ-domain GTPase activating protein 1 (IQGAP1) is a scaffold protein and functions as an oncogene in various human malignancies. However, the underlying effect and molecular mechanisms of IQGAP1 on paclitaxel (PTX) resistance and ferroptosis in ESCC remain to be elucidated. In this study, we found that IQGAP1 was highly expressed in ESCC tissues and could as a potential biomarker for diagnosis and predicting the prognosis of ESCC. Functional studies revealed that IQGAP1 overexpression reduced the sensitivity of ESCC cells to PTX by enhancing ESCC cell viability and proliferation and inhibiting cell death, and protected ESCC cells from ferroptosis, whereas IQGAP1 knockdown exhibited contrary effects. Importantly, reductions of chemosensitivity and ferroptosis caused by IQGAP1 overexpression were reversed with ferroptosis inducer RSL3, while the increases of chemosensitivity and ferroptosis caused by IQGAP1 knockdown were reversed with ferroptosis inhibitor ferrostatin-1 (Fer-1) in ESCC cells, indicating that IQGAP1 played a key role in resistance to PTX through regulating ferroptosis. Mechanistically, we demonstrated that IQGAP1 overexpression upregulated the expression of Yes-associated protein (YAP), the central mediator of the Hippo pathway. YAP inhibitor Verteporfin (VP) could reverse the effects of IQGAP1 overexpression on ESCC chemoresistance and ferroptosis. Taken together, our findings suggest that IQGAP1 promotes chemoresistance by blocking ferroptosis through targeting YAP. IQGAP1 may be a novel therapeutic target for overcoming chemoresistance in ESCC.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-ß-lactamase 1.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-ß-lactamase 1 (NDM-1), a bacterial enzyme that degrades ß-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

Honokiol ameliorates cigarette smoke-induced damage of airway epithelial cells via the SIRT3/SOD2 signalling pathway.

Cigarette smoking can cause damage of airway epithelial cells and contribute to chronic obstructive pulmonary disease (COPD). Honokiol is originally isolated from Magnolia obovata with multiple biological activities. Here, we investigated the protective effects of honokiol on cigarette smoke extract (CSE)-induced injury of BEAS-2B cells. BEAS-2B cells were treated with 300 mg/L CSE to construct an in vitro cell injury model, and cells were further treated with 2, 5 and 10 µM honokiol, then cell viability and LDH leakage were analysed by CCK-8 and LDH assay kits, respectively. Apoptosis was detected by flow cytometry analysis. ELISA was used to measure the levels of tumour necrosis factor (TNF)-ɑ, IL-1ß, IL-6, IL-8 and MCP-1. The results showed that honokiol (0.5-20 µM) showed non-toxic effects on BEAS-2B cells. Treatment with honokiol (2, 5 and 10 µM) reduced CSE (300 mg/L)-induced decrease in cell viability and apoptosis in BEAS-2B cells. Honokiol also decreased CSE-induced inflammation through inhibiting expression and secretion of inflammatory cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-8 and MCP-1. Moreover, honokiol repressed CSE-induced reactive oxygen species (ROS) production, decrease of ATP content and mitochondrial biogenesis, as well as mitochondrial membrane potential. Mechanistically, honokiol promoted the expression of SIRT3 and its downstream target genes, which are critical regulators of mitochondrial function and oxidative stress. Silencing of SIRT3 reversed the protective effects of honokiol on CSE-induced damage and mitochondrial dysfunction in BEAS-2B cells. These results indicated that honokiol attenuated CSE-induced damage of airway epithelial cells through regulating SIRT3/SOD2 signalling pathway.

Estimating the CO2 emissions of Chinese cities from 2011 to 2020 based on SPNN-GNNWR.

Global warming is a serious threat to human survival and health. Facing increasing global warming, the issue of CO2 emissions has attracted more attention. China is a major contributor of anthropogenic CO2 emissions and so it is essential to accurately estimate China's CO2 emissions and analyze their changing characteristics. This study recalculates CO2 emissions from Chinese cities from 2011 to 2020 using the SPNN-GNNWR model and multiple factors to reduce the uncertainty in emission estimates. The SPNN-GNNWR model has excellent predictions (R2: 0.925, 10-fold CV R2: 0.822) when cross-validation is used. The results indicate that the total CO2 emissions in China calculated by the model are close to those accounted for by other authorities in the world, with the total CO2 emissions increasing from 9.122 billion tonnes in 2011 to 9.912 billion tonnes in 2020. The city with the largest increase in CO2 emissions is Tianjin, and the city with the largest decrease is Beijing. The study also reveals the regional differences in CO2 emissions in Chinese mainland, including emissions, emission intensity and per capita emissions. Capturing and understanding the emissions and the related socioeconomic characteristics of different cities can help to develop effective emission mitigation strategies.

Intervention of Dietary Protein Levels on Muscle Quality, Antioxidation, and Autophagy in the Muscles of Triploid Crucian Carp (Carassius carassius Triploid).

The aim of this study is to investigate the effect of dietary protein levels on flesh quality, oxidative stress, and autophagy status in the muscles of triploid crucian carp (Carassius carassius triploid), and the related molecular mechanisms. Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. It could be found that the whole-body ash content significantly increased in high protein level groups (p < 0.05). The 29% dietary protein level group exhibited the highest muscle moisture, although there was an inconspicuous decrease in the chewiness of the muscles when compared with the other groups. The dietary protein level influenced the content of free amino acids and nucleotides, especially the content of flavor amino acids, which exhibited an increasing tendency along with the increasing protein level, such as alanine and glutamic acid, while the flavor nucleotides showed different fluctuation trends. Moreover, the genes related to muscle development were shown to be influenced by the dietary protein level, especially the expression of MRF4, which was up-regulated with the increasing dietary protein levels. The 29% dietary protein level promoted the majority of analyzed muscle genes expression to the highest level when compared to other dietary levels, except the Myostain, whose expression reached its highest at 38% dietary protein levels. Furthermore, the effect of dietary protein levels on antioxidant signaling pathway genes were also examined. High protein levels would boost the expression of GSTα; GPX1 and GPX4α mRNA expression showed the highest level at the 32% dietary protein group. The increasing dietary protein level decreased both mRNA and protein expressions of Nrf2 by up-regulating Keap1. Autophagy-related gene expression levels reached the peak at 32% dietary protein level, as evidenced by a similar change in protein expression of FoxO1. In summary, muscle nutritional composition, antioxidative pathways, and autophagy levels were affected by the dietary protein levels. A total of 29-32% dietary protein level would be the appropriate level range to improve muscle quality and promote the antioxidant and autophagy capacity of triploid crucian carp muscles.

Black-White disparity in severe cardiovascular maternal morbidity: A systematic review and meta-analysis.

BACKGROUND: To synthesize existing evidence on Black-White disparities in the prevalence of severe cardiovascular maternal morbidity. METHODS: We searched MEDLINE, EMBASE, and CINAHL for observational studies published before July 31, 2021 that compared the risk of severe cardiovascular maternal morbidity between Black and White women. The outcome was severe cardiovascular maternal morbidity, including acute myocardial infarction, peripartum cardiomyopathy, and stroke during pregnancy, delivery, or postpartum. We extracted relevant information including adjusted and unadjusted effect estimates. We used random-effects models to estimate the pooled association between race and severe cardiovascular maternal morbidity, presented as odds ratios with 95% confidence intervals for the comparison of Black women relative to White women. RESULTS: We included 18 studies that met the eligibility criteria for systematic review and meta-analysis. All studies were conducted in the United States and included a total of 7,656,876 Black women and 26,412,600 White women. Compared with White women, Black women had an increased risk of any severe cardiovascular maternal morbidity (adjusted odds ratio, 1.90; 95% confidence interval, 1.54-2.33). Black women were at risk of acute myocardial infarction (adjusted odds ratio, 1.38; 95% confidence interval, 1.14-1.68), peripartum cardiomyopathy (adjusted odds ratio, 1.71; 95% confidence interval, 1.51-1.94), and stroke (adjusted odds ratio, 2.13; 95% confidence interval, 1.39-3.26). CONCLUSIONS: Black women have a considerably higher risk of severe cardiovascular maternal morbidity than White women, including acute myocardial infarction, peripartum cardiomyopathy, and stroke. Reducing inequality in adverse cardiovascular outcomes of pregnancy between Black and White women should be prioritized.

Preparation and Characterization of Degradable Cellulose-Based Paper with Superhydrophobic, Antibacterial, and Barrier Properties for Food Packaging.

A great paradigm for foremost food packaging is to use renewable and biodegradable lignocellulose-based materials instead of plastic. Novel packages were successfully prepared from the cellulose paper by coating a mixture of polylactic acid (PLA) with cinnamaldehyde (CIN) as a barrier screen and nano silica-modified stearic acid (SA/SiO2) as a superhydrophobic layer. As comprehensively investigated by various tests, results showed that the as-prepared packages possessed excellent thermal stability attributed to inorganic SiO2 incorporation. The excellent film-forming characteristics of PLA improved the tensile strength of the manufactured papers (104.3 MPa) as compared to the original cellulose papers (70.50 MPa), enhanced by 47.94%. Benefiting from the rough nanostructure which was surface-modified by low surface energy SA, the contact angle of the composite papers attained 156.3°, owning superhydrophobic performance for various liquids. Moreover, the composite papers showed excellent gas, moisture, and oil bacteria barrier property as a result of the reinforcement by the functional coatings. The Cobb300s and WVP of the composite papers were reduced by 100% and 88.56%, respectively, and their antibacterial efficiency was about 100%. As the novel composite papers have remarkable thermal stability, tensile strength, and barrier property, they can be exploited as a potential candidate for eco-friendly, renewable, and biodegradable cellulose paper-based composites for the substitute of petroleum-derived packages.

Polyhedral oligomeric silsesquioxane grafted silica-based core-shell microspheres for reversed-phase high-performance liquid chromatography.

Polyhedral oligomeric silsesquioxane (POSS) was used to modify spherical silica to fabricate core-shell POSS@SiO2 microspheres. The material was characterized by Fourier transform infrared experiments, scanning electron microscopy, thermogravimetric analysis and elemental analysis. The material was also used as a stationary phase for HPLC separation. The POSS@SiO2 column exhibits a reverse-phase liquid chromatography (RPLC) retention mechanism. The column efficiency of alkylbenzenes reaches 67,200 plates·m-1. The POSS@SiO2 column was also utilized for separation of basic anilines and polycyclic aromatic hydrocarbons. Compared with the commercial C8 column, the POSS@SiO2 column exhibits enhanced separation selectivity. The column was also used for the separation of synthetic cytokinins 6-benzylaminopurine and 6-furfurylaminopurine in bean sprout after extraction. In addition, the methacrylate groups on the surface of the POSS@SiO2 microsphere were further functionalized so as to facilitate the fabrication of versatile stationary phases with various separation mechanisms. Graphical abstract Schematic presentation of the two-step fabrication of polyhedral oligomeric silsesquioxane grafted silica-based (POSS@SiO2) core-shell microspheres for use in HPLC.

Total Synthesis of (-)-Himeradine†A.

(-)-Himeradine A is a complex lycopodium alkaloid with seven rings and ten stereogenic centers that shows anticancer activity against lymphoma L1210 cells. A total synthesis has been developed that builds off prior work on (+)-fastigiatine. A 2,4,6-trisubstitited piperidine ring forms the core of the quinolizidine segment, and was prepared by diastereoselective reduction of a pyridine and classic resolution of an intermediate. The remaining secondary amine was introduced with a catalyst-controlled Overman rearrangement. The piperidine segment was coupled in a B-alkyl Suzuki reaction with a bicyclic bromoenone, which was a key intermediate for the synthesis of (+)-fastigiatine. The final transformation featured a transannular Mannich reaction and cyclization to complete the quinolizidine. Five bonds and four new rings were generated in this one-pot procedure. (-)-Himeradine A was prepared in 17 steps in the longest linear sequence.

Lipid Dysregulation Induced by Gasoline and Diesel Exhaust Exposure and the Interaction with Age.

Limited knowledge exists regarding gasoline and diesel exhaust effects on lipid metabolism. This study collected gasoline and diesel exhaust under actual driving conditions and conducted inhalation exposure on male young and middle-aged C57BL/6J mice for 4 h/day for 5 days to simulate commuting exposure intensity. Additionally, PM2.5 from actual roadways, representing gasoline and diesel vehicles, was generated for exposure to human umbilical vein endothelial cells (HUVECs) and normal liver cells (LO2) for 24, 48, and 72 h to further investigate exhaust particle toxicity. Results showed that diesel exhaust reduced total cholesterol and low-density lipoprotein cholesterol levels in young mice, indicating disrupted lipid metabolism. Aspartate aminotransferase and alanine aminotransferase levels increased by 53.7% and 21.7%, respectively, suggesting potential liver injury. Diesel exhaust exposure decreased superoxide dismutase and increased glutathione peroxidase levels. Cell viability decreased, and reactive oxygen species levels increased in HUVECs and LO2 following exposure to exhaust particles, with dose- and time-dependent effects. Diesel exhaust particles exhibited more severe inhibition of cell proliferation and oxidative damage compared to gasoline exhaust particles. These findings provide novel evidence of the risk of disrupted lipid metabolism due to gasoline and diesel exhaust, emphasizing the toxicity of diesel exhaust.

A novel capsid-XL32-derived adeno-associated virus serotype prompts retinal tropism and ameliorates choroidal neovascularization.

Gene therapy has been adapted, from the laboratory to the clinic, to treat retinopathies. In contrast to subretinal route, intravitreal delivery of AAV vectors displays the advantage of bypassing surgical injuries, but the viral particles are more prone to be nullified by the host neutralizing factors. To minimize such suppression of therapeutic effect, especially in terms of AAV2 and its derivatives, we introduced three serine-to-glycine mutations, based on the phosphorylation sites identified by mass spectrum analysis, to the XL32 capsid to generate a novel serotype named AAVYC5. Via intravitreal administration, AAVYC5 was transduced more effectively into multiple retinal layers compared with AAV2 and XL32. AAVYC5 also enabled successful delivery of anti-angiogenic molecules to rescue laser-induced choroidal neovascularization and astrogliosis in mice and non-human primates. Furthermore, we detected fewer neutralizing antibodies and binding IgG in human sera against AAVYC5 than those specific for AAV2 and XL32. Our results thus implicate this capsid-optimized AAVYC5 as a promising vector suitable for a wide population, particularly those with undesirable AAV2 seroreactivity.

Expansive discovery of chemically diverse structured macrocyclic oligoamides.

Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.

Generalized biomolecular modeling and design with RoseTTAFold All-Atom.

Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.

Discovering Geographical Flock Patterns of CO2 Emissions in China Using Trajectory Mining Techniques.

Carbon dioxide (CO2) emissions are considered a significant factor that results in climate change. To better support the formulation of effective policies to reduce CO2 emissions, specific types of important emission patterns need to be considered. Motivated by the flock pattern that exists in the domain of moving object trajectories, this paper extends this concept to a geographical flock pattern and aims to discover such patterns that might exist in CO2 emission data. To achieve this, a spatiotemporal graph (STG)-based approach is proposed. Three main parts are involved in the proposed approach: generating attribute trajectories from CO2 emission data, generating STGs from attribute trajectories, and discovering specific types of geographical flock patterns. Generally, eight different types of geographical flock patterns are derived based on two criteria, i.e., the high-low attribute values criterion and the extreme number-duration values criterion. A case study is conducted based on the CO2 emission data in China on two levels: the province level and the geographical region level. The results demonstrate the effectiveness of the proposed approach in discovering geographical flock patterns of CO2 emissions and provide potential suggestions and insights to assist policy making and the coordinated control of carbon emissions.

Precisely patterned nanofibres made from extendable protein multiplexes.

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha-helical coiled coils have such symmetric, extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibres can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibres can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.