Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

Delineating the shape of COat Protein complex-II coated membrane bud.

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Path analysis for controlling climate change in global aviation.

The aviation industry's emissions have had a significant impact on global climate change. This study focuses on carbon emission trading schemes, sustainable aviation fuels (SAFs), and hydrogen energy, as vital means for the aviation industry to reduce emissions. To evaluate the climate effects of global routes under four scenarios (24 sub-scenarios) until 2100, this study proposes the Aviation-FAIR (Aviation-Finite Amplitude Impulse Response) method. The findings reveal that while CO2 emissions and concentrations are significant, other emissions, such as N2O and CH4, have a greater effective radiative forcing (ERF) and contribute significantly to climate change. Moreover, SAFs are more effective in mitigating airline pollutant emissions than relying solely on carbon trading schemes. The effectiveness of hydrogen fuel cells may be hindered by technical limitations compared to hydrogen turbine engines. The findings of this study provide reference for the global aviation industry to adopt emission reduction measures.

Research on nondestructive detection of sweet-waxy corn seed varieties and mildew based on stacked ensemble learning and hyperspectral feature fusion technology.

The variety and quality of corn seeds are crucial factors affecting crop yield and farmers' economic benefits. This study adopts an innovative method based on a hyperspectral imaging system combined with stacked ensemble learning, aiming to achieve varieties classification and mildew detection of sweet-waxy corn seeds. First, data interference is eliminated by extracting the spectral and texture information of each corn sample and preprocessing the data. Secondly, a stacked ensemble learning model (Stack) was constructed by stacking base models and meta-models. Its results were compared with those of the base models, including Gradient Boosting Decision Tree (GBDT), Extreme Gradient Boosting (XGBoost), Light Gradient Boosting Machine (LightGBM), and Random Forest (RF).Finally, the overall performance of the model is improved through the information fusion strategy of hyperspectral data and texture information. The research results indicate that the GBDT-Stack model, which integrates spectral and texture data, demonstrated optimal performance in the comprehensive classification of both corn seed varieties and mold detection. On the test set, the model achieved an average prediction accuracy of 97.01%. Specifically, the model achieved a test set accuracy ranging from 94.49% to 97.58% for different corn seed varieties and a test set accuracy of 98.89% for mildew detection. This model not only classifies corn seed varieties but also accurately detects mildew, demonstrating its wide applicability. The method has huge potential and is of great significance for improving crop yield and quality.

Electronic Polarization Leads to a Drier Dewetted State for Hydrophobic Gating in the Big Potassium Channel.

In the hydrophobic gating mechanism proposed for some ion channels, ion permeation is not blocked by the physical dimension of the channel pore but by its dewetted state which constitutes the energetic bottleneck. A major source of uncertainty in the mechanism is that the dewetted state was not observed in experiments and only probed in simulations using nonpolarizable force fields, which do not accurately represent the properties of confined water. Here we analyze hydration of the central cavity in the pore-gate domain of the Big Potassium channel using molecular dynamics and grand canonical Monte Carlo simulations with enhanced sampling techniques. Including polarization leads to a much drier dewetted state and a higher barrier for the transition to the wet state, suggesting more effective hydrophobic gating. The simulations also identify two backbone carbonyls at the bottom of the selectivity filter as good candidates for characterizing the dewetted state using infrared spectroscopies.

A parameterized two-domain thermodynamic model explains diverse mutational effects on protein allostery.

New experimental findings continue to challenge our understanding of protein allostery. Recent deep mutational scanning study showed that allosteric hotspots in the tetracycline repressor (TetR) and its homologous transcriptional factors are broadly distributed rather than spanning well-defined structural pathways as often assumed. Moreover, hotspot mutation-induced allostery loss was rescued by distributed additional mutations in a degenerate fashion. Here, we develop a two-domain thermodynamic model for TetR, which readily rationalizes these intriguing observations. The model accurately captures the in vivo activities of various mutants with changes in physically transparent parameters, allowing the data-based quantification of mutational effects using statistical inference. Our analysis reveals the intrinsic connection of intra- and inter-domain properties for allosteric regulation and illustrate epistatic interactions that are consistent with structural features of the protein. The insights gained from this study into the nature of two-domain allostery are expected to have broader implications for other multi-domain allosteric proteins.

Maritime Freight Carbon Emission in the U.S. using AIS data from 2018 to 2022.

Global maritime emissions, a 3% contributor to greenhouse gases, anticipate a surge of 90-130% by 2050. Regulatory challenges persist due to international governance gaps. Legislative strides, including the EU Emission Trading System, highlight global efforts. In the U.S., despite legislative commitment, consensus hurdles impede cross-regional carbon management. Prevailing top-down emissions estimation methods warrant scrutiny. This paper unveils U.S. maritime emissions intricacies, focusing on carbon accounting, transfer, and compensation for cargo and tanker vessels. Leveraging AIS data (2018-2022), an activity-based/bottom-up approach navigates emissions calculations, aiming to reshape understanding and foster strategic reductions. The study bridges gaps in U.S. maritime emission research, promising insights into transfer and compensation dynamics. By concentrating on high-impact vessel types, it contributes to emissions mitigation strategies, steering towards a sustainable U.S. maritime future.

Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

The effect of polymer coating on nanoparticles' interaction with lipid membranes studied by coarse-grained molecular dynamics simulations.

Nanoparticles' (NPs) permeation through cell membranes, whether it happens via passive or active transport, is an essential initial step for their cellular internalization. The NPs' surface coating impacts the way they translocate through the lipid bilayer and the spontaneity of the process. Understanding the molecular details of NPs' interaction with cell membranes allows the design of nanosystems with optimal characteristics for crossing the lipid bilayer: computer simulations are a powerful tool for this purpose. In this work, we have performed coarse-grained molecular dynamics simulations and free energy calculations on spherical titanium dioxide NPs conjugated with polymer chains of different chemical compositions. We have demonstrated that the hydrophobic/hydrophilic character of the chains, more than the nature of their terminal group, plays a crucial role in determining the NPs' interaction with the lipid bilayer and the thermodynamic spontaneity of NPs' translocation from water to the membrane. We envision that this computational work will be helpful to the experimental community in terms of the rational design of NPs for efficient cell membrane permeation.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

24-hour average PM2.5 concentration caused by aircraft in Chinese airports from Jan. 2006 to Dec. 2023.

Since 2006, the rapid development of China's aviation industry has been accompanied by a significant increase in one of its emissions, namely, PM2.5, which poses a substantial threat to human health. However, little data is describing the PM2.5 concentration caused by aircraft activities. This study addresses this gap by initially computing the monthly PM2.5 emissions of the landing-take-off (LTO) stage from Jan. 2006 to Dec. 2023 for 175 Chinese airports, employing the modified BFFM2-FOA-FPM method. Subsequently, the study uses the Gaussian diffusion model to measure the 24-hour average PM2.5 concentration resulting from flight activities at each airport. This study mainly draws the following conclusions: Between 2006 and 2023, the highest recorded PM2.5 concentration data at all airports was observed in 2018, reaching 5.7985 micrograms per cubic meter, while the lowest point was recorded in 2022, at 2.0574 micrograms per cubic meter. Moreover, airports with higher emissions are predominantly located in densely populated and economically vibrant regions such as Beijing, Shanghai, Guangzhou, Chengdu, and Shenzhen.

Flexibility of Binding Site is Essential to the Ca2+ Selectivity in EF-Hand Calcium-Binding Proteins.

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site.

Sequence Sensitivity in Membrane Remodeling by Polyampholyte Condensates.

Intrinsically disordered peptides (IDPs) have been found to undergo liquid-liquid phase separation (LLPS) and produce complex coacervates that play numerous regulatory roles in the cell. Recent experimental studies have discovered that LLPS at or near the membrane surface helps in the biomolecular organization during signaling events and can significantly alter the membrane morphology. However, the molecular mechanism and microscopic details of such processes still remain unclear. Here we study the effect of polyampholyte and polyelectrolyte condensation on two different anionic membranes, as they represent a majority of naturally occurring IDPs. The polyampholytes are fifty-residue polymers, made of glutamate(E) and lysine(K) with different charge patterns. The polyelectrolytes are separate chains of E25 and K25. We first calibrate the MARTINI v3.0 force field and then perform long-time-scale coarse-grained molecular dynamics simulations. We find that condensates formed by all the polyampholytes get adsorbed on the membrane. However, the strong polyampholytes (i.e., blocky sequences) can remodel the membranes more prominently than the weaker ones (i.e., scrambled sequences). Condensates formed by the blocky sequences induce a significant negative curvature (∼0.1 nm-1) and local demixing of lipids, whereas those by the scrambled sequences tend to wet the membrane to a greater extent without generating significant curvature or demixing. We perform several microscopic analyses to characterize the nature of the interaction between membranes and these condensates. Our analyses of interaction energetics reveal that membrane remodeling and/or wetting are favored by enhanced interactions between polyampholytes with lipids and the counterions.

Efficient Sampling of Cavity Hydration in Proteins with Nonequilibrium Grand Canonical Monte Carlo and Polarizable Force Fields.

Prediction of the hydration levels of protein cavities and active sites is important to both mechanistic analysis and ligand design. Due to the unique microscopic environment of these buried water molecules, a polarizable model is expected to be crucial for an accurate treatment of protein internal hydration in simulations. Here we adapt a nonequilibrium candidate Monte Carlo approach for conducting grand canonical Monte Carlo simulations with the Drude polarizable force field. The GPU implementation enables the efficient sampling of internal cavity hydration levels in biomolecular systems. We also develop an enhanced sampling approach referred to as B-walking, which satisfies detailed balance and readily combines with grand canonical integration to efficiently calculate quantitative binding free energies of water to protein cavities. Applications of these developments are illustrated in a solvent box and the polar ligand binding site in trypsin. Our simulation results show that including electronic polarization leads to a modest but clear improvement in the description of water position and occupancy compared to the crystal structure. The B-walking approach enhances the range of water sampling in different chemical potential windows and thus improves the accuracy of water binding free energy calculations.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

The impact of Chinese airport infrastructure on airline pollutant emissions: A hybrid stochastic-neural network approach based on utility functions.

With China being the world's largest emitter of greenhouse gases and its aviation sector burgeoning, the environmental performance of Chinese airlines has global significance. Amidst rising demands for eco-friendly practices from both customers and regulators, the interplay between airport infrastructure and environmental performance becomes pivotal. This research offers an innovative methodology to gauge the environmental performance of Chinese airlines, emphasizing the distance traveled between airports using weighted additive utility functions. Leveraging neural networks, the study investigates the impact of various airport infrastructural characteristics on environmental performance. Noteworthy findings indicate that ground control measures, automatic information services at origin airports, surface concrete on runways at both ends, and a centerline lighting system in destination airports positively influence environmental performance. In contrast, longer and wider runways at origin airports, increased distances to control towers, and asphalt runways at destination airports adversely affect it. These insights not only underscore the importance of strategic infrastructure enhancements for reducing carbon footprints but also hold profound policy implications. As global climate change remains at the forefront, fostering sustainable airport infrastructure in China can significantly contribute to worldwide mitigation efforts.

Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR.

Modulating allosteric coupling offers unique opportunities for biomedical applications. Such efforts can benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of cooperativity between distant sites. We demonstrate that effects of allostery hotspot mutations can be evaluated qualitatively and semiquantitatively by molecular dynamics simulations in a bacterial tetracycline repressor (TetR). The simulations recapitulate the effects of these mutations on abolishing the induction function of TetR and provide a rationale for the different rescuabilities observed to restore allosteric coupling of the hotspot mutations. We demonstrate that the same noninducible phenotype could be the result of perturbations in distinct structural and energetic properties of TetR. Our work underscores the value of explicitly computing the functional free energy landscapes to effectively evaluate and rank hotspot mutations despite the prevalence of compensatory interactions and therefore provides quantitative guidance to allostery modulation for therapeutic and engineering applications.

B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Study on active ingredient and mechanism in preventing vascular dementia of Tianzhusan coming from Tujia medicine / 中国中药杂志

To make clear of the absorbed components of Tianzhusan (TZS) and its possible mechanism in preventing vascular dementia (VD), the rats' models of VD were prepared by a permanent ligation of the bilateral common carotid arteries. After 60 days, rats were administrated with TZS for 0.1 g x kg(-1), and the volume is 0.02 mL x g(-1). After 3 days, the medicated serum was prepared and detected by UPLC, and then we predicted the possible chemical structure of the absorbed components of TZS. According to the absorbed components, the potential targets of TZS were found by ligand profiling of Discovery Studio 3.5. All of these target genes were submitted to DAVID onine for gene set enrichment analysis (GSEA). The 5 absorbed components of TZS have been predicted, and four of them have been identified as parishin B, parishin C, parishin, pennogenin-3-O-alpha-L-rhamnopyranosy-(1-->2)-beta-D-glucoside. Through reverse finding targets, we got 861 pharmacophore models and 9 pathways from KEGG, BIOCARTA after document verification. These results showed that the efficacy mechanism of TZS on VD perhaps were be related with these absorbed components and pathways. If the traditional herbs could be proved effective by efficacy tests, the serum pharmacochemistry, computer-aided drug design, system biology and other technologies can be used in the next experiments, which will be beneficial to fast discovery of material basis and mechanisms of traditional medicine coming form ethnic minorities.