Flexible filament winding strategy to prepare COF@polyionic liquid-coated fibers for non-selective exclusion of macromolecules in electro-enhanced solid-phase microextraction.

BACKGROUND: Accurate quantitative analysis of small molecule metabolites in biological samples is of great significance. Hydroxypolycyclic aromatic hydrocarbons (OH-PAHs) are metabolic derivatives of emerging pollutants, reflecting exposure to polycyclic aromatic hydrocarbons (PAHs). Macromolecules such as proteins and enzymes in biological samples will interfere with the accurate quantification of OH-PAHs, making direct analysis impossible, requiring a series of complex treatments such as enzymatic hydrolysis. Therefore, the development of matrix-compatible fiber coatings that can exclude macromolecules is of great significance to improve the ability of solid-phase microextraction (SPME) technology to selectively quantify small molecules in complex matrices and achieve rapid and direct analysis. RESULTS: We have developed an innovative coating with a stable macromolecular barrier using electrospinning and flexible filament winding (FW) technologies. This coating, referred to as the hollow fibrous covalent organic framework@polyionic liquid (F-COF@polyILs), demonstrates outstanding conductivity and stability. It accelerates the adsorption equilibrium time (25 min) for polar OH-PAHs through electrically enhanced solid-phase microextraction (EE-SPME) technology. Compared to the powder form, F-COF@polyILs coating displays effective non-selective large-size molecular sieving. Combining gas chromatography-tandem triple quadrupole mass spectrometry (GC-MS/MS), we have established a simple, efficient quantitative analysis method for OH-PAHs with a low detection limit (0.008-0.05 ng L-1), wide linear range (0.02-1000 ng L-1), and good repeatability (1.0%-7.3 %). Experimental results show that the coated fiber exhibits good resistance to matrix interference (2.5%-16.7 %) in complex biological matrices, and has been successfully used for OH-PAHs analysis in human urine and plasma. SIGNIFICANCE: FW technology realizes the transformation of the traditional powder form of COF in SPME coating to a uniform non-powder coating, giving its ability to exclude large molecules in complex biological matrices. A method for quantitatively detecting OH-PAHs in real biological samples was also developed. Therefore, the filament winding preparation method for F-COF@polyILs coated fibers, along with fibrous COFs' morphology control, has substantial implications for efficiently extracting target compounds from complex matrices.

Discovering optimal kinetic pathways for self-assembly using automatic differentiation.

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.-time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred.

Identifying and avoiding radiation damage in macromolecular crystallography.

Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.

Bridging structural and cell biology with cryo-electron microscopy.

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

Exposure to environmental pharmaceuticals affects the macromolecular composition of mussels digestive glands.

Human pharmaceuticals represent a major challenge in natural environment. A better knowledge on their mechanisms of action and adverse effects on cellular pathways is fundamental to predict long-term consequences for marine wildlife. The FTIRI Imaging (FTIRI) spectroscopy represents a vibrational technique allowing to map specific areas of non-homogeneous biological samples, providing a unique biochemical and ultrastructural fingerprint of the tissue. In this study, FTIRI technique has been applied, for the first time, to characterize (i) the chemical building blocks of digestive glands of Mytilus galloprovincialis, (ii) alterations and (iii) resilience of macromolecular composition, after a 14-days exposure to 0.5 µg/L of carbamazepine (CBZ), valsartan (VAL) and their mixture, followed by a 14-days recovery period. Spectral features of mussels digestive glands provided insights on composition and topographical distribution of main groups of biological macromolecules, such as proteins, lipids, and glycosylated compounds. Pharmaceuticals caused an increase in the total amount of protein and a significant decrease of lipids levels. Changes in macromolecular features reflected the modulation of specific molecular and biochemical pathways thus supporting our knowledge on mechanisms of action of such emerging pollutants. Overall, the applied approach could represent an added value within integrated strategies for the effects-based evaluation of environmental contaminants.

Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding.

This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNA-mediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

Biochemical and biophysical interaction of rare earth elements with biomacromolecules: A comprehensive review.

The growing utilization of rare earth elements (REEs) in industrial and technological applications has captured global interest, leading to the development of high-performance technologies in medical diagnosis, agriculture, and other electronic industries. This accelerated utilization has also raised human exposure levels, resulting in both favourable and unfavourable impacts. However, the effects of REEs are dependent on their concentration and molecular species. Therefore, scientific interest has increased in investigating the molecular interactions of REEs with biomolecules. In this current review, particular attention was paid to the molecular mechanism of interactions of Lanthanum (La), Cerium (Ce), and Gadolinium (Gd) with biomolecules, and the biological consequences were broadly interpreted. The review involved gathering and evaluating a vast scientific collection which primarily focused on the impact associated with REEs, ranging from earlier reports to recent discoveries, including studies in human and animal models. Thus, understanding the molecular interactions of each element with biomolecules will be highly beneficial in elucidating the consequences of REEs accumulation in the living organisms.

Therapeutic applications of biological macromolecules and scaffolds for skeletal muscle regeneration: A review.

Skeletal muscle (SM) mass and strength maintenance are important requirements for human well-being. SM regeneration to repair minor injuries depends upon the myogenic activities of muscle satellite (stem) cells. However, losses of regenerative properties following volumetric muscle loss or severe trauma or due to congenital muscular abnormalities are not self-restorable, and thus, these conditions have major healthcare implications and pose clinical challenges. In this context, tissue engineering based on different types of biomaterials and scaffolds provides an encouraging means of structural and functional SM reconstruction. In particular, biomimetic (able to transmit biological signals) and several porous scaffolds are rapidly evolving. Several biological macromolecules/biomaterials (collagen, gelatin, alginate, chitosan, and fibrin etc.) are being widely used for SM regeneration. However, available alternatives for SM regeneration must be redesigned to make them more user-friendly and economically feasible with longer shelf lives. This review aimed to explore the biological aspects of SM regeneration and the roles played by several biological macromolecules and scaffolds in SM regeneration in cases of volumetric muscle loss.

Current research status and development prospects of embolic microspheres containing biological macromolecules and others.

Transcatheter arterial embolization (TACE) has been used in the treatment of malignant tumors, sudden hemorrhage, uterine fibroids, and other diseases, and with advances in imaging techniques and devices, materials science, and drug release technology, more and more embolic agents that are drug-carrying, self-imaging, or have multiple functions are being developed. Microspheres provide safer and more effective therapeutic results as embolic agents, with their unique spherical appearance and good embolic properties. Embolic microspheres are the key to arterial embolization, blocking blood flow and nutrient supply to the tumor target. This review summarizes some of the currently published embolic microspheres, classifies embolic microspheres according to matrix, and summarizes the characteristics of the microsphere materials, the current status of research, directions, and the value of existing and potential applications. It provides a direction to promote the development of embolic microspheres towards multifunctionalization, and provides a reference to promote the research and application of embolic microspheres in the treatment of tumors.

Modulation of bacterial membranes and cellular macromolecules by dimethyl sulfoxide: A dose-dependent study providing novel insights.

Using Escherichia coli as a model, this manuscript delves into the intricate interactions between dimethyl sulfoxide (DMSO) and membranes, cellular macromolecules, and the effects on various aspects of bacterial physiology. Given DMSO's wide-ranging use as a solvent in microbiology, we investigate the impacts of both non-growth inhibitory (1.0 % and 2.5 % v/v) and slightly growth-inhibitory (5.0 % v/v) concentrations of DMSO. The results demonstrate that DMSO causes alterations in bacterial membrane potential, influences the electrochemical characteristics of the cell surface, and exerts substantial effects on the composition and structure of cellular biomolecules. Genome-wide gene expression data from DMSO-treated E. coli was used to further investigate and bolster the results. The findings of this study provide valuable insights into the complex relationship between DMSO and biological systems, with potential implications in drug delivery and cellular manipulation. However, it is essential to exercise caution when utilizing DMSO to enhance the solubility and delivery of bioactive compounds, as even at low concentrations, DMSO exerts non-inert effects on cellular macromolecules and processes.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Controlled Supramolecular Polymerization via Bioinspired, Liquid-Liquid Phase Separation of Monomers.

Dynamic supramolecular assemblies, driven by noncovalent interactions, pervade the biological realm. In the synthetic domain, their counterparts, supramolecular polymers, endowed with remarkable self-repair and adaptive traits, are often realized through bioinspired designs. Recently, controlled supramolecular polymerization strategies have emerged, drawing inspiration from protein self-assembly. A burgeoning area of research involves mimicking the liquid-liquid phase separation (LLPS) observed in proteins to create coacervate droplets and recognizing their significance in cellular organization and diverse functions. Herein, we introduce a novel perspective on synthetic coacervates, extending beyond their established role in synthetic biology as dynamic, membraneless phases to enable structural control in synthetic supramolecular polymers. Drawing parallels with the cooperative growth of amyloid fibrils through LLPS, we present metastable coacervate droplets as dormant monomer phases for controlled supramolecular polymerization. This is achieved via a π-conjugated monomer design that combines structural characteristics for both coacervation through its terminal ionic groups and one-dimensional growth via a π-conjugated core. This design leads to a unique temporal LLPS, resulting in a metastable coacervate phase, which subsequently undergoes one-dimensional growth via nucleation within the droplets. In-depth spectroscopic and microscopic characterization provides insights into the temporal evolution of disordered and ordered phases. Furthermore, to modulate the kinetics of liquid-to-solid transformation and to achieve precise control over the structural characteristics of the resulting supramolecular polymers, we invoke seeding in the droplets, showcasing living growth characteristics. Our work thus opens up new avenues in the exciting field of supramolecular polymerization, offering general design principles and controlled synthesis of precision self-assembled structures in confined environments.

The effect of konjac glucomannan on enzyme kinetics and fluorescence spectrometry of digestive enzymes: An in vitro research from the perspective of macromolecule crowding.

Konjac glucomannan (KGM) can significantly prolong gastrointestinal digestion. However, it is still worth investigating whether the macromolecular crowding (MMC) induced by KGM is correlated with digestion. In this paper, the MMC effect was quantified by fluorescence resonance energy transfer and microrheology, and the digests of starch, protein, and oil were determined. The digestive enzymes were analyzed by enzyme reaction kinetic and fluorescence quenching. The results showed that higher molecular weight (604.85 âˆ¼ 1002.21 kDa) KGM created a larger MMC (>0.8), and influenced the digestion of macronutrients; the digests of starch, protein, and oil all decreased significantly. MMC induced by KGM decreased the Michaelis-Menten constants (Km and Vmax) of pancreatic α-amylase (PPA), pepsin (PEP), and pancreatic lipase (PPL). The larger MMC (>0.8) induced by KGM resulted in the decrease of fluorescence quenching constants (Ksv) in PPA and PPL, and the increase of Ksv in PEP. Therefore, varying degrees of MMC induced by KGM could play a role in regulating digestion and the inhibitory effect on digestion was more significant in a relatively more crowded environment induced by KGM. This study provides theoretical support for the strategies of nutrient digestion regulation from the perspective of MMC caused by dietary fiber.

Scaling and merging macromolecular diffuse scattering with mdx2.

Diffuse scattering is a promising method to gain additional insight into protein dynamics from macromolecular crystallography experiments. Bragg intensities yield the average electron density, while the diffuse scattering can be processed to obtain a three-dimensional reciprocal-space map that is further analyzed to determine correlated motion. To make diffuse scattering techniques more accessible, software for data processing called mdx2 has been created that is both convenient to use and simple to extend and modify. mdx2 is written in Python, and it interfaces with DIALS to implement self-contained data-reduction workflows. Data are stored in NeXus format for software interchange and convenient visualization. mdx2 can be run on the command line or imported as a package, for instance to encapsulate a complete workflow in a Jupyter notebook for reproducible computing and education. Here, mdx2 version 1.0 is described, a new release incorporating state-of-the-art techniques for data reduction. The implementation of a complete multi-crystal scaling and merging workflow is described, and the methods are tested using a high-redundancy data set from cubic insulin. It is shown that redundancy can be leveraged during scaling to correct systematic errors and obtain accurate and reproducible measurements of weak diffuse signals.

A validation strategy to assess the role of phase separation as a determinant of macromolecular localization.

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.

Oriented docking of the template for improved imprinting efficiency toward peptide with modifications.

Molecular imprinting polymers (MIPs) are synthetic receptors as biomimetic materials for various applications ranging from sensing to separation and catalysis. However, currently existing MIPs are stuck to some of the issues including the longer preparation steps and poor performance. In this report, a facile and one-pot strategy by integrating the in-situ growth of magnetic nanoparticles and reversed phase microemulsion oriented molecularly imprinting strategy to develop magnetic molecular imprinted nanocomposites was proposed. Through self-assembling of the template, it brought up highly ordered and uniform arrangement of the imprinting structure, which offered faster adsorption kinetic as adsorption equilibrium was achived within 15 min, higher adsorption capacity (Qmax = 48.78 ± 1.54 µmol/g) and high affinity (Kd = 127.63 ± 9.66 µM) toward paradigm molecule-adenosine monophosphate (AMP) compared to the conventional bulk imprinting. The developed MIPs offered better affinity and superior specificity which allowed the specific enrichment toward targeted phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Interestingly, different types of MIPs can be developed which could targetly enrich the specific phosphorylated peptides for mass spectrometry analysis by simply switching the templates, and this strategy also successfully achieved imprinting of macromolecular peptides. Collectively, the approach showed broad applicability to target specific enrichment from metabolites to phosphorylated peptides and providing an alternative choice for selective recognition and analysis from complex biological systems.

High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

A mean-field theory for predicting single polymer collapse induced by neutral crowders.

Macromolecular crowding can induce the collapse of a single long polymer into a globular form due to depletion forces of entropic nature. This phenomenon has been shown to play a significant role in compacting the genome within the bacterium Escherichia coli into a well-defined region of the cell known as the nucleoid. Motivated by the biological significance of this process, numerous theoretical and computational studies have searched for the primary determinants of the behavior of polymer-crowder phases. However, our understanding of this process remains incomplete and there is debate on a quantitatively unified description. In particular, different simulation studies with explicit crowders have proposed different order parameters as potential predictors for the collapse transition. In this work, we present a comprehensive analysis of published simulation data obtained from different sources. Based on the common behavior we find in this data, we develop a unified phenomenological model that we show to be predictive. Finally, to further validate the accuracy of the model, we conduct new simulations on polymers of various sizes, and investigate the role of jamming of the crowders.