Potent microtubule-depolymerizing activity of a mitotic Kif18b-MCAK-EB network.

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation.

Tandem Integration of Biological and Electrochemical Catalysis for Efficient Polyester Upcycling under Ambient Conditions.

Excessive production of waste polyethylene terephthalate (PET) poses an ecological challenge, which necessitates developing technologies to extract the values from end-of-life PET. Upcycling has proven effective in addressing the low profitability of current recycling strategies, yet existing upcycling technologies operate under energy-intensive conditions. Here we report a cascade strategy to steer the transformation of PET waste into glycolate in an overall yield of 92.6% under ambient conditions. The cascade approach involves setting up a robust hydrolase with 95.6% PET depolymerization into ethylene glycol (EG) monomer within 12 h, followed by an electrochemical process initiated by a CO-tolerant Pd/Ni(OH)2 catalyst to convert the EG intermediate into glycolate with high Faradaic efficiency of 97.5%. Techno-economic analysis and life cycle assessment indicate that, compared with the widely adopted electrochemical technology that heavily relies on alkaline pretreatment for PET depolymerization, our designed enzymatic-electrochemical approach offers a cost-effective and low-carbon pathway to upgrade PET.

Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeleton.

Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic ß cells, a long-standing model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating ß cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhance glucose-stimulated insulin secretion. Using scanning electron microscopy, we directly visualize the cortical cytoskeleton, allowing us to validate the effect of these filament-disrupting chemicals. We find the state of the cortical actin layer does not correlate with levels of insulin secretion, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton.

Conformational Selection in Enzyme-Catalyzed Depolymerization of Bio-based Polyesters.

Enzymatic degradation of polymers holds promise for advancing towards a bio-based economy. However, bulky polymers presents challenges in accessibility for biocatalysts, hindering depolymerization reactions. Beyond the impact of crystallinity, polymer chains can reside in different conformations affecting binding efficiency to the enzyme. We previously showed that the gauche and trans chain conformers associated with crystalline and amorphous regions of the synthetic polyethylene terephthalate (PET) display different affinity to PETase, thus affecting the depolymerization rate. However, structural-function relationships for biopolymers remain poorly understood in biocatalysis. In this study, we explored biodegradation of by-us previously synthesized bio-polyesters made from a rigid bicyclic chiral terpene-based diol and copolymerized with various renewable diesters. Herein, four of those polyesters spanning from semi-aromatic to aliphatic were subjected to enzymatic degradations in concert with induced-fit docking (IFD) analyses. Our findings demonstrate the importance of conformational selection in enzymatic depolymerization of biopolymers. A straight or twisted conformation of the polymer chain is crucial in biocatalytic degradation by showing different affinities to enzyme ground-state conformers. This work highlights the importance of considering the conformational match between the polymer and the enzyme to optimize the biocatalytic degradation efficiency of biopolymers, providing valuable insights for the development of sustainable bioprocesses.

Recent Advances and Challenges in Enzymatic Depolymerization and Recycling of PET Wastes.

Poly (ethylene terephthalate) (PET) is one of the most commonly used plastics in daily life and various industries. Enzymatic depolymerization and recycling of post-consumer PET (pc-PET) provides a promising strategy for the sustainable circular economy of polymers. Great protein engineering efforts have been devoted to improving the depolymerization performance of PET hydrolytic enzymes (PHEs). In this review, we first discuss the mechanisms and challenges of enzymatic PET depolymerization. Subsequently, we summarize the state-of-the-art engineering of PHEs including rational design, machine learning, and directed evolution for improved depolymerization performance, and highlight the advances in screening methods of PHEs. We further discuss several factors that affect the enzymatic depolymerization efficiency. We conclude with our perspective on the opportunities and challenges in bio-recycling and bio-upcycling of PET wastes.

Microtubule depolymerization induces ferroptosis in neuroblastoma cells.

Estramustine (EM), a clinically successful hormone-refractory anti-prostate cancer drug, exhibited potent anti-proliferative activity, depolymerized microtubules, blocked cells at mitosis, and induced cell death in different cancer cells. Altered iron metabolism is a feature of cancer cells. Using EM, we examined the plausible relationship between microtubule depolymerization and induction of ferroptosis in human neuroblastoma (SH-SY5Y and IMR-32) cells. EM reduced glutathione (GSH) levels and induced reactive oxygen species (ROS) generation. The pre-treatment of neuroblastoma cells with ROS scavengers (N-acetyl cysteine and dithiothreitol) reduced the anti-proliferative effects of EM. EM treatment increased labile iron pool (LIP), depleted glutathione peroxidase 4 (GPX4) levels, and lipid peroxidation, hallmark features of ferroptosis, highlighting ferroptosis induction. Ferroptosis inhibitors (deferoxamine mesylate and liproxstatin-1) abrogated the cytotoxic effects of EM, further confirming ferroptosis induction. Vinblastine and nocodazole also increased LIP and induced lipid peroxidation in neuroblastoma cells. This study provides evidence for the coupling of microtubule integrity to ferroptosis. The results also suggest that microtubule-depolymerizing agents may be considered for developing pro-ferroptosis chemotherapeutics.

Valorization of Corn Straw for Production of Glucose by Two-Step Depolymerization.

Depolymerization of the cellulose part in lignocellulose to glucose is a significant step for lignocellulose valorization. As one of the main by-products of agricultural biomass in crop-producing filed, valorization of corn straw has attracted considerable attention. In this study, a two-step depolymerizing strategy of high-pressure CO2-H2O pretreatment and oxidation-hydrolysis was applied for selective depolymerization of the cellulose component of corn straw to glucose production. Most part of the hemicellulose component could be removed through high-pressure CO2-H2O pretreatment in the presence of low concentration of acetic acid, and then as high as 32.2 % yield of glucose was achieved in water at 170 °C for 6 h without additional catalyst. The active acid sites generated during the partial oxidation of hydroxymethyl groups to carboxyl groups on glucose units of cellulose was shown to be crucial for the efficient valorization of corn straw for glucose production.

Photocatalytic, Oxidative Cleavage of C-C Bond in Lignin Models and Native Lignin.

It is challenging to realize the selective C-C bond cleavage of lignin ß-O-4 linkages for production of high-value aromatic chemicals due to its intrinsic inertness and complex structure. Here we report a light-driven, chlorine-radical-based protocol to realize the oxidative C-C bond cleavage in various lignin model compounds catalyzed by commercially available TPT and CaCl2, achieving high conversion and good to high product yields at room temperature. Mechanistic studies reveal that the preferential activation of Cß-H bond facilitates the oxidation and C-C bond cleavage of lignin ß-O-4 model via chlorine radical. Furthermore, this method is also applicable to the depolymerization of natural lignin extracts, furnishing the aromatic oxygenates from the cleavage of Cα-Cß bonds. This study provides experimental foundations to the depolymerization and valorization of lignin into high value-added aromatic compounds.

Artificial Processive Catalytic Systems.

Processive catalysts remain attached to a substrate and perform multiple rounds of catalysis. They are abundant in nature. This review highlights artificial processive catalytic systems, which can be divided into (A) catalytic rings that move along a polymer chain, (B) catalytic pores that hold polymer chains and decompose them, (C) catalysts that remain attached to and move around a cyclic substrate via supramolecular interactions, and (D) anchored catalysts that remain in contact with a substrate via multiple catalytic interactions (see frontispiece).

Naked mole-rat TMEM2 lacks physiological hyaluronan-degrading activity.

Mouse transmembrane protein 2 (mTMEM2) has been identified as a hyaluronidase, which has extracellularly G8 and GG domains and PbH1 repeats; however, our previously study showed that human TMEM2 (hTMEM2) is not a catalytic hyaluronidase due to the absence of the critical amino acid residues (His248/Ala303) in the GG domain. Naked mole-rats (NMRs) accumulate abundant high-molecular weight hyaluronan (HA) in their tissues, suggesting decreased HA degradation. Therefore, we aimed to evaluate the HA-degrading activity of NMR TMEM2 (nmrTMEM2) and compare it with those of mTMEM2 and hTMEM2. The amino acid residues of nmrTMEM2 (Asn247/Val302) are similar to Asn248/Phe303 of hTMEM2, and nmrTMEM2-expressing HEK293T cells showed negligible activity. We confirmed the significance of these amino acid residues using an inactive chimeric TMEM2 with the human GG domain, which acquired catalytic activity when Asn248/Phe303 was substituted with His248/Ala303. Semi-quantitative comparison of the activities of the membrane-fractions derived from m/h/nmrTMEM2-expressing HEK293T cells revealed that at least 20- and 14-fold higher amounts of nmr/hTMEM2 were required to degrade HA to the same extent as by mTMEM2. Thus, unlike mTMEM2, nmrTMEM2 is not a physiological hyaluronidase. The inability of nmrTMEM2 to degrade HA might partially account for the high-molecular-weight HA accumulation in NMR tissues.

Electrochemical C-H/C-C Bond Oxygenation: A Potential Technology for Plastic Depolymerization.

Herein, we provide eco-friendly and safely operated electrocatalytic methods for the selective oxidation directly or with water, air, light, metal catalyst or other mediators serving as the only oxygen supply. Heavy metals, stoichiometric chemical oxidants, or harsh conditions were drawbacks of earlier oxidative cleavage techniques. It has recently come to light that a crucial stage in the deconstruction of plastic waste and the utilization of biomass is the selective activation of inert C(sp3 )-C/H(sp3 ) bonds, which continues to be a significant obstacle in the chemical upcycling of resistant polyolefin waste. An appealing alternative to chemical oxidations using oxygen and catalysts is direct or indirect electrochemical conversion. An essential transition in the chemical and pharmaceutical industries is the electrochemical oxidation of C-H/C-C bonds. In this review, we discuss cutting-edge approaches to chemically recycle commercial plastics and feasible C-C/C-H bonds oxygenation routes for industrial scale-up.

Modeling study of kinesin-13 MCAK microtubule depolymerase.

Mitotic centromere-associated kinesin (MCAK) motor protein is a typical member of the kinesin-13 family, which can depolymerize microtubules from both plus and minus ends. A critical issue for the MCAK motor is how it performs the depolymerase activity. To address the issue, the pathway of the MCAK motor moving on microtubules and depolymerizing the microtubules is presented here. On the basis of the pathway, the dynamics of both the wild-type and mutant MCAK motors is studied theoretically, which include the full-length MCAK, the full-length MCAK with mutations in the α4-helix of the motor domain, the mutant full-length MCAK with a neutralized neck, the monomeric MCAK and the mutant monomeric MCAK with a neutralized neck. The studies show that a single dimeric MCAK motor can depolymerize microtubules in a processive manner, with either one tubulin or two tubulins being removed per times. The theoretical results are in agreement with the available experimental data. Moreover, predicted results are provided.

Cytomorphological Disparities in Invasive Breast Cancer Cells following Neoadjuvant Endocrine Therapy and Chemotherapy.

INTRODUCTION: Neoadjuvant endocrine therapy (NAE) offers a breast-conserving surgery rate and clinical response rate similar to those of neoadjuvant chemotherapy (NAC), while presenting fewer adverse events and lower pathological complete response rates. The assessment of pathological response determines degenerative changes and predicts the prognosis of breast cancer treated with NAC. This study clarified the degenerative changes occurring in breast cancer following NAE. METHODS: Our study encompassed two groups: NAE, consisting of 15 patients, and NAC, comprising 18 patients. Tissue samples were obtained from core needle biopsies and surgeries. Nuclear and cell areas were calculated using Autocell analysis. Furthermore, we assessed markers associated with microtubule depolymerization (KIF2A) and initiators of apoptosis (caspase-9). RESULTS: In the NAC group, we observed significant increases in both cytoplasmic and cell areas. These changes in cytoplasm and cells were notably more pronounced in the NAC group compared to the NAE group. After treatment, KIF2A exhibited a decrease, with the magnitude of change being greater in the NET group than in the NAC group. However, no discernible differences were found in caspase-9 expression between the two groups. CONCLUSION: Our findings indicate that NAE induces condensation in cancer cells via cell cycle arrest or apoptosis. Conversely, NAC leads to cell enlargement due to the absence of microtubule depolymerization. These discrepancies underscore the importance of accounting for these distinctions when establishing criteria for evaluating pathological responses.

How Depolymerization-Based Plasticization Affects the Process of Cold Crystallization in Poly(P-Dioxanone).

The self-plasticization, i.e., the increase in the polymer chains' mobility by including its monomer, has a major impact on a polymer's structural, thermal, and mechanical properties. In this study, differential scanning calorimetry (DSC), optical and Raman microscopies, thermo-mechanical analysis (TMA), size exclusion chromatography equipped with a multi-angle light scattering detector (SEC-MALS), and X-ray diffraction analysis (XRD) are used to investigate the effect of thermally induced self-plasticization of poly-(p-dioxanone), PDX, on the crystal growths from the amorphous and molten states. Significant changes in the crystallization behavior and mechanical properties of PDX are found only for samples self-plasticized at the depolymerization temperature (Td) above 150 °C. The intense self-plasticization leads to the decrease of the crystallization temperature, increase of the crystal growth rapidity, disappearance of the distinct α→α' polymorphic transition, reduction of the overall melting temperature, and segregation of the redundant monomer. Although the morphology of the crystalline phase has a major impact on the mechanical properties of PDX, the self-plasticization itself does not seem to result in any major changes in the magnitude, localization, or morphology of formed crystallites (these are primarily driven by the temperature of crystal growth). The manifestation of the variable activation energy concept is discussed for the present crystallization data.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

Enzymatic degradation of polylactic acid (PLA).

Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: • Enzymes can be promisingly utilized for PLA upcycling. • Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. • Approaches to improve enzymatic degradation of PLA are discussed.

Purification and Structural Analyses of Sulfated Polysaccharides from Low-Value Sea Cucumber Stichopus naso and Anticoagulant Activities of Its Oligosaccharides.

Three polysaccharides (SnNG, SnFS and SnFG) were purified from the body wall of Stichopus naso. The physicochemical properties, including monosaccharide composition, molecular weight, sulfate content, and optical rotation, were analyzed, confirming that SnFS and SnFG are sulfated polysaccharides commonly found in sea cucumbers. The highly regular structure {3)-L-Fuc2S-(α1,}n of SnFS was determined via a detailed NMR analysis of its oxidative degradation product. By employing ß-elimination depolymerization of SnFG, tri-, penta-, octa-, hendeca-, tetradeca-, and heptadeca-saccharides were obtained from the low-molecular-weight product. Their well-defined structures confirmed that SnFG possessed the backbone of {D-GalNAc4S6S-ß(1,4)-D-GlcA}, and each GlcA residue was branched with Fuc2S4S. SnFS and SnFG are both structurally the simplest version of natural fucan sulfate and fucosylated glycosaminoglycan, facilitating the application of low-value sea cucumbers S. naso. Bioactivity assays showed that SnFG and its derived oligosaccharides exhibited potent anticoagulation and intrinsic factor Xase (iXase) inhibition. Moreover, a comparative analysis with the series of oligosaccharides solely branched with Fuc3S4S showed that in oligosaccharides with lower degrees of polymerization, such as octasaccharides, Fuc2S4S led to a greater increase in APTT prolongation and iXase inhibition. As the degree of polymerization increases, the influence from the sulfation pattern diminishes, until it is overshadowed by the effects of molecular weight.

Discrimination and simultaneous quantification of poly(ethylene terephthalate) and poly(butylene terephthalate) microplastics in environmental samples via gas chromatography-tandem mass spectrometry.

A method has been developed to quantify PET and PBT microplastics (MPs) based on depolymerization and detection of depolymerization products by gas chromatography-tandem mass spectrometry (GC-MS/MS) without a complex separation process from environmental samples. Under the optimal depolymerization conditions, PET and PBT were efficiently converted to ethylene glycol (78%) and 1,4-butanediol (87%), respectively. Subsequently, the linear curves were constructed between signal intensities of depolymerization products and polymer masses by GC-MS/MS, and the correlation coefficients of PET and PBT were 0.996 and 0.997, respectively. The spiking and recovery experiments of PET and PBT in the environmental samples showed that the recovery was stable in the range 89-100%, and the limit of detection was 4.95 µg and 1.39 µg of PET and PBT, respectively. The method has been proven to be capable of simultaneous identification and quantification of PBT and PET MPs in real environmental water samples without complex separation process, which provided a scheme for the determination of microplastics.

A new strategy for PET depolymerization: Application of bimetallic MOF-74 as a selective catalyst.

Large-volume production of poly(ethylene terephthalate) (PET), especially in the form of bottles and food packaging containers, causes problems with polymer waste management. Waste PET could be recycled thermally, mechanically or chemically and the last method allows to obtain individual monomers, but most often it is carried out in the presence of homogeneous catalysts, that are difficult to separate and reuse. In view of this, this work reports for the first time, application of bimetallic MOF-74 - as heterogeneous catalyst - for depolymerization of PET with high monomer (bishydroxyethyl terephthalate, BHET) recovery. The effect of type and amount of second metal in the MOF-74 (Mg/M) was systematically investigated. The results showed increased activity of MOF-74 (Mg/M) containing Co2+, Zn2+ and Mn2+ as a second metal, while the opposite correlation was observed for Cu2+ and Ni2+. It was found that the highest catalytic activity was demonstrated by the introduction of Mg-Mn into MOF-74 with ratio molar 1:1, which resulted in complete depolymerization of PET and 91.8% BHET yield within 4 h. Furthermore, the obtained catalyst showed good stability in 5 reaction cycles and allowed to achieve high-purity BHET, which was confirmed by HPLC analysis. The as-prepared MOF-74 (Mg/Mn) was easy to separate from the post-reaction mixture, clean and reuse in the next depolymerization reaction.

Degradation Product-Promoted Depolymerization Strategy for Chemical Recycling of Poly(bisphenol A carbonate).

The accumulation of waste plastics has a severe impact on the environment, and therefore, the development of efficient chemical recycling methods has become an extremely important task. In this regard, a new strategy of degradation product-promoted depolymerization process was proposed. Using N,N'-dimethyl-ethylenediamine (DMEDA) as a depolymerization reagent, an efficient chemical recycling of poly(bisphenol A carbonate) (BPA-PC or PC) material was achieved under mild conditions. The degradation product 1,3-dimethyl-2-imidazolidinone (DMI) was proven to be a critical factor in facilitating the depolymerization process. This strategy does not require catalysts or auxiliary solvents, making it a truly green process. This method improves the recycling efficiency of PC and promotes the development of plastic reutilization.