Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

Association between per- and polyfluoroalkyl substances and sex hormone levels in males based on human studies.

BACKGROUND: Per- and poly-fluoroalkyl substances (PFAS) are ubiquitous chemicals in the environment and our daily lives. Several epidemiological studies have revealed that PFAS exposure is linked to male sex hormone levels; however, the conclusions are inconsistent across studies. Consequently, we performed a meta-analysis to systematically evaluate the association between PFAS exposure and male sex hormones. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) standards were followed during the meta-analysis. PubMed, Wed of Science, Embase, Cochrane Library, and Ovid databases were used to identify suitable articles before June 2023. The 95% CI and ß values were calculated to assess the association between male sex hormone levels and PFAS exposure. Heterogeneity among the included studies was tested using inconsistency statistics (I2). RESULTS: The literature search identified 12 published articles that met our search criteria, involving 7506 participants. Our results revealed that perfluorononanoic acid (PFNA) and perfluorooctanoic acid (PFOA) exposures were negatively correlated with testosterone (ß = -0.05; 95% CI: -0.09, -0.02, P = 0.003) and (ß = -0.04; 95% CI: -0.08, 0.00, P = 0.049), respectively. CONCLUSION: Exposure to PFNA and PFOA is negatively correlated with changes in male testosterone levels. This correlation suggests that we need to pay attention in the future to whether they are potential risk factors for male reproductive health.

Enhanced removal of mixed VOCs with different hydrophobicities by Tween 20 in a biotrickling filter: Kinetic analysis and biofilm characteristics.

Mass transfer limitation usually causes the poor performance of biotrickling filters (BTFs) for the treatment of hydrophobic volatile organic compounds (VOCs) during long-term operation. In this study, two identical lab-scale BTFs were established to remove a mixture of n-hexane and dichloromethane (DCM) gases using non-ionic surfactant Tween 20 by Pseudomonas mendocina NX-1 and Methylobacterium rhodesianum H13. A low pressure drop (≤110 Pa) and a rapid biomass accumulation (17.1 mg g-1) were observed in the presence of Tween 20 during the startup period (30 d). The removal efficiency (RE) of n-hexane was enhanced by 15.0%- 20.5% while DCM was completely removed with the inlet concentration (IC) of 300 mg·m-3 at different empty bed residence times in the Tween 20 added BTF. The viable cells and the relative hydrophobicity of the biofilm were increased under the action of Tween 20, which facilitated the mass transfer and enhanced the metabolic utilization of pollutants by microbes. Besides, Tween 20 addition enhanced the biofilm formation processes including the increased extracellular polymeric substance (EPS) secretion, biofilm roughness and biofilm adhesion. The kinetic model simulated the removal performance of the BTF with Tween 20 for the mixed hydrophobic VOCs, and the goodness-of-fit was above 0.9.

Mid-infrared HgTe Colloidal Quantum Dot LEDs.

Mid-infrared HgTe colloidal quantum dot electroluminescent devices are demonstrated. With emission at 4 µm, devices achieved an external quantum efficiency of ∼10-3 and power conversion efficiency of ∼10-4 under biases of a few volts. The power conversion efficiency benefited from lowering the transparent electrode resistance through the incorporation of a metal conductive grid. The average power emitted was about 16 µW at 2 V bias with 50% duty cycle and a 1 mm2 device. The room-temperature electroluminescence efficiency at low current was limited by the photoluminescence efficiency of the quantum dots, while the diode structure provided efficient electron-hole recombination.

Extracellular Calcium Ion Concentration Regulates Chondrocyte Elastic Modulus and Adhesion Behavior.

Extracellular calcium ion concentration levels increase in human osteoarthritic (OA) joints and contribute to OA pathogenesis. Given the fact that OA is a mechanical problem, the effect of the extracellular calcium level ([Ca2+]) on the mechanical behavior of primary human OA chondrocytes remains to be elucidated. Here, we measured the elastic modulus and cell-ECM adhesion forces of human primary chondrocytes with atomic force microscopy (AFM) at different extracellular calcium ion concentration ([Ca2+]) levels. With the [Ca2+] level increasing from the normal baseline level, the elastic modulus of chondrocytes showed a trend of an increase and a subsequent decrease at the level of [Ca2+], reaching 2.75 mM. The maximum increment of the elastic modulus of chondrocytes is a 37% increase at the peak point. The maximum unbinding force of cell-ECM adhesion increased by up to 72% at the peak point relative to the baseline level. qPCR and immunofluorescence also indicated that dose-dependent changes in the expression of myosin and integrin ß1 due to the elevated [Ca2+] may be responsible for the variations in cell stiffness and cell-ECM adhesion. Scratch assay showed that the chondrocyte migration ability was modulated by cell stiffness and cell-ECM adhesion: as chondrocyte's elastic modulus and cell-ECM adhesion force increased, the migration speed of chondrocytes decreased. Taken together, our results showed that [Ca2+] could regulate chondrocytes stiffness and cell-ECM adhesion, and consequently, influence cell migration, which is critical in cartilage repair.

Annulative Methods in the Synthesis of Complex Meroterpene Natural Products.

From the venerable Robinson annulation to the irreplaceable Diels-Alder cycloaddition, annulation reactions have fueled the progression of the field of natural product synthesis throughout the past century. In broader terms, the ability to form a cyclic molecule directly from two or more simpler fragments has transformed virtually every aspect of the chemical sciences from the synthesis of organic materials to bioconjugation chemistry and drug discovery. In this Account, we describe the evolution of our meroterpene synthetic program over the past five years, enabled largely by the development of a tailored anionic annulation process for the synthesis of hydroxylated 1,3-cyclohexanediones from lithium enolates and the reactive ß-lactone-containing feedstock chemical diketene.First, we provide details on short total syntheses of the prototypical polycyclic polyprenylated acylphloroglucinol (PPAP) natural products hyperforin and garsubellin A, which possess complex bicyclo[3.3.1]nonane architectures. Notably, these molecules have served as compelling synthetic targets for several decades and induce a number of biological effects of relevance to neuroscience and medicine. By merging our diketene annulation process with a hypervalent iodine-mediated oxidative ring expansion, bicyclo[3.3.1]nonane architectures can be easily prepared from simple 5,6-fused bicyclic diketones in only two chemical operations. Leveraging these two key chemical reactions in combination with various other stereoselective transformations allowed for these biologically active targets to be prepared in racemic form in only 10 steps.Next, we extend this strategy to the synthesis of complex fungal-derived meroterpenes generated biosynthetically from the coupling of 3,5-dimethylorsellinic acid (DMOA) and farnesyl pyrophosphate. A Ti(III)-mediated radical cyclization of a terminal epoxide was used to rapidly prepare a 6,6,5-fused tricyclic ketone which served as an input for our annulation/rearrangement process, ultimately enabling a total synthesis of protoaustinoid A, an important biosynthetic intermediate in DMOA-derived meroterpene synthesis, and its oxidation product berkeleyone A. Through a radical-based, abiotic rearrangement process, the bicyclo[3.3.1]nonane cores of these natural products could again be isomerized, resulting in the 6,5-fused ring systems of the andrastin family and ultimately delivering a total synthesis of andrastin D and preterrenoid. Notably, these isomerization transformations proved challenging when employing classic, acid-induced conditions for carbocation generation, thus highlighting the power of radical biomimicry in total synthesis. Finally, further oxidation and rearrangement allowed for access to terrenoid and the lactone-containing metabolite terretonin L.Overall, the merger of annulative diketene methodology with an oxidative rearrangement transformation has proven to be a broadly applicable strategy to synthesize bicyclo[3.3.1]nonane-containing natural products, a class of small molecules with over 1000 known members.

Thermodynamic Control over Molecular Aggregate Assembly Enables Tunable Excitonic Properties across the Visible and Near-Infrared.

Specific molecular arrangements within H-/J-aggregates of cyanine dyes enable extraordinary photophysical properties, including long-range exciton delocalization, extreme blue/red shifts, and excitonic superradiance. Despite extensive literature on cyanine aggregates, design principles that drive the self-assembly to a preferred H- or J-aggregated state are unknown. We tune the thermodynamics of self-assembly via independent control of the solvent/nonsolvent ratio, ionic strength, or dye concentration, obtaining a broad range of conditions that predictably stabilize the monomer (H-/J-aggregate). Diffusion-ordered spectroscopy, cryo-electron microscopy, and atomic force microscopy together reveal a dynamic equilibrium between monomers, H-aggregated dimers, and extended J-aggregated 2D monolayers. We construct a model that predicts the equilibrium composition for a range of standard Gibbs free energies, providing a vast aggregation space which we access using the aforementioned solvation factors. We demonstrate the universality of this approach among several sheet-forming cyanine dyes with tunable absorptions spanning visible, near, and shortwave infrared wavelengths.

Synthesis of nanospindle-akaganéite and its photocatalytic degradation for methyl orange.

Iron oxyhydroxides as important catalysts and environmental mineral materials have drawn significant interest for their potential applications in the field of wastewater treatment. In this work, we investigated the influence of nonionic surfactant Brij30 or glucose (0.01 wt%) on the formation of iron precipitates in iron(III) chloride solutions for 3 days at 40, 60 and 80 °C. The results showed that the presence of glucose or Brij30 could promote the nanospindle-akaganéite formation and the akaganéite with a length of 300-500 nm obtained at 60 °C was the optimal catalyst for organic photocatalysis degradation. Further, we investigated the capacity of C60 akaganéite for degradation removal of methyl orange (MO) under the action of hydrogen peroxide (H2O2) addition and/or UV irradiation, and in the presence of different radical scavengers at pH 4.5. We also researched the effects of various levels of H2O2 and catalyst, and the reaction pH values. It was found that akaganéites could remove almost 100% of MO under 100 mg·L-1 of catalyst and H2O2 at pH 4.5. Akaganéite maintained 86% of MO removal capacity after four successive cycles. Our results can be used as a reference for the synthesis of environmentally functional material and the application in photocatalytic degradation of organic pollutant.

Programmable meroterpene synthesis.

The bicyclo[3.3.1]nonane architecture is a privileged structural motif found in over 1000 natural products with relevance to neurodegenerative disease, bacterial and parasitic infection, and cancer among others. Despite disparate biosynthetic machinery, alkaloid, terpene, and polyketide-producing organisms have all evolved pathways to incorporate this carbocyclic ring system. Natural products of mixed polyketide/terpenoid origins (meroterpenes) are a particularly rich and important source of biologically active bicyclo[3.3.1]nonane-containing molecules. Herein we detail a fully synthetic strategy toward this broad family of targets based on an abiotic annulation/rearrangement strategy resulting in a 10-step total synthesis of garsubellin A, an enhancer of choline acetyltransferase and member of the large family of polycyclic polyprenylated acylphloroglucinols. This work solidifies a strategy for making multiple, diverse meroterpene chemotypes in a programmable assembly process involving a minimal number of chemical transformations.

Allylative Approaches to the Synthesis of Complex Guaianolide Sesquiterpenes from Apiaceae and Asteraceae.

With hundreds of unique members isolated to date, guaianolide lactones represent a particularly prolific class of terpene natural products. Given their extensive documented therapeutic properties and fascinating chemical structures, these metabolites have captivated the synthetic chemistry community for many decades. As a result of divergent biosynthetic pathways, which produce a wide array of stereochemical and oxidative permutations, a unifying synthetic pathway to this broad family of natural products is challenging. Herein we document the evolution of a chiral-pool-based synthetic program aimed at accessing an assortment of guaianolides, particularly those from the plant family Apiaceae as well as Asteraceae, members of which possess distinct chemical substructures and necessitate deviating synthetic platforms. An initial route employing the linear monoterpene linalool generated a lower oxidation state guaianolide but was not compatible with the majority of family members. A double-allylation disconnection using a carvone-derived fragment was then developed to access first an Asteraceae-type guaianolide and then various Apiaceae congeners. Finally, using these findings in conjunction with a tandem polyoxygenation cascade, we developed a pathway to highly oxygenated nortrilobolide. A variety of interesting observations in metal-mediated aldehyde allylation and alkene polyoxygenation are reported and discussed.

MeV photoelectron spectrometer for ultraintense laser interactions with atoms and molecules.

Traditional laser-matter spectroscopy techniques fail to accurately analyze photoelectrons and ions from ultrahigh intensity studies with terawatt and petawatt laser systems. We present a magnetic deflection, photoelectron spectrometer for ultrahigh intensity laser interactions with atoms and molecules in the single atom/molecule limit. Spectrometer fabrication and calibration, and noise background are presented as well as example photoelectron spectra for argon and chloromethane over an energy range from 20 keV to 2 MeV.

In situ fabrication of a composite hydrogel with tunable mechanical properties for cartilage tissue engineering.

A composite hydrogel with tunable mechanical properties has been fabricated and characterized in this study. We investigated its swelling degree, morphology, structure and thermal stability. Moreover, the effect of strontium chloride concentration on both the dynamic rheology and nanomechanical properties of the composite hydrogels was confirmed in this work. To eliminate the viscoelastic influences of hydrogels during nanomechanical tests, we first analyzed the elastic modulus of strontium alginate (Alg-Sr) and strontium alginate/chondroitin sulfate (Alg/CS-Sr) hydrogels via atomic force microscopy (AFM) using the rate-jump method. Chondrocytes were cultured with the Alg-Sr and Alg/CS-Sr hydrogels respectively. Cell viability assay reveals that the Alg/CS-Sr hydrogel possesses good cytocompatibility. Flow cytometry, qPCR and western blotting analysis suggest that the Alg/CS-Sr hydrogel exerts a positive effect on the inhibition of apoptosis and may exert anti-inflammatory effects in articular cartilage related applications. Furthermore, the preliminary in vivo study shows that the Alg/CS-Sr composite hydrogel facilitates the repair of cartilage in rabbit cartilage defect. Taken together, it is indicated that the Alg/CS-Sr composite hydrogel might be a promising scaffold to promote the repair of cartilage defects.

Nanocrystals of a new camptothecin derivative WCN-21 enhance its solubility and efficacy.

WCN-21 is a new camptothecin derivative we synthesized and has desirable anti-tumor efficacy, but its aqueous solubility is very low and hurdles the further evaluation and development. In this study, we prepared nanocrystals of WCN-21 through a bottom-up approach to enhance its solubility and obtained WCN-21 nanorods (WND) and nanospheres (WNP). We investigated the crystallization of WND and WNP in different temperature and solvents and found that both temperature and solvents affect the crystal shapes and sizes. We prepared WND at 50°C and DMSO : H2O 1: 50 and WNP at 25°C and DMSO : H2O 1: 100 and found they were dispersed evenly in water with average hydrodynamic diameters 337 and 231 nm, respectively. WND and WNP increased the solubility of WCN-21 from extreme insolubility to more than 9 and 11 mM in H2O or PBS, respectively. In vitro studies showed that WND and WNP enhanced the uptake of WCN-21 in tumor cells by 3 and 9 folds, and increased cytotoxicity of WCN-21 in comparison with free WCN-21 by 5 and 6 folds, respectively. In xenograft tumor mice, intravenous injection of WND and WNP enhanced the accumulation of WCN-21 in tumor tissues and improved the anti-tumor efficacy. In addition, WND and WNP did not increase the toxicity of WCN-21 in mice. Therefore, nanocrystal is a robust tool to improve the solubility of insoluble drugs and holds a great potential in the application of drug development.

Total Synthesis of (-)-Spinosyn A via Carbonylative Macrolactonization.

Spinosyn A (1), a complex natural product featuring a unique 5,6,5,12-fused tetracyclic core structure, is the major component of spinosad, an organic insecticide and an FDA-approved agent used worldwide. Herein, we report an efficient total synthesis of (-)-spinosyn A with 15 steps in the longest linear sequence and 23 steps total from readily available compounds 14 and 23. The synthetic approach features several important catalytic transformations including a chiral amine-catalyzed intramolecular Diels-Alder reaction to afford 22 in excellent diastereoselectivity, a one-step gold-catalyzed propargylic acetate rearrangement to convert 28 to α-iodoenone 31, an unprecedented palladium-catalyzed carbonylative Heck macrolactonization to form the 5,12-fused macrolactone in one step, and a gold-catalyzed Yu glycosylation to install the challenging ß-forosamine. This total synthesis is highly convergent and modular, thus offering opportunities to synthesize spinosyn analogues in order to address the emerging cross-resistance problems.

Copper-Catalyzed Cyclopropanol Ring Opening Csp(3)-Csp(3) Cross-Couplings with (Fluoro)Alkyl Halides.

Novel and general copper-catalyzed cyclopropanol ring opening cross-coupling reactions with difluoroalkyl bromides, perfluoroalkyl iodides, monofluoroalkyl bromides, and 2-bromo-2-alkylesters to synthesize various ß-(fluoro)alkylated ketones are reported. The reactions feature mild conditions and excellent functional group compatibility and can be scaled up to gram scale. Preliminary mechanistic studies suggest the involvement of radical intermediates. The difluoroalkyl-alkyl cross-coupling products can also be readily converted to more valuable and diverse gem-difluoro-containing compounds by taking advantage of the carbonyl group resulting from cyclopropanol ring opening.