Synthesis of a Zn(II)-2-aminoimidazole Framework as an Efficient Carbonic Anhydrase Mimic.

Carbonic anhydrase (CA) plays a crucial role in the CO2 capture processes by catalyzing the hydration of CO2. In this study, we synthesized a bioinspired carbonic anhydrase Zn-MOF (metal-organic framework) incorporating 2-aminoimidazole and Zn2+ as initial constituents. The synthesized Zn-MOF exhibited promising potential for efficiently catalyzing the CO2 hydration. Structural analyses such as SEM, XRD, and BET confirmed that the Zn-MOF crystal consisted of stacked grains with an average size of approximately 36 nm, forming a micron-sized spherical structure. Functionally, Zn-MOF exhibited effective catalytic activity toward both CO2 hydration and ester hydrolysis. The introduction of amino groups significantly enhanced the esterase activity of Zn-MOF to 0.28 U/mg at ambient temperature, which was twice that of ZIF-8. Furthermore, the introduction of amino groups resulted in remarkable hydrothermal stability, with the esterase activity reaching 0.72 U/mg after undergoing hydrothermal treatment at 80 °C for 12 h. Additionally, Zn-MOF exhibited enhanced capability in CO2 hydration at a pH value exceeding 8.5. After six repeated uses, ZIF-8 and Zn-MOF retained approximately 68 and 65% of their initial enzyme activity, respectively, underscoring the potential practical applicability of Zn-MOF in industrial CO2 capture processes. This work showcases the development of a novel Zn-MOF crystal as an efficient CA mimic, effectively emulating the active sites of natural CA using 2-aminoimidazole as a coordinating ligand for Zn2+ coordination. These findings not only advance the field of innovative enzyme mimics but also pave the way for further exploration of industrial CO2 capture catalysts.

Carbonic anhydrase encapsulation using bamboo cellulose scaffolds for efficient CO2 capture and conversion.

Utilizing carbonic anhydrase (CA) to catalyze CO2 hydration offers a sustainable and potent approach for carbon capture and utilization. To enhance CA's reusability and stability for successful industrial applications, enzyme immobilization is essential. In this study, delignified bamboo cellulose served as a renewable porous scaffold for immobilizing CA through oxidation-induced cellulose aldehydation followed by Schiff base linkage. The catalytic performance of the resulting immobilized CA was evaluated using both p-NPA hydrolysis and CO2 hydration models. Compared to free CA, immobilization onto the bamboo scaffold increased CA's optimal temperature and pH to approximately 45 °C and 9.0, respectively. Post-immobilization, CA activity demonstrated effective retention (>60 %), with larger scaffold sizes (i.e., 8 mm diameter and 5 mm height) positively impacting this aspect, even surpassing the activity of free CA. Furthermore, immobilized CA exhibited sustained reusability and high stability under thermal treatment and pH fluctuation, retaining >80 % activity even after 5 catalytic cycles. When introduced to microalgae culture, the immobilized CA improved biomass production by ∼16 %, accompanied by enhanced synthesis of essential biomolecules in microalgae. Collectively, the facile and green construction of immobilized CA onto bamboo cellulose block demonstrates great potential for the development of various CA-catalyzed CO2 conversion and utilization technologies.

The extracellular polymeric substances (EPS) accumulation of Spirulina platensis responding to Cadmium (Cd2+) exposure.

Spirulina platensis can secrete extracellular polymeric substances (EPS) helping to protect damage from stress environment, such as cadmium (Cd2+) exposure. However, the responding mechanism of S. platensis and the secreted EPS to exposure of Cd2+ is still unclear. This research focuses on the effects of Cd2+ on the composition and structure of the EPS and the response mechanism of EPS secretion from S. platensis for Cd2+ exposure. S. platensis can produce 261.37 mg·g-1 EPS when exposing to 20 mg·L-1 CdCl2, which was 2.5 times higher than the control group. The S. platensis EPS with and without Cd2+ treatment presented similar and stable irregularly fibrous structure. The monosaccharides composition of EPS in Cd2+ treated group are similar with control group but with different monosaccharides molar ratios, especially for Rha, Gal, Glc and Glc-UA. And the Cd2+ treatment resulted in a remarkable decline of humic acid and fulvic acid content. The antioxidant ability of S. platensis EPS increased significantly when exposed to 20 mg·L-1 CdCl2, which could be helpful for S. platensis protecting damage from high concentration of Cd2+. The transcriptome analysis showed that sulfur related metabolic pathways were up-regulated significantly, which promoted the synthesis of sulfur-containing amino acids and the secretion of large amounts of EPS.

A peptide-engineered alginate aerogel with synergistic blood-absorbing and platelet-binding capabilities to rapidly stop bleeding.

Polysaccharide matrix infused with hemostasis-stimulating chemistry represents a critical medical need of bleeding management. Herein, we describe the development of a polysaccharide-peptide conjugate platform, an alginate engineered with fibrinogen-derived platelet-binding peptides (APE). The alginate backbone was found to allow for multivalent grafting of the peptides. Processing APE conjugate into crosslinked aerogels promoted platelet accumulation, leading to a significant reduction in the coagulation time of whole rabbit blood and improving the stability of the formed clot. The APE aerogels also exhibited a high porosity and fluid uptake capacity (>90 in weight ratio) as well as good biocompatibility in hemostasis. Furthermore, in vivo studies conducted in rat models of tail cut and hepatic hemorrhage showed that APE aerogels reduced bleeding time by >58 % and blood loss by >61 %. The platelet-enrichment capacity of the APE construct synergized by high absorbency in its aerogel form offers a prototype for customized polysaccharide hemostats.

Simultaneous carbon dioxide sequestration and nitrate removal by Chlorella vulgaris and Pseudomonas sp. consortium.

Carbon dioxide and nitrogen oxides are the main components of fossil flue gas causing the most serious environmental problems. Developing a sustainable and green method to treat carbon dioxide and nitrogen oxides of flue gas is still challenging. Here, a co-cultured microalgae/bacteria system, Chlorella vulgaris and Pseudomonas sp., was developed for simultaneous sequestration of CO2 and removal of nitrogen oxides from flue gas, as well as producing valuable microalgae biomass. The co-cultured Chlorella vulgaris and Pseudomonas sp. showed the highest CO2 fixation and NO3--N removal rate of 0.482 g L-1d-1 and 129.6 mg L-1d-1, the total chlorophyll accumulation rate of 65.6 mg L-1 at the initial volume ratio of Chlorella vulgaris and Pseudomonas sp. as 1:10. The NO3--N removal rate can be increased to 183.5 mg L-1d-1 by continuous addition of 0.6 g L-1d-1 of glucose, which was 37% higher than that of co-culture system without the addition of glucose. Photosynthetic activity and carbonic anhydrase activity of Chlorella vulgaris were significantly increased when co-cultured with Pseudomonas sp. Excitation-emission matrix (EEM) fluorescence spectroscopy indicated that the humic acid-like substances released from Pseudomonas sp. could increase the growth of microalgae. This work provides an attractive way to simultaneously treatment of CO2 and NOX from flue gas to produce valuable microalgal biomass.

Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors.

G-protein-coupled receptor (GPCR) density at the cell surface is thought to regulate receptor function. Spatially resolved measurements of local-density effects on GPCRs are needed but technically limited by density heterogeneity and mobility of membrane receptors. We now develop a deep-learning (DL)-enhanced diffusion imaging assay that can measure local-density effects on ligand-receptor interactions in the plasma membrane of live cells. In this method, the DL algorithm allows the transformation of 100 ms exposure images to density maps that report receptor numbers over any specified region with ∼95% accuracy by 1 s exposure images as ground truth. With the density maps, a diffusion assay is further established for spatially resolved measurements of receptor diffusion coefficient as well as to express relationships between receptor diffusivity and local density. By this assay, we scrutinize local-density effects on chemokine receptor CXCR4 interactions with various ligands, which reveals that an agonist prefers to act with CXCR4 at low density while an inverse agonist dominates at high density. This work suggests a new insight into density-dependent receptor regulation as well as provides an unprecedented assay that can be applicable to a wide variety of receptors in live cells.

Carbon Dot Blinking Fingerprint Uncovers Native Membrane Receptor Organizations via Deep Learning.

Oligomeric organization of G protein-coupled receptors is proposed to regulate receptor signaling and function, yet rapid and precise identification of the oligomeric status especially for native receptors on a cell membrane remains an outstanding challenge. By using blinking carbon dots (CDs), we now develop a deep learning (DL)-based blinking fingerprint recognition method, named deep-blinking fingerprint recognition (BFR), which allows automatic classification of CD-labeled receptor organizations on a cell membrane. This DL model integrates convolutional layers, long-short-term memory, and fully connected layers to extract time-dependent blinking features of CDs and is trained to a high accuracy (∼95%) for identifying receptor organizations. Using deep blinking fingerprint recognition, we found that CXCR4 mainly exists as 87.3% monomers, 12.4% dimers, and <1% higher-order oligomers on a HeLa cell membrane. We further demonstrate that the heterogeneous organizations can be regulated by various stimuli at different degrees. The receptor-binding ligands, agonist SDF-1α and antagonist AMD3100, can induce the dimerization of CXCR4 to 33.1 and 20.3%, respectively. In addition, cytochalasin D, which inhibits actin polymerization, similarly prompts significant dimerization of CXCR4 to 30.9%. The multi-pathway organization regulation will provide an insight for understanding the oligomerization mechanism of CXCR4 as well as for elucidating their physiological functions.

Amino Acid-Mediated Synthesis of the ZIF-8 Nanozyme That Reproduces Both the Zinc-Coordinated Active Center and Hydrophobic Pocket of Natural Carbonic Anhydrase.

The zeolitic imidazolate framework-8 (ZIF-8) nanozyme has been synthesized using hydrophobic amino acid (AA) to regulate crystal growth. The as-synthesized ZIF-8 reproduces both the structural and functional properties of natural carbonic anhydrase (CA). Structurally, Zn2+/2-methylimidazole coordinated units mimic very well the active center of CA while the hydrophobic microdomains of the adsorbed AA simulate the CA hydrophobic pocket. Functionally, the nanozymes show excellent CA-like esterase activity by giving specific enzyme activity of 0.22 U mg-1 at 25 °C in the case of Val-ZIF-8. More strikingly, such nanozymes are superior to natural CA by having excellent hydrothermal stability, which can give highly enhanced esterase activity with increasing temperature. The specific enzyme activity of Val-ZIF-8 at 80 °C is about 25 times higher than that at 25 °C. In addition, AA-ZIF-8 also shows an excellent catalytic efficiency toward carbon dioxide (CO2) hydration. This study puts forward the important role of hydrophobic microdomains in biomimetic nanozymes for the first time and develops a facile and mild method for the synthesis of nanozymes with controlled morphology and size to achieve excellent catalytic efficiency.

Oligomerization-Enhanced Receptor-Ligand Binding Revealed by Dual-Color Simultaneous Tracking on Living Cell Membranes.

GPCR oligomerization plays a critical role in cellular signaling, yet the stoichiometry of the interactions between oligomers and binding ligands in living cells remains a longstanding challenge. Here, by developing a dual-color simultaneous tracking system based on a total internal reflection fluorescence microscope (TIRFM), the CCR5-CCL5 interactions are visualized and quantitatively assessed in real time. Results show that each oligomeric state of CCR5 could bind with CCL5 but with different binding affinities; CCR5 dimers have a 3.5-fold higher binding affinity than the monomers. The dimerization may cause an asymmetric conformational change which makes the first binding pocket have a 3.5-fold higher binding affinity and the second have only a half compared with the monomeric CCR5. This study is the first example to directly scrutinize the CCR5-CCL5 interactions at the single-molecule level on living cell membranes and will offer great potential for the interaction stoichiometry study of diverse surface proteins.

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane.

Single-molecule fluorescence imaging is a powerful tool to study protein function by tracking molecular position and distribution, but the precise and rapid identification of dynamic molecules remains challenging due to the heterogeneous distribution and interaction of proteins on the live cell membrane. We now develop a deep-learning (DL)-assisted single-molecule imaging method that can precisely distinguish the monomer and complex for rapid and real-time tracking of protein interaction. This DL-based model, which comprises convolutional layers, max pooling layers, and fully connected layers, is trained to reach an accuracy of >98% for identifying monomer and complex. We use this method to investigate the dynamic process of chemokine receptor CXCR4 on the live cell membrane during the early signaling stage. The results show that, upon ligand activation, the CXCR4 undergoes a dynamic process of forming a receptor complex. We further demonstrate that the CXCR4 complex tends to be internalized at 2.5-fold higher rate into the cell interior than the monomer via the clathrin-dependent pathway. This study is the first example to scrutinize the early signaling process of CXCR4 at the single-molecule level on the live cell membrane. We envision that this DL-assisted imaging method would be a broadly useful technique to study more protein families for elucidating their physiological and pathological functions.

Nonblinking carbon dots for imaging and tracking receptors on a live cell membrane.

Blinking occurs with nearly all fluorophores including organic dyes, fluorescent proteins, semiconductor quantum dots and carbon dots (CDs). We developed non-blinking and photoresistant fluorescent CDs by introducing multiple aromatic domains onto a single carbon dot and demonstrated their great potential for imaging and tracking of receptors on a live cell membrane.

Protein Nanobarrel for Integrating Chlorophyll a Molecules and Its Photochemical Performance.

Taking inspiration from biology's effectiveness in nanoscale organization of chlorophylls for photosynthesis, we describe here a design for chlorophyll-protein conjugates that exploits the central hydrophobic cavity of GroEL protein nanobarrel as a binding pocket for chlorophyll. We found water-soluble conjugates of chlorophyll with GroEL could be easily generated via detergent dialysis. The number of chlorophyll units bound to GroEL is tunable by varying the equilibrium concentration of chlorophyll during dialysis. Meanwhile, it is shown that an increase in the entrapped chlorophyll amount leads to an improvement of chlorophyll-GroEL photostability. Using methyl viologen as an electron acceptor, we demonstrate that chlorophyll-GroEL has photoreduction activity, which is also switchable in on/off illumination mode. Finally, it is shown that chlorophyll-GroEL-sensitized solar cells have good photoelectric properties, yielding a high photoelectric conversion efficiency of ∼0.9%. The current strategy may be adopted for integrating other photosensitizing dyes or for other photocatalytic reactions.

Reactive Oxygen Species-Induced Aggregation of Nanozymes for Neuron Injury.

Nanozymes show excellent enzyme activity and robust catalytic properties, but the targeting capability to disease organs is limited because of lack of specificity. Herein, we developed an ultrasmall (∼3 nm) organic nanozyme that can gradually aggregate under a reactive oxygen species (ROS)-rich environment via a spontaneous reaction, namely, ROS-induced aggregation. The size of nanozymes is 75 and 100 times higher than the original size under •OH and H2O2 environments without losing enzyme activity. In vitro experiments confirm that nanozymes prefer to aggregate in mitochondria under ROS-rich conditions. Importantly, the nanozymes show in situ ROS-induced aggregation in the brain, ∼9 times higher uptake than ordinary nanozymes, indicating their potential for treating ROS-related diseases in the central nervous system.

The in vitro synergistic denaturation effect of heat and surfactant on photosystem I isolated from Arthrospira Platensis.

Photosystem I (PSI) generates the most negative redox potential found in nature, and the performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermal stability of PSI. Thus, understanding thermal denaturation is an important prerequisite for the use of PSI at elevated temperatures. To assess the thermal stability of surfactant-solubilized PSI from cyanobacteria Arthrospira Platensis, the synergistic denaturation effect of heat and surfactant was studied. At room temperature, surfactant n-dodecyl-ß-D-maltoside solubilized PSI trimer gradually disassembles into PSI monomers and free pigments over long time. In the solubilizing process of PSI particles, surfactant can uncouple pigments of PSI, and the high concentration of surfactant causes the pigment to uncouple more; after the surfactant-solubilizing process, the uncoupling is relatively slow. During the heating process, changes were monitored by transmittance T800nm, ellipticity θ686nm and θ222nm, upon slow heating (1.5 °C per minute) of samples in Tris buffer (20 mM, pH 7.8) from 20 to 95 °C. The thermal denaturation of surfactant-solubilized PSI is a much more complicated process, which includes the uncoupling of pigments by surfactants, the disappearance of surrounding surfactants, and the unfolding of PSI α-helices. During the heating process, the uncoupling chlorophyll a (Chla) and converted pheophytin (Pheo) can form excitons of Chla-Pheo. The secondary structure α-helix of PSI proteins is stable up to 87-92 °C in the low-concentration surfactant solubilized PSI, and high-concentration surfactant and pigments uncoupling can accelerate the α-helical unfolding.

Conductive Core-Shell Aramid Nanofibrils: Compromising Conductivity with Mechanical Robustness for Organic Wearable Sensing.

One-dimensional organic nanomaterials with a combination of electric conductivity, flexibility, and mechanical robustness are highly in demand in a variety of flexible electronic devices. Herein, conducting polymers were combined with robust Kevlar nanofibrils (aramid nanofibrils, abbreviated as ANFs) via in situ polymerization. Owing to the strong interactions between ANFs and conjugated polymers, the resultant core-shell ANFs showed high electric conductivity in combination with flexibility, robustness, physical stability, and endurance to bending and solvents, in sharp contrast to many inorganic conductive nanomaterials. Due to their responsivity of conductivity to different stimuli (e.g., humidity and strain), their membranes were capable not only of sensing human motions and speech words, but also of showing high sensitivity to variation of environmental humidity. In such a way, these core-shell ANFs may pave the way for combining both conductivity and mechanical properties applicable for diverse wearable devices.

Cell-Free Expression of Unnatural Amino Acid Incorporated Aquaporin SS9 with Improved Separation Performance in Biomimetic Membranes.

Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from Photobacterium profundum SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid (p-propargyloxyphenylalanine, pPpa) (P-AQP SS9) in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg2+ and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (Pf) of 7.46×10-4 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (Pf=1.31×10-4 m/s) and 12.1 times higher than that of the DOPC liposomes (Pf=6.15×10-5m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.

Denitrifying sulfide removal process on high-tetracycline wastewater.

Antibiotics wastewater from tetracycline (TC) production unit can have high levels of chemical oxygen demand, ammonium and sulfate and up to a few hundreds of milligrams per liter of TC. Denitrifying sulfide removal (DSR) process is set up for simultaneously removal of sulfur, carbon and nitrogen from waters. The DSR process was for the first time studied for treating TC wastewaters. The TC stress has no adverse effects on removal rates of nitrate and acetate; however, it moderately deteriorated sulfide removal rates and S(0) accumulation rates when the concentration is higher than 100mgL(-1) TC. The Thauera sp., and Pseudomonas sp. present the heterotrophs and Sulfurovum sp. presented the autotroph for the present DSR reactions. The high tolerance of TC stress by the tested consortium was explained by the excess production of extracellular polymeric substances at high TC concentration, which can bind with TC for minimizing its inhibition effects.

Enhanced photocurrent production by bio-dyes of photosynthetic macromolecules on designed TiO2 film.

The macromolecular pigment-protein complex has the merit of high efficiency for light-energy capture and transfer after long-term photosynthetic evolution. Here bio-dyes of A. platensis photosystem I (PSI) and spinach light-harvesting complex II (LHCII) are spontaneously sensitized on three types of designed TiO2 films, to assess the effects of pigment-protein complex on the performance of bio-dye sensitized solar cells (SSC). Adsorption models of bio-dyes are proposed based on the 3D structures of PSI and LHCII, and the size of particles and inner pores in the TiO2 film. PSI shows its merit of high efficiency for captured energy transfer, charge separation and transfer in the electron transfer chain (ETC), and electron injection from FB to the TiO2 conducting band. After optimization, the best short current (JSC) and photoelectric conversion efficiency (η) of PSI-SSC and LHCII-SSC are 1.31 mA cm(-2) and 0.47%, and 1.51 mA cm(-2) and 0.52%, respectively. The potential for further improvement of this PSI based SSC is significant and could lead to better utilization of solar energy.

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis.

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-ß-D-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of (1)O2, such as 1 mM NaN3, may be added into the mixture.

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis.

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-ß-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.