In Situ Grown Silver-Polymer Framework with Coordination Complexes for Functional Artificial Tissues.

Self-sensing actuators are critical to artificial robots with biomimetic proprio-/exteroception properties of biological neuromuscular systems. Existing add-on approaches, which physically blend heterogeneous sensor/actuator components, fall short of yielding satisfactory solutions, considering their suboptimal interfaces, poor adhesion, and electronic/mechanical property mismatches. Here, a single homogeneous material platform is reported by creating a silver-polymer framework (SPF), thus realizing the seamless sensing-actuation unification. The SPF-enabled elastomer is highly stretchable (1200%), conductive (0.076 S m-1 ), and strong (0.76 MPa in-strength), where the stretchable polymer matrix synthesis and in situ silver nanoparticles reduction are accomplished simultaneously. Benefiting from the multimodal sensing capability from its architecture itself (mechanical and thermal cues), self-sensing actuation (proprio-deformations and external stimuli perceptions) is achieved for the SPF-based pneumatic actuator, alongside an excellent load-lifting attribute (up to 3700 times its own weight), substantiating its advantage of the unified sensing-actuation feature in a single homogenous material. In view of its human somatosensitive muscular systems imitative functionality, the reported SPF bodes well for use with next-generation functional tissues, including artificial skins, human-machine interfaces, self-sensing robots, and otherwise dynamic materials.

Single-Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions.

The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron-sulfur protein containing a unique Fe2 S2 (His)1 (Cys)3 metal cluster with a single Fe-N(His87) coordinating bond. This labile Fe-N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe-N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM-SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single-molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe-N bond.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy.

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene. It plays an essential role in many biological systems as a vital driving force for protein folding, stability, and receptor-ligand interaction/recognition. To date, the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional (3D) protein structures and corresponding computational calculations. However, their experimental verification and quantification remain sparse at the molecular level, mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins. Here, we use atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of protein neutrophil gelatinase-associated lipocalin (also known as NGAL, siderocalin, lipocalin 2) that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols. Based on a site-specific cysteine engineering and anchoring method, we first characterized the stability and unfolding pathways of apo-NGAL. Then, the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized. AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites, F122 and F133. Here, NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect. Thus, our work demonstrates high-precision detection of the weak cation-π interaction in NGAL. Electronic Supplementary Material: Supplementary material (additional SDS-PAGE, UV-vis, protein sequences, and more experimental methods) is available in the online version of this article at 10.1007/s12274-021-4065-9.

OaAEP1-mediated PNA-protein conjugation enables erasable imaging of membrane proteins.

We report the use of a protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation demands only an N-terminal GL dipeptide in the target protein and a C-terminal NGL tripeptide in the PNA. We demonstrate the versatility of this approach by attaching a PNA strand to three different proteins. Lastly, we show that erasable imaging of EGFR on HEK293 cell membranes is achieved with DNA origami nanostructures and toehold-mediated strand displacement.

One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy.

Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N 3-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results.

Enzymatic Protein-Protein Conjugation through Internal Site Verified at the Single-Molecule Level.

Enzymes are widely used for protein ligation because of their efficient and site-specific connections under mild conditions. However, many enzymatic ligations are restricted to connections between protein termini while protein-protein conjugation at a specific internal site is limited. Previous work has found that Sortase A (SrtA) conjugates small molecules/peptides to a pilin protein at an internal lysine site via an isopeptide bond. Herein, we apply this noncanonical ligation property of SrtA for protein-protein conjugation at a designed YPKH site. Both a small protein domain, I27, and a large protein, GFP, were ligated at the designed internal site. Moreover, besides characterization by classic methods at the ensemble level, the specific ligation site at the interior YPKH motif is unambiguously verified by atomic force microscopy-based single-molecule force spectroscopy, showing the characteristic unfolding signature at the single-molecule level. Finally, steered molecular dynamics simulations also agreed with the results.

Highly Dynamic Polynuclear Metal Cluster Revealed in a Single Metallothionein Molecule.

Human metallothionein (MT) is a small-size yet efficient metal-binding protein, playing an essential role in metal homeostasis and heavy metal detoxification. MT contains two domains, each forming a polynuclear metal cluster with an exquisite hexatomic ring structure. The apoprotein is intrinsically disordered, which may strongly influence the clusters and the metal-thiolate (M-S) bonds, leading to a highly dynamic structure. However, these features are challenging to identify due to the transient nature of these species. The individual signal from dynamic conformations with different states of the cluster and M-S bond will be averaged and blurred in classic ensemble measurement. To circumvent these problems, we combined a single-molecule approach and multiscale molecular simulations to investigate the rupture mechanism and chemical stability of the metal cluster by a single MT molecule, focusing on the Zn4S11 cluster in the α domain upon unfolding. Unusual multiple unfolding pathways and intermediates are observed for both domains, corresponding to different combinations of M-S bond rupture. None of the pathways is clearly preferred suggesting that unfolding proceeds from the distribution of protein conformational substates with similar M-S bond strengths. Simulations indicate that the metal cluster may rearrange, forming and breaking metal-thiolate bonds even when MT is folded independently of large protein backbone reconfiguration. Thus, a highly dynamic polynuclear metal cluster with multiple conformational states is revealed in MT, responsible for the binding promiscuity and diverse cellular functions of this metal-carrier protein.

Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease.

The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the OaAEP1 ligation, we achieved a stepwise polymerization of the protein on the surface. The produced polyprotein can be verified by protein unfolding scenario using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides an alternative method for polyprotein engineering and immobilization.

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Verification of sortase for protein conjugation by single-molecule force spectroscopy and molecular dynamics simulations.

Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker. As a non-natural linker with several flexible glycine residues, it is unknown whether it affects the properties of the conjugated protein. To verify the use of sortase for protein-protein conjugation, we combined single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to characterize sortase-conjugated polyprotein I27 with three different linkers. We found that the I27 with classic linkers "LPETGGG" and "LPETG" from sortase ligation were of normal stability. However, a protein with a longer artificial linker "LPETGGGG" showed a 15% lower unfolding force. MD simulations revealed that the 4G linker showed a high probability of a closed conformation, in which the adjacent monomer has transient protein-protein interaction. Thus, we verify the use of sortase for protein conjugation, and a longer linker with a higher glycine content should be used with caution.

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level.

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.

Thermodynamically stable whilst kinetically labile coordination bonds lead to strong and tough self-healing polymers.

There is often a trade-off between mechanical properties (modulus and toughness) and dynamic self-healing. Here we report the design and synthesis of a polymer containing thermodynamically stable whilst kinetically labile coordination complex to address this conundrum. The Zn-Hbimcp (Hbimcp = 2,6-bis((imino)methyl)-4-chlorophenol) coordination bond used in this work has a relatively large association constant (2.2 × 1011) but also undergoes fast and reversible intra- and inter-molecular ligand exchange processes. The as-prepared Zn(Hbimcp)2-PDMS polymer is highly stretchable (up to 2400% strain) with a high toughness of 29.3 MJ m-3, and can autonomously self-heal at room temperature. Control experiments showed that the optimal combination of its bond strength and bond dynamics is responsible for the material's mechanical toughness and self-healing property. This molecular design concept points out a promising direction for the preparation of self-healing polymers with excellent mechanical properties. We further show this type of polymer can be potentially used as energy absorbing material.

Simultaneous nutrient removal and biomass/lipid production by Chlorella sp. in seafood processing wastewater.

Microalgae cultivation in wastewater has received increasing attention in recent years due to its many advantages. In this work, microalgae were cultured in seafood processing wastewater (SPW) for algal biomass and lipid production as well as nutrient removal. The biomass yield of Chlorella sp. achieved in the batch cultivation was 896 mg L-1, indicating that SPW contains a certain amount of nutrients which can be used for the growth of microalgae. However, the maximum specific growth rate of Chlorella sp. cultured in SPW throughout the whole cultivation period was only 0.040 d-1, suggesting that the growth of algal cells was inhibited during the culture process. High concentration of unionized ammonia in the SPW was found to be a factor inhibiting the growth of Chlorella sp. Aerated SPW (ASPW) and diluted SPW (DSPW) proved to be better culture media than SPW without pretreatment. The maximum specific growth rates of Chlorella sp. cultured in ASPW and DSPW during the culture interval were 0.156 and 0.091 d-1, respectively. Aeration pretreatment of SPW reduced the amount of toxic unionized ammonia, while most of the nutrients were retained in the wastewater. Therefore, higher biomass productivity (77.7 mg L-1 d-1) and higher lipid productivity (20.4 mg L-1 d-1) of microalgae were achieved in ASPW. Additionally, improved nutrient removal rates from ASPW were also achieved due to the faster growth of microalgae. The average nutrient removal rates in ASPW during the whole cultivation period were 4.98 and 1.91 mg L-1 d-1 for nitrogen and phosphorus, respectively.

[Study on the effect of electric field on the secondary structure of SOD by FTIR spectroscopy].

After the SOD (superoxide dismutase) was treated with different strength of electric field, the effect of electric field on the secondary structure of SOD was studied by FTIR (Fourier-transformation infrared spectroscopy). The results have shown that different electric field strength has different effects on the secondary structure contents of the SOD. As compared with the contrast, the relative contents of alpha-helix and beta-sheet were decreased, while beta-turn content increased. The random coil contents were increased except for the 1.0 kV x cm(-1). The electric treatment tends to transform the alpha-helix and beta-sheet into beta-turn and random coil, and different strength of electric field has a different effect on the transformation. The changes in SOD activity have a direct relationship to the contents of beta-sheet.

Environmental pollution and shipping feasibility of the Nicaragua Canal.

In recent years, the Nicaraguan government's renewed interest in constructing this interoceanic canal has once again aroused widespread concern, particularly in the global shipping industry. The project's immense ecological risks, coupled with the recent expansions of both the Panama Canal and the Suez Canal, have raised questions among scientists and experts about its viability. Whether the Nicaragua Canal is really feasible for international shipping, given its high marine pollution risks, requires the further study. This paper discusses and analyses the feasibility of the Nicaragua Canal in the context of its environmental impact and value as a shipping service. This paper aims to provide an important information reference to inform strategic decision-making among policymakers and stakeholders. Our research results indicate that the environmental complexity, economic costs and safety risks of building a new transoceanic canal are simply too high to justify the project.

[An experimental study of the effect of structure sealing of infrared CO2 sensor on its accuracy].

OBJECTIVE: To study the effect of structure sealing of infrared CO2 sensor on its accuracy. METHOD: Two experiments were designed. The IR CO2 sensor based on infrared principle and no comparing lighting path with only one beam of light were calibrated in one experiment, and in the other experiment the sensors were placed in an environment simulating the practical condition. The temperature, pressure and standard gas were kept same in the two experiments while CO2 concentrations in the dead volume were varied. RESULT: Readings from the sensors varied with the variation of the CO2 concentration in the dead volume of the sensors. CONCLUSION: The structure sealing of the IR CO2 sensor has great influence on its accuracy. A sealed dead volume in the IR CO2 sensor can decrease or eliminate the effect.

[Environmental control and life support system of spacecraft].

Environment control and life support system is a very complicated and important subsystem of manned spacecraft. It is a key technology must be broken through for the realization of manned space flight. Its functions, technical requirements and main technology were reviewed in this paper. Its test verification was also discussed.

Concentrated microalgae cultivation in treated sewage by membrane photobioreactor operated in batch flow mode.

This study investigated the microalgae biomass production and nutrients removal efficiency from treated sewage by newly developed membrane photobioreactor in which Chlorella vulgaris was cultured in batch flow mode. Its performance was compared with conventional photobioreactor. The results show that the volumetric microalgae productivity was 39.93 and 10.36 mg L(-1)d(-1) in membrane photobioreactor and conventional photobioreactor, respectively. The nutrients removal rate in membrane photobioreactor was 4.13 mg N L(-1)d(-1) and 0.43 mg P L(-1)d(-1), which was obviously higher than that in conventional photobioreactor (0.59 mg N L(-1)d(-1) and 0.08 mg P L(-1)d(-1)). The better performance of membrane photobioreactor was due to the submerged membrane module in the reactor which acted as a solid-liquid separator and thereby enabled the reactor to operate with higher supply flow rate of cultivation medium. Moreover, in the outflow stage of the membrane photobioreactor, the microalgae culture liquor in the reactor could be further concentrated.

[Treatment characteristics of saline domestic wastewater by constructed wetland].

A series of experiments were conducted to evaluate the feasibility of using constructed wetland (CW) to remove pollutants from saline domestic sewage. The experimental results indicated that the effects of salinity on the contaminant removal were insignificant when the influent salinities of the CWs were less than or equal to 1.5%. For the influent salinity of 0%, 0.5%, 1.0% and 1.5%, the average removal rates of the CWs were found to be above 68.3% for COD and above 66.1% for NH4(+) -N. When the influent salinity was increased to 2.0%, the individual numbers of microorganisms in the CW reduced obviously. It was similar to the change of the soil enzyme activity in the CW. Then the removal efficiency of the CW also dropped significantly. The average removal rate of COD and NH4(+) -N dropped to 52.9% and 50.3%, respectively. The effects of HRT on the treatment performance of CW under the saline condition of 1.5% were also investigated in this study. And the results showed that nitrogen removal was more greatly affected by HRT than organic matter removal. The NH4(+) -N removal efficiency in CW decreased from 65.1% -78.2% to 47.1% when the HRT of the CW varied from 3-5 d to 2 d.