A wafer scale thin film of ultra-small Sc2O3 nanocrystals on a 2D COF with high rigidity.

Scandium oxide (Sc2O3) has a wide range of applications in metallurgy, chemical industry, electronics and many other high-tech fields. However, most Sc2O3 materials exist in the powder or bulk form, while nanostructured Sc2O3 has rarely been reported as there is a lack of a common method to control its dimensionality, hindering the understanding of new properties and potential applications of nano-Sc2O3 materials. In this paper, we establish a procedure to synthesize a two-dimensional (2D) Sc2O3-covalent organic framework (COF) composite film where the crystal size of Sc2O3 domains is as small as ∼3 nm. The composite film is prepared by a Schiff base condensation reaction at the sharp n-pentane/water interface using a combination of surfactant-monolayer-assisted interfacial synthesis and laminar assembly polymerization methods. Then the conditions of nucleation and uniform film formation of the 2D Sc2O3/COF are explored further. Meanwhile, an atomic force microscopy indentation test shows that the material has a high Young's modulus of 89.1 ± 3.8 GPa, which is much higher than those of the majority of reported 2D polymer materials. We further extended this synthesis method to the preparation of Yb2O3 (ytterbium oxide) and/or Er2O3 (erbium oxide)-incorporated 2D COF composite films, verifying the universality of this strategy. This work provides an opportunity to vary the dimensionality of many kinds of metal oxides and explore the potential applications of low-dimensional Sc2O3 materials.

Exploring the molecular mechanism of Chlorella vulgaris in response to androstenedione exposure based on genes continuously up-regulated in transcription analysis.

Androstenedione (ADSD) is one of the widely detected androgens in diverse aquatic environments. However, there were few reports on the molecular mechanism of Chlorella vulgaris exposure to ADSD. In our previous research, we have investigated the genes associated with chlorophyll metabolism in Chlorella vulgaris response to ADSD. In this study, we focus on continuously up-regulated genes to explore the mechanism underlying Chlorella vulgaris resistance to ADSD toxicity. Chlorella vulgaris was exposed to ADSD with five concentration gradients. The continuously up-regulated genes were enriched by Series Test of Cluster (STC) analysis and verified by qRT-PCR. Microalgae Super Oxidase Dimutase (SOD) and Microalgae Malonic dialdehyde (MDA), two indicators of oxidative stress, were determined by ELISA after exposure to ADSD. The results showed that ADSD can stimulate the production of extracellular polymeric substances (EPS) and lead to enlargement in the cell body of Chlorella vulgaris. In addition, steroid biosynthesis and oxidoreductase activity processes were consistently up-regulated upon exposure to ADSD. In conclusion, our study highlighted the crucial role of phenotypic modification, hormone synthesis, and redox mechanisms in protecting Chlorella vulgaris cells from the harmful effects of ADSD contamination.

Machine Learning in Bioelectrocatalysis.

At present, the global energy crisis and environmental pollution coexist, and the demand for sustainable clean energy has been highly concerned. Bioelectrocatalysis that combines the benefits of biocatalysis and electrocatalysis produces high-value chemicals, clean biofuel, and biodegradable new materials. It has been applied in biosensors, biofuel cells, and bioelectrosynthesis. However, there are certain flaws in the application process of bioelectrocatalysis, such as low accuracy/efficiency, poor stability, and limited experimental conditions. These issues can possibly be solved using machine learning (ML) in recent reports although the combination of them is still not mature. To summarize the progress of ML in bioelectrocatalysis, this paper first introduces the modeling process of ML, then focuses on the reports of ML in bioelectrocatalysis, and ultimately makes a summary and outlook about current issues and future directions. It is believed that there is plenty of scope for this interdisciplinary research direction.

The material-microorganism interface in microbial hybrid electrocatalysis systems.

This review presents a comprehensive summary of the material-microorganism interface in microbial hybrid electrocatalysis systems. Microbial hybrid electrocatalysis has been developed to combine the advantages of inorganic electrocatalysis and microbial catalysis. However, electron transfer at the interfaces between microorganisms and materials is a very critical issue that affects the efficiency of the system. Therefore, this review focuses on the electron transfer at the material-microorganism interface and the strategies for building efficient microorganism and material interfaces. We begin with a brief introduction of the electron transfer mechanism in both the bioanode and biocathode of bioelectrochemical systems to understand the material-microorganism interface. Next, we summarise the strategies for constructing efficient material-microorganism interfaces including material design and modification and bacterial engineering. We also discuss emerging studies on the bio-inorganic hybrid electrocatalysis system. Understanding the interface between electrode/active materials and the microorganisms, especially the electron transfer processes, could help to drive the evolution of material-microorganism hybrid electrocatalysis systems towards maturity.

Cation affinity purification of histidine-tagged proteins.

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1-3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. KEY POINTS: • Histidine-tagged proteins can be precipitated by low-concentrations common cations • A novel nonchromatographic protein purification method was developed • Purified protein can be obtained in pellet form by only three centrifugations.

Effect of SpyTag/SpyCatcher cyclization on stability and refolding of green fluorescent protein.

To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its derivative to act as a linear structure control (L-SRGFP). SRGFP and L-SRGFP showed similar fluorescence characteristics to the wild-type GFP, while compared with GFP and L-SRGFP, the thermal stability and denaturation resistance of SRGFP were improved. The refolding efficiencies of these three denatured proteins were investigated under different pH, temperature and initial protein concentration conditions, and it was found that SRGFP was superior to GFP and L-SRGFP in terms of refolding yield and refolding speed. In the pH range of 8.0-8.5, SRGFP could basically recover all fluorescence, while GFP and L-SRGFP recovered only about 87.52% and 88.58%. When refolded at a high temperature (37 °C), SRGFP still recovered 85.27% of the fluorescence, whereas GFP and L-SRGFP recovered only around 69.43% and 68.45%. At a high initial protein concentration (5 mg/mL), the refolding yield of SRGFP was about 15% higher than that of both GFP and L-SRGFP. These results suggest that the introduction of SpyRing structure (head-to-tail cyclization via SpyTag and SpyCatcher) improved the protein's stability and facilitated the refolding of denatured protein.

Encapsulation of Nitrilase in Zeolitic Imidazolate Framework-90 to Improve Its Stability and Reusability.

In this study, nitrilase (Nit) was immobilized in zeolite imidazole framework-90 (ZIF-90) by one-pot biomimetic mineralization strategy. The structure, morphology and functional groups of ZIF-90 and immobilized enzyme Nit@ZIF-90 were characterized by scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). Circular dichroism (CD) proved that the immobilized method of encapsulation in ZIF-90 could effectively maintain the intrinsic conformation of Nit. Meanwhile, the stability and reusability of Nit@ZIF-90 were systematically evaluated. Compared with the free enzyme, the thermal, pH and organic solvents stability of Nit@ZIF-90 were significantly increased. Further, Nit@ZIF-90 exhibited better reusability during the hydrolysis of acrylonitrile and retained 48.34% of the initial activity after 10 cycles. Besides, the Ni@ZIF-90 had preferable storage stability, which showed a high degree of residual activity (more than 64 %) after storage at 4 °C for 7 d. The improved stability and reusability of the Nit@ZIF-90 implied that it could be used as a potential effective biocatalyst for hydrolysis of nitrile compounds in industrial application.

SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli.

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.

One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry.

Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes.

During enzyme immobilization, enzyme activity and protein distribution are affected by various factors such as enzyme load, temperature, and pH. In general, two types of protein distribution patterns (heterogeneous or homogeneous) are observed inside a porous carrier, owing to differences in preparation parameters. During the immobilization of a fusion protein (CCApH) of cephalosporin C acylase (CCA) and pHluorin (a pH-sensitive mutant of green fluorescent protein), different shaking speeds induced obvious differences in protein distribution on an epoxy carrier, LX-1000EPC. Enzyme immobilization with a homogeneous distribution pattern was observed at a low shaking speed (120 rpm) with an operational stability of 10 batches at 37°C. The operational stability of an immobilisate with heterogeneous protein distribution prepared at a high shaking speed (200 rpm) was six batches. Given the pH-sensitive characteristics of pHluorin in the fusion protein, the intraparticle pH of CCApH immobilisates during catalysis was monitored using confocal laser scanning microscopy. The microenvironmental pH of the immobilisate with heterogeneous protein distribution sharply decreased by about 2 units; this decrease in the pH may be detrimental to the life-span of immobilized CCA. Thus, this work demonstrates the good operational stability of pH-sensitive proton-forming immobilized enzymes with homogeneous protein distribution.

Differentially proteomic analysis of the hemocytes against Aeromonas hydrophila infection in oriental river prawn Macrobrachium nipponense by iTRAQ approach.

As the direct executors of biological function, the expression level of proteins in host will reveal the molecular mechanisms regulating bacteria infection more directly. In the present study, the differential proteomes of Macrobrachium nipponense hemocytes response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography electrospray ionization tandem mass spectrometry. The hemocyte proteins from the unchallenged and A. hydrophila challenged prawn, M. nipponense, at 12, 24 and 36 h post infection were compared. From this, a total of 3372 proteins were identified and 1014 proteins were considered differentially expressed, of which 117 common differentially expressed proteins were indicated between the time points. Hierarchical clustering, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the general characterization of overall enriched proteins. Cytoskeletal proteins including myosin heavy chain, myosin regulatory light chain, actin, tubulin alpha/beta chain, troponin I and troponin T as well as antioxidant enzymes such as catalase and cytosolic MnSOD were found significantly up-regulated in hemocytes, indicating that the phagocytosis process and ROS system were induced after challenge with A. hydrophila. And other proteins such as integrin ß, innexin inx2-like and heat shock protein 60 also participate in prawn immune response against bacteria. Parallel reaction monitoring analyses were carried out for validation of the expression levels of differentially expressed proteins, which indicated high reliability of the proteomic results. This is the first report on proteome of M. nipponense hemocytes against A. hydrophila infection, which contributes to better understanding on the molecular mechanisms of prawns.

High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli.

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

SpyTag/SpyCatcher cyclization enhances the thermostability and organic solvent tolerance of L-phenylalanine aldolase.

OBJECTIVES: To improve the thermostability and organic solvent tolerance of L-phenylserine aldolase, the in vivo SpyTag/SpyCatcher cyclization strategy was applied in this work. RESULTS: The in vivo cyclization of L-phenylserine aldolase was achieved by fusing the tags of SpyCatcher and SpyTag to the N- and C-termini of the enzyme, respectively. The kcat values and the circular dichroism spectra of the linear and cyclized LPAs are very similar, indicating that the cyclized LPA can be folded appropriately like the wild type. The cyclized enzyme has better thermostability and organic solvent tolerance than does the wild type. The half-life of L-phenylserine aldolase after cyclization was increased by 8.3 times at 70 °C, and the T50 also increased from 56.8 to 67.1 °C. The cyclized enzyme showed a remarkably higher tolerance to organic solvents (e.g., methanol, ethanol and acetone). CONCLUSIONS: These results suggest that the in vivo cyclization using SpyTag/SpyCatcher is an effective strategy to improve the stability of enzymes, which potentially could be applied in industrial bioconversion.

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors.

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.

Quantification Challenges for Atom Probe Tomography of Hydrogen and Deuterium in Zircaloy-4.

Analysis and understanding of the role of hydrogen in metals is a significant challenge for the future of materials science, and this is a clear objective of recent work in the atom probe tomography (APT) community. Isotopic marking by deuteration has often been proposed as the preferred route to enable quantification of hydrogen by APT. Zircaloy-4 was charged electrochemically with hydrogen and deuterium under the same conditions to form large hydrides and deuterides. Our results from a Zr hydride and a Zr deuteride highlight the challenges associated with accurate quantification of hydrogen and deuterium, in particular associated with the overlap of peaks at a low mass-to-charge ratio and of hydrogen/deuterium containing molecular ions. We discuss possible ways to ensure that appropriate information is extracted from APT analysis of hydrogen in zirconium alloy systems that are important for nuclear power applications.

Ti and its alloys as examples of cryogenic focused ion beam milling of environmentally-sensitive materials.

Hydrogen pick-up leading to hydride formation is often observed in commercially pure Ti (CP-Ti) and Ti-based alloys prepared for microscopic observation by conventional methods, such as electro-polishing and room temperature focused ion beam (FIB) milling. Here, we demonstrate that cryogenic FIB milling can effectively prevent undesired hydrogen pick-up. Specimens of CP-Ti and a Ti dual-phase alloy (Ti-6Al-2Sn-4Zr-6Mo, Ti6246, in wt.%) were prepared using a xenon-plasma FIB microscope equipped with a cryogenic stage reaching -135 °C. Transmission electron microscopy (TEM), selected area electron diffraction, and scanning TEM indicated no hydride formation in cryo-milled CP-Ti lamellae. Atom probe tomography further demonstrated that cryo-FIB significantly reduces hydrogen levels within the Ti6246 matrix compared with conventional methods. Supported by molecular dynamics simulations, we show that significantly lowering the thermal activation for H diffusion inhibits undesired environmental hydrogen pick-up during preparation and prevents pre-charged hydrogen from diffusing out of the sample, allowing for hydrogen embrittlement mechanisms of Ti-based alloys to be investigated at the nanoscale.

The Laplace Project: An integrated suite for preparing and transferring atom probe samples under cryogenic and UHV conditions.

We present sample transfer instrumentation and integrated protocols for the preparation and atom probe characterization of environmentally-sensitive materials. Ultra-high vacuum cryogenic suitcases allow specimen transfer between preparation, processing and several imaging platforms without exposure to atmospheric contamination. For expedient transfers, we installed a fast-docking station equipped with a cryogenic pump upon three systems; two atom probes, a scanning electron microscope / Xe-plasma focused ion beam and a N2-atmosphere glovebox. We also installed a plasma FIB with a solid-state cooling stage to reduce beam damage and contamination, through reducing chemical activity and with the cryogenic components as passive cryogenic traps. We demonstrate the efficacy of the new laboratory protocols by the successful preparation and transfer of two highly contamination- and temperature-sensitive samples-water and ice. Analysing pure magnesium atom probe data, we show that surface oxidation can be effectively suppressed using an entirely cryogenic protocol (during specimen preparation and during transfer). Starting with the cryogenically-cooled plasma FIB, we also prepared and transferred frozen ice samples while avoiding significant melting or sublimation, suggesting that we may be able to measure the nanostructure of other normally-liquid or soft materials. Isolated cryogenic protocols within the N2 glove box demonstrate the absence of ice condensation suggesting that environmental control can commence from fabrication until atom probe analysis.

Comparative proteomic analysis between two haemocyte subpopulations in shrimp Fenneropenaeus chinensis.

In our previous work, granulocytes and hyalinocytes were successfully separated by immunomagnetic bead (IMB) method using monoclonal antibodies (mAbs) against granulocytes of shrimp (Fenneropenaeus chinensis). In order to elucidate the proteomic differentiation between granulocytes and hyalinocytes, in this paper, the differentially expressed proteins were analyzed between non-fixed/un-permeabilized (NFP) haemocytes and fixed/permeabilized (FP) haemocytes using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS). Then the FP haemocytes were separated into two haemocyte subpopulations using IMB method, and the comparative proteome between granulocytes and hyalinocytes was investigated. The results showed that 10 differentially expressed protein spots were detected and identified as 4 proteins in the NFP haemocytes. Twenty one differentially expressed proteins were successfully identified between granulocytes and hyalinocytes, which include 4 unique expressed proteins in granulocytes, 4 significantly highly expressed proteins in granulocytes, and 13 significantly high expressed proteins in hyalinocytes. According to Gene Ontology annotation, the identified proteins between granulocytes and hyalinocytes were classified into six categories, including binding proteins, proteins involved in catalytic activity, enzyme regulator activity, structural molecule activity, translation regulator activity, and ungrouped proteins. Furthermore, quantitative PCR confirmed that the trend of transcription levels of three selected genes were consistent with the proteomic data from 2-DE. The results may lead to better understanding of the functions of haemocyte subpopulations.

Separation of haemocyte subpopulations in shrimp Fenneropenaeus chinensis by immunomagnetic bead using monoclonal antibody against granulocytes.

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp (Fenneropenaeus chinensis) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F. chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60-0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12-0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis.