Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes.

BACKGROUND: The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult. RESULTS: We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. CONCLUSIONS: Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications.

Correction: A super-high brightness and excellent colour quality laser-driven white light source enables miniaturized endoscopy.

Correction for 'A super-high brightness and excellent colour quality laser-driven white light source enables miniaturized endoscopy' by Shuxing Li et al., Mater. Horiz., 2023, 10, 4581-4588, https://doi.org/10.1039/D3MH01170D.

A super-high brightness and excellent colour quality laser-driven white light source enables miniaturized endoscopy.

A laser-driven white light source promises intrinsic advantages for miniaturized endoscopic illumination. However, it remains a great challenge to simultaneously achieve high brightness and excellent colour rendition due to the shortage of highly efficient and thermally robust red-emitting laser phosphor converters. Here, we designed CaAlSiN3:Eu@Al (CASN@Al) converters with neglectable efficiency loss by tightly bonding all-inorganic phosphor films on an aluminium substrate. A layer-by-layer phosphor converter (LuAG/CASN@Al), i.e., stacking a green-emitting Lu3Al5O12:Ce (LuAG) layer on CASN@Al, was constructed to enhance light conversion efficiency and reduce reabsorption loss under blue laser excitation, which thus produces an excellent white light source with a luminous efficacy of 258 lm W-1 and a colour rendering index of 91. A miniaturized endoscopy with a coupling efficiency twice that of the commercial white LEDs was demonstrated by using the laser-driven white light and showed a central illuminance as high as 52 730 lx, more vivid images and long-term reliability.

Black carbon derived from pyrolysis of maize straw and polystyrene microplastics affects soil biodiversity.

Understanding the environmental correlation of microbial community under external stimulation is significant for ecological restoration. However, few studies focused on the response of soil biodiversity induced by black carbon (BC) derived from pyrolysis of straw and microplastics (MPs) due to their widespread existence in natural environment. In this study, polystyrene MPs (PS) and maize straw with different mass ratios were used as raw materials to prepare BC by pyrolysis. The surface morphology, chemical composition and sequential variations of different functional groups of BC were systematically analyzed. The leachate from BC was identified by three-dimensional excitation emission matrice (3D-EEM). The corresponding results showed that yield, value of O/C and N element content of BC decreased with more PS. The changed C content and oxygen-containing functional groups occurred. The order of functional groups of BC formed by co-pyrolysis was: C=C > C-O > C-H > Si-O-Si. The main component of leaching from BC was humic-like and fulvic-like acid. Simultaneously, the input of exogenous BC into soil affected abundance, composition and metabolic pathways of microorganisms. The study helps to understand environmental implication of BC which was pyrolyzed from maize straw and MPs, providing an idea for improving biogeochemical cycle process in soil.

Dissolved organic matter derived from biodegradable microplastic promotes photo-aging of coexisting microplastics and alters microbial metabolism.

Dissolved organic matter (DOM) leaching from biodegradable microplastics (BMPs) and its characteristics and corresponding environmental implication are rarely investigated. In this study, the main component of DOM leachate from the two BMPs (polyadipate/butylene terephthalate (PBAT)/polycaprolactone (PCL)) was verified by using excitation-emission matrix-parallel factor analysis (EEM-PARAFAC). The PBAT-DOM (PBOM) was aromatized and terrestrial. Comparatively, PCL-DOM (PLOM) had low molecular weight. PBOM contained protein-like components while PLOM contained tryptophan and tyrosine components. Interestingly, both PBOM and PLOM could accelerate the decomposition and oxidation of coexisting polystyrene (PS) under light irradiation. Further, the difference in composition and the properties of BMPs-DOM significantly affected its photochemical activity. The high territoriality and protein-like component of PBOM significantly promoted the generation of 1O2 and O2•-, which caused faster disruptions to the backbone of PS. Simultaneously, the microbial community's richness, diversity, and metabolism were obviously improved under the combined pressure of aged PS and BMPs-DOM. This study threw light on the overlooked contribution of DOM derived from BMPs in the aging process of NMPs and their impact on the microbial community and provided a promising strategy for better understanding of combined MPs' fate and environmental risk.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G.

An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5'-AA-3' dinucleotides, and to a lesser extent, 5'-GA-3' dinucleotides. APOBEC3G binds to the common 3'A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5'A or 5'G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.

A general method for rapid synthesis of refractory carbides by low-pressure carbothermal shock reduction.

Refractory carbides are attractive candidates for support materials in heterogeneous catalysis because of their high thermal, chemical, and mechanical stability. However, the industrial applications of refractory carbides, especially silicon carbide (SiC), are greatly hampered by their low surface area and harsh synthetic conditions, typically have a very limited surface area (<200 m2 g-1), and are prepared in a high-temperature environment (>1,400 °C) that lasts for several or even tens of hours. Based on Le Chatelier's principle, we theoretically proposed and experimentally verified that a low-pressure carbothermal reduction (CR) strategy was capable of synthesizing high-surface area SiC (569.9 m2 g-1) at a lower temperature and a faster rate (∼1,300 °C, 50 Pa, 30 s). Such high-surface area SiC possesses excellent thermal stability and antioxidant capacity since it maintained stability under a water-saturated airflow at 650 °C for 100 h. Furthermore, we demonstrated the feasibility of our strategy for scale-up production of high-surface area SiC (460.6 m2 g-1), with a yield larger than 12 g in one experiment, by virtue of an industrial viable vacuum sintering furnace. Importantly, our strategy is  also applicable to the rapid synthesis of refractory metal carbides (NbC, Mo2C, TaC, WC) and even their emerging high-entropy carbides (VNbMoTaWC5, TiVNbTaWC5). Therefore, our low-pressure CR method provides an alternative strategy, not merely limited to temperature and time items, to regulate the synthesis and facilitate the upcoming industrial applications of carbide-based advanced functional materials.

New insight into transformation of tetracycline in presence of Mn(II): Oxidation versus photolysis.

Tetracycline (TC) and Mn(II) is a common antibiotic and metal ion respectively. Nevertheless, literatures involving in the effects of Mn(II) on TC transformation are still insufficient. In this study, the kinetic experiment, spectral analysis, complexation experiment and electrochemical analysis, theoretical calculation and products detection were carried out to probe into oxidation and photolysis of TC with Mn(II). Mn(II) greatly accelerated TC oxidation, preferably tending to complex with TC at O10 - O12 or O2 - O3 site. There were a TC-Mn(II)/TC-Mn(III) redox couple and electron transfer process. Conversely, Mn(II) inhibited photolysis of TC. The photolysis of excited TC could compete with energy dissipation reactions. The electron transfer and complexation reaction easily made excited TC energy transfer, thus slowing down photolysis process. During the TC transformation, the intensity of functional groups was significantly decreased. Simultaneously, the degradation pathways mainly included eight reactions. It is a very interesting and probably overlooked phenomenon, which identifies new transformation of TC with Mn(II). This study helps to further understand fate and environmental behavior of antibiotics and metal ion.

The aging behavior of polyvinyl chloride microplastics promoted by UV-activated persulfate process.

Microplastics (MPs) experienced different aging processes in environment. Literatures about effect of artificially-accelerated aging on MPs behavior are still insufficient. The accelerated process induced by ultraviolet(UV)-activated persulfate (PS) is a promising technology for obtaining different aged MPs to understand long-term aging behavior. In the work, the aging behavior of polyvinyl chloride (PVC) accelerated by UV/PS system were investigated. It exhibited a dechlorination with 58.495 ± 6.090 mg/L Cl- release after 35 h UV-activated PS (0.01 M) process. The treatment led to significant alternations on surface morphology and chemical feature of PVC. The crystallinity was increased, and average size was reduced from 154.11 µm to 119.28 µm with aging time. Subsequently, many smaller size particles were produced. Furthermore, the process induced the breaking of backbone. Simultaneously, more oxygen-containing functional groups were identified. The oxidation reaction accelerated by sulfate radical (SO4•-) and reactive oxygen species (ROS) was predominant, which immensely promoted aging process. Sustained high levels of free radical contributed to production of alcohols and carboxylic acids short chain organics. The study explored aging behavior of PVC accelerated by UV/PS system, which could be helpful for understanding environmental behavior and providing further information to assess potential risks of MPs.

Characteristics and removal efficiency of microplastics in sewage treatment plant of Xi'an City, northwest China.

Wastewater treatment plants (WWTP) are the important source of microplastics (MPs) in the environment, but the distribution and property of MPs in WWTPs are not fully understood. The study systematically investigated the transportation, characteristics and fate of MPs in both sewage and sludge of WWTP in Xi'an, a western region of China to complement the research on the fate of MPs in WWTP in inland China. The consequence was abundance, size, shape, color and type of MPs in sewage and sludge of different treatment processes were analyzed and compared by the metallurgical microscope and Fourier Transform Infrared (FTIR) spectrometer. Results showed that the MP abundance decreased from 288.5 n/L of the influent to 22.9 n/L of the effluent, corresponding to the removal rate of 92.1%. Among them, the secondary treatment has the best removal effect, with a removal rate of 72.1%. With the treatment process, the distribution of MPs with different characteristics is constantly changing, which leads to differences in the distribution of MPs in sludge and sewage. It is speculated from the characteristics of MPs that domestic sewage is still the main source of microplastics in Beishiqiao WWTP. Restricting the use of plastic products in daily life or finding alternatives is of great significance to the construction of MPs pollution.

The structure of APOBEC1 and insights into its RNA and DNA substrate selectivity.

APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded ß-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates.

A solid-state colorimetric fluorescence Pb2+-sensing scheme: mechanically-driven CsPbBr3 nanocrystallization in glass.

Highly toxic Pb2+ poses a great threat to the health of human beings and ecosystems, urgently calling for an efficient Pb2+ detection method. Herein, we demonstrated a brand-new solid-state fluorescence Pb2+-sensing scheme based on a type of Pb2+-responsive borate glass powder that is able to precipitate CsPbBr3 nanocrystals on the glass surface upon grinding with Pb2+ sources, following a mechanically driven glass crystallization mechanism. Pb2+ sensing is achieved via the Pb2+ concentration-dependent green emission of CsPbBr3 as an indicator signal and independent red emission of Eu3+ as a reference signal. Under UV light irradiation, the obvious emissive color variation from red to green as Pb2+ concentration varies enables the intuitive Pb2+-sensing by naked eyes. With the aid of a spectrometer and smartphone, Pb2+ concentration can be quantitatively determined with the detection limit as low as ∼70 ppm and ∼400 ppm, respectively. The semi-quantitative Pb2+ detection is also possible by comparing the emissive color with the calibrated color card. Hopefully, the proposed solid-state fluorescence Pb2+-sensing strategy with high selectivity can be used for portable and quick Pb2+ analysis in daily life.

Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G.

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.

The Methylation Patterns and Transcriptional Responses to Chilling Stress at the Seedling Stage in Rice.

Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2-3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)-COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

Warm White Light with a High Color-Rendering Index from a Single Gd3Al4GaO12:Ce3+ Transparent Ceramic for High-Power LEDs and LDs.

Transparent ceramics (TCs) are promising for high-power (hp) white light-emitting diode (WLED) and laser diode (LD) lighting. However, comfortable warm white light has not been achieved only using a single TC in hp-WLEDs/LDs. Herein, highly transparent Gd3Al4GaO12:Ce3+ (GAGG:Ce3+) TCs (transmittance, T = 55.9-80.2%) were prepared via a solid-state reaction. Ce3+ as a doped activator center in grains plays a positive role in luminescence based on the microstructural investigations by scanning electron microscopy and the cathodoluminescence system. T decreases upon increasing the Ce3+ concentration and/or the ceramic thickness, whereas the luminous efficacy of hp-WLEDs/LDs goes up. For blue hp-LEDs driven at 350 mA or LDs of 2 W, warm white light with a low correlated-color temperature of ∼3000 K was achieved by a single GAGG:Ce3+ TC, benefiting from its broad emission band (full width at half maximum, FWHM = 133-137 nm) and abundant red components (peaking at about 568-574 nm). The color-rendering index of hp-WLEDs reaches 78.9. These results are much better than the performance of the traditional Y3Al5O12:Ce3+ (YAG:Ce3+) TC, indicating that GAGG:Ce3+ TCs are promising color converters for hp-WLEDs/LDs with a comfortable warm white light.

Unique Color Converter Architecture Enabling Phosphor-in-Glass (PiG) Films Suitable for High-Power and High-Luminance Laser-Driven White Lighting.

As a next-generation high-power lighting technology, laser lighting has attracted great attention in high-luminance applications. However, thermally robust and highly efficient color converters suitable for high-quality laser lighting are scarce. Despite its versatility, the phosphor-in-glass (PiG) has been seldom applied in laser lighting because of its low thermal conductivity. In this work, we develop a unique architecture in which a phosphor-in-glass (PiG) film was directly sintered on a high thermally conductive sapphire substrate coated by one-dimensional photonic crystals. The designed color converter with the composite architecture exhibits a high internal quantum efficiency close to that of the original phosphor powders and an excellent packaging efficiency up to 90%. Furthermore, the PiG film can even be survived under the 11.2 W mm-2 blue laser excitation. Combining blue laser diodes with the YAG-PiG-on-sapphire plate, a uniform white light with a high luminance of 845 Mcd m-2(luminous flux: 1839 lm), luminous efficacy of 210 lm W-1, and correlated color temperature of 6504 K was obtained. A high color rendering index of 74 was attained by adding a robust orange or red phosphor layer to the architecture. These outstanding properties meet the standards of vehicle regulations, enabling the PiG films with the composite architecture to be applied in automotive lighting or other high-power and high-luminance laser lighting.

Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H.

APOBEC3H (A3H) is a member of the APOBEC3 subfamily of DNA cytosine deaminases that are important for innate immune defense and have been implicated in cancer biogenesis. To understand the structural basis for A3H biochemical function, we determined a high-resolution structure of human A3H and performed extensive biochemical analysis. The 2.49 Å crystal structure reveals a uniquely long C-terminal helix 6 (h6), a disrupted ß5 strand of the canonical five-stranded ß-sheet core, and a long loop 1 around the Zn-active center. Mutation of a loop 7 residue, W115, disrupted the RNA-mediated dimerization of A3H yielding an RNA-free monomeric form that still possessed nucleic acid binding and deaminase activity. A3H expressed in HEK293T cells showed RNA dependent HMW complex formation and RNase A-dependent deaminase activity. A3H has a highly positively charged surface surrounding the Zn-active center, and multiple positively charged residues within this charged surface play an important role in the RNA-mediated HMW formation and deaminase inhibition. Furthermore, these positively charged residues affect subcellular localization of A3H between the nucleus and cytosol. Finally, we have identified multiple residues of loop 1 and 7 that contribute to the overall deaminase activity and the methylcytosine selectivity.

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA.

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

Effect on intensity of treadmill running on learning, memory and expressions of cell cycle-related proteins in rats with cerebral ischemia.

OBJECTIVE: We discussed the intensity of treadmill running on learning, memory and expression of cell cycle-related proteins in rats with cerebral ischemia. METHOD: Eighty healthy male SD rats were randomly divided into normal group, model group, intensity I group and intensity II group, with 20 rats in each group. The four-vessel occlusion method of Pulsinelli (4-VO) was used to induce global cerebral ischemia. Brain neuronal morphology was observed by hematoxylin-eosin (HE) staining at 3h, 6h, 24h and 48h after modeling, respectively. Hippocampal expressions of cyclin A and cyclin E were detected by immunohistochemistry. At 48h after modeling, the learning and memory performance of rats was tested by water maze experiment. RESULT: Compared with the normal group, the other three groups had a significant reduction in surviving neurons, prolonging of escape latency and decreased number of passes over the former position of the platform (P<0.05). The number of surviving neurons and the number of passes over the former position of the platform were obviously lower in the model group than in intensity I group (P<0.05), but significantly higher compared with intensity II group (P<0.05). Escape latency of the model group was obviously prolonged as compared with intensity I group (P<0.05), but much shorter than that of intensity II group (P<0.05). Compared with the normal group, the expressions of cyclin A and cyclin E were significantly upregulated at different time points after modeling (P<0.05). The expression of the model group was higher than that of intensity I group, but lower than that of intensity II group (P<0.05). CONCLUSION: Moderate intensity of treadmill running can help protect brain neurons and improve learning and memory performance of rats with global cerebral ischemia. But high intensity of treadmill running has a negative impact, possibly through the regulation of cell cycle-related proteins in ischemia/reperfusion injury.