Enhanced molecular stability of Apxâ ¡ antigen during secretion in Corynebacterium glutamicum by rational design.

ApxⅡ is a vaccine antigen used to protect against porcine contagious pleuropneumonia, which is a significant threat to the pig industry. Here, we aimed to improve the proteolytic degradation stability of ApxⅡ during its secretion by establishing a complete screening process of stable variants through bioinformatics and site-directed mutagenesis. We employed a combination of semi-rational and rational design strategies to create 34 single-point variants of ApxⅡ. Among them, R114E and T115D variants exhibited better stability without compromising antigen activity. Furthermore, we constructed a multi-site variant, R114E/T115D, which demonstrated the best stability, activity, and yield. Protein stability and molecular dynamic analysis indicated that the greater solubility and lower structural expansion coefficient might explain the increased stability of R114E/T115D. Additionally, site T115 was identified as a key point of truncated ApxⅡ stability. The R114E/T115D variant, with its proven stability and intact antigenic activity, holds promising prospects for industrial-scale applications in the prevention of porcine contagious pleuropneumonia.

Microbial conversion of ethanol to high-value products: progress and challenges.

Industrial biotechnology heavily relies on the microbial conversion of carbohydrate substrates derived from sugar- or starch-rich crops. This dependency poses significant challenges in the face of a rising population and food scarcity. Consequently, exploring renewable, non-competing carbon sources for sustainable bioprocessing becomes increasingly important. Ethanol, a key C2 feedstock, presents a promising alternative, especially for producing acetyl-CoA derivatives. In this review, we offer an in-depth analysis of ethanol's potential as an alternative carbon source, summarizing its distinctive characteristics when utilized by microbes, microbial ethanol metabolism pathway, and microbial responses and tolerance mechanisms to ethanol stress. We provide an update on recent progress in ethanol-based biomanufacturing and ethanol biosynthesis, discuss current challenges, and outline potential research directions to guide future advancements in this field. The insights presented here could serve as valuable theoretical support for researchers and industry professionals seeking to harness ethanol's potential for the production of high-value products.

CRLNet: A Multimodal Peach Detection Network Based on Cooperative Asymptotic Enhancement and the Fusion of Granularity Refinement.

Accurate peach detection is essential for automated agronomic management, such as mechanical peach harvesting. However, ubiquitous occlusion makes identifying peaches from complex backgrounds extremely challenging. In addition, it is difficult to capture fine-grained peach features from a single RGB image, which can suffer from light and noise in scenarios with dense small target clusters and extreme light. To solve these problems, this study proposes a multimodal detector, called CRLNet, based on RGB and depth images. First, YOLOv9 was extended to design a backbone network that can extract RGB and depth features in parallel from an image. Second, to address the problem of information fusion bias, the Rough-Fine Hybrid Attention Fusion Module (RFAM) was designed to combine the advantageous information of different modes while suppressing the hollow noise at the edge of the peach. Finally, a Transformer-based Local-Global Joint Enhancement Module (LGEM) was developed to jointly enhance the local and global features of peaches using information from different modalities in order to enhance the percentage of information about the target peaches and remove the interference of redundant background information. CRLNet was trained on the Peach dataset and evaluated against other state-of-the-art methods; the model achieved an mAP50 of 97.1%. In addition, CRLNet also achieved an mAP50 of 92.4% in generalized experiments, validating its strong generalization capability. These results provide valuable insights for peach and other outdoor fruit multimodal detection.

Origins of Diastereoselectivity in the Addition of Enoxysilanes to Vinyl Diazonium Salts.

The addition of enoxysilanes to vinyl diazonium ions occurs with varying levels of diastereoselectivity. To understand the origins of the stereoselectivity, we studied these transformations using density functional theory (DFT) calculations. The selectivity stems from a stabilizing cation-π interaction that orients the nucleophile and the diazonium ion.

Organocatalyzed Photoelectrochemistry for the Generation of Acyl and Phosphoryl Radicals through Hydrogen Atom-Transfer Process.

An organocatalyzed photoelectrochemical method for the generation of acyl and phosphoryl radicals from formamides, aldehydes, and phosphine oxides has been developed. This protocol utilizes 9,10-phenanthrenequinone (PQ) as both a molecular catalyst and a hydrogen atom-transfer (HAT) reagent, eliminating the requirement for external metal-based reagents, HAT reagents, and oxidants. The generated acyl radicals can be applied to a range of radical-mediated transformation reactions, including C-H carbamoylation of heteroarenes, intermolecular tandem radical cyclization of CF3-substituted N-arylacrylamides, as well as intramolecular cyclization reactions. The use of acyl radicals in these transformations offers an efficient and sustainable approach to accessing structurally diverse carbonyl compounds.

HFIP-Promoted Divergent Cycloadditions of Difluoroenoxysilanes with 2-Indolylmethanols: Synthesis of Fluoro 2H-Pyrano[3,4-b]indoles and gem-Difluoro Cyclopenta[b]indoles.

An oxa-6π-electrocyclization of difluoroenoxysilanes with diaryl 2-indolylmethanols has been developed. In addition, a rarely reported C3-nucleophilic [3+2] cycloaddition of difluoroenoxysilanes with dialkyl 2-indolylmethanols has been disclosed. This divergent cycloaddition approach affording readily available difluoroenoxysilanes as three-atom and C2 synthons provides rapid access to fluoro 2H-pyrano[3,4-b]indoles and gem-difluoro cyclopenta[b]indoles in good to excellent yields with good functional group tolerance. The metal-free and mild conditions using only HFIP as the solvent without any external acid catalyst illuminate practical and environmentally benign advantages.

Synergistic synthesis of gold nanoflowers as upconversion near-infrared nanoprobe energy acceptor and recognition unit for improved hydrogen sulfide sensing.

A highly sensitive and selective upconversion near-infrared (NIR) fluorescence and colorimetric dual readout hydrogen sulfide (H2S) nanoprobe was constructed based on the excellent NIR fluorescence emission performance of upconversion nanomaterials (UCNPs), the specific recognition effect of synergistically synthesized gold nanoflowers (trypsin-stabled AuNFs (Try-AuNFs)) and the effective NIR fluorescence quenching capability. In this assay, the sensing strategy included three processes. First of all, the synthesized UCNPs can emit 803 nm NIR fluorescence when they were excited by 980 nm excitation light. Secondly, as a result of the principle of fluorescence resonance energy transfer (FRET), Try-AuNFs can effectively quench the NIR fluorescence of UCNPs at 803 nm, which can effectively improve the signal-to-background ratio of nanoprobes, thereby improving the sensitivity of the probes. Thirdly, in the presence of H2S, the Try protective layer on the surface of Try-AuNFs was specifically penetrated, which will subsequently cleave Try-AuNFs via the strong S-Au bond. As such, the NIR fluorescence of UCNPs will be restored, achieving high selectivity and sensitivity detection of H2S. Under optimized conditions, the linear response range of H2S was 0.1-300 µM, and the detection limit was 53 nM. It is worth noting that the Try on the surface of Try-AuNFs via the synergistic effect can increase the steric hindrance of the probe, and this can effectively prevent the interaction between the probe with biothiols (cysteine (Cys), homocysteine (Hcy)) and other natural amino acids (non-thiol-containing) with resultant in the high selectivity regarding the detection of H2S in human serum, which is unlikely to be achieved by AuNFs synthesized by the gold seed method (Se-AuNFs). This work not only provided a new type of UCNPs fluorescence quencher and recognition unit, but also exemplified that the use of the physical properties (steric hindrance) of protein ligands on the surface of nanoflowers can improve the specificity of the probe. This will provide new ideas for the design of other nanoprobes.

La(OTf)3-Catalyzed [3+2] Cycloaddition Reactions for the Synthesis of Benzo[d]oxazoles/Benzofurans.

The La(OTf)3-catalyzed [3+2] cycloaddition reactions for the synthesis of benzo[d]oxazoles/benzofurans via quinones and 1,2-di-tert-butyl-3-(cyanimino)diaziridine (1,3-di-tert-butyl-2-cyanoguanidine)/vinyl azides have been explored. A series of 5-hydroxybenzofuran-4-carboxylic acid derivatives and 5-hydroxybenzo[d]oxazole-4-carboxylic acid derivatives were conveniently obtained with high yields and good stereoselectivities, which could be used for further transformations to valuable compounds.

Synthesis of Fluorine-Containing Multisubstituted Oxa-Spiro[4,5]cyclohexadienones via a Fluorinated Alcohol-Catalyzed One-Pot Sequential Cascade Strategy.

In recent years, the application of fluorinated alcohols as solvents, cosolvents, or additives has become important in modern organic synthesis. However, their potential as efficient catalysts in organic synthesis has not been well-explored. In this article, we report on the development of a one-pot sequential cascade reaction of p-quinone methides with difluoroenoxysilanes using hexafluoroisopropanol as catalyst. This reaction allows for the preparation of fluorinated multisubstituted oxa-spiro[4,5]cyclohexadienones. By using 50 mol % 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), the reaction proceeds smoothly to yield 1,6-conjugated products, which are then subjected to oxidative dearomatization/hemiacetalization using PhI(OAc)2. The overall process affords moderate to high yields and excellent diastereoselectivities.

Highly Diastereoselective [3 + 2] Cycloaddition of Aziridines with Difluorinated Silyl Enol Ethers: Divergent Synthesis of 4,4-Difluoropyrrolidines and 4-Fluoropyrroles.

A highly diastereoselective [3 + 2] cycloaddition of aziridines with difluorinated silyl enol ethers has been developed. This approach provides a facile methodology for highly functionalized gem-difluorinated pyrrolidines in good to excellent yields with good functional group tolerance. A one-pot, two-step approach for synthesis of structurally interesting fluorinated pyrroles has also been developed through a cycloaddition/aromatization/desulfonation sequence. Moreover, readily available substrates, mild reaction conditions, and easy scale-up synthesis show practical advantages.

Hyper-production of porcine contagious pleuropneumonia subunit vaccine proteins in Escherichia coli by developing a bicistronic T7 expression system.

The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease affecting the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli; however, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The expression of three vaccine antigens Oml1, Oml7, and ApxII in the four strongest bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates (MWP) led to improved production. Finally, the production yields reached unprecedented levels of 2.43 g L-1 of Oml1, 2.59 g L-1 of Oml7, and 1.21 g L-1 of ApxII, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study establishes an efficient bicistronic T7 expression system that can be used to express recombinant proteins in E. coli and achieves the hyper-production of PCP vaccine proteins.

Four uncommon cycloamphilectane-type diterpenoids with antibacterial activity from the South China Sea soft coral Sinularia brassica.

The chemical investigation on the soft coral Sinularia brassica collected off Xuwen Country, Guangdong Province, China, has resulted in the isolation and characterization of three uncommon cycloamphilectane-type diterpenoids, namely sinucycloamtin A-C (1-3), along with two known analogues (5 and 6). In addition, compounds 2 and 3 were hydrolyzed and their hydrolytic derivative sinucycloamtin D (4) was obtained. The structures of these previously undescribed compounds were established on the basis of extensive spectroscopic analysis, X-ray diffraction analysis, chemical conversion, as well as the comparison with the literature reported data. Compounds 1-3 represented the first examples of benzene-containing cycloamphilectane-type diterpenoids isolated from soft coral of genus Sinularia. In the in vitro bioassays, all the isolated and derived diterpenoids exhibited significant antibacterial activities against the fish pathogenic bacteria Phoyobacterium damselae FP2244 and Streptococcus parauberis SPOF3K with MIC90 values ranging from 3.7 to 9.1 µM.