Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Aggregation-Induced Emission Luminogens: A New Possibility for Efficient Visualization of RNA in Plants.

RNAs play important roles in regulating biological growth and development. Advancements in RNA-imaging techniques are expanding our understanding of their function. Several common RNA-labeling methods in plants have pros and cons. Simultaneously, plants' spontaneously fluorescent substances interfere with the effectiveness of RNA bioimaging. New technologies need to be introduced into plant RNA luminescence. Aggregation-induced emission luminogens (AIEgens), due to their luminescent properties, tunable molecular size, high fluorescence intensity, good photostability, and low cell toxicity, have been widely applied in the animal and medical fields. The application of this technology in plants is still at an early stage. The development of AIEgens provides more options for RNA labeling. Click chemistry provides ideas for modifying AIEgens into RNA molecules. The CRISPR/Cas13a-mediated targeting system provides a guarantee of precise RNA modification. The liquid-liquid phase separation in plant cells creates conditions for the enrichment and luminescence of AIEgens. The only thing that needs to be looked for is a specific enzyme that uses AIEgens as a substrate and modifies AIEgens onto target RNA via a click chemical reaction. With the development and progress of artificial intelligence and synthetic biology, it may soon be possible to artificially synthesize or discover such an enzyme.

Cocrystallization-driven Formation of fcc-based Ag110 Nanocluster with Chinese Triple Luban Lock Shape.

Luban locks with mortise and tenon structure have structural diversity and architectural stability, and it is extremely challenging to synthesize Luban lock-like structures at the molecular level. In this work, we report the cocrystallization of two structurally related atom-precise fcc silver nanoclusters Ag110 (SPhF)48 (PPh3 )12 (Ag110 ) and Ag14 (µ6 -S)(SPhF)12 (PPh3 )8 (Ag14 ). It is worth noting that the Ag110 cluster is the first compound to simulate the complex Luban lock structure at the molecular level. Meanwhile, Ag110 is the largest known fcc-based silver nanocluster, so far, there is no precedent for fcc silver nanocluster with more than 100 silver atoms. DFT calculations show that Ag110 is a 58-electron superatom with an electronically closed shell1S2 1P6 1D10 2S2 1F14 2P6 1G18 . Ag110 ⋅Ag14 can rapidly catalyze the reduction of 4-nitrophenol within 4 minutes. In addition, Ag110 presents clear structural evidence to reveal the critical size and mechanism of the transformation of metal core from fcc stacking to quasi-spherical superatom. This research work provides an important structural model for studying the nucleation mechanism and structural assembly of silver nanoclusters.

Molecular characterization, expression analysis, and biological effects of interleukin-8 in grass carp Ctenopharyngodon idellus.

Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus. A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR(-)CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600) and L2 (GenBank ID: AB470924), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro. Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish.

Self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin.

We have recently reported that calreticulin (CRT), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant CRT (rCRT) exhibits extraordinarily strong immunological activities. We herein further demonstrate that rCRT fragments 18-412 (rCRT/18-412), rCRT/39-272, rCRT/120-308 and rCRT/120-250 can self-oligomerize in solution and are 50-100 fold more potent than native CRT (nCRT, isolated from mouse livers) in activating macrophages in vitro. We narrowed down the active site of CRT to residues 150-230, the activity of which also depends on dimerization. By contrast, rCRT/18-197 is almost completely inactive. When rCRT/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. Additionally, rCRT/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. Inhibition of macrophage endocytosis partially blocks the stimulatory effect of rCRT/18-412. We conclude that the immunologically active site of CRT maps between residues 198-230 and that soluble CRT could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization.

[Expression of SIRT1 in right auricle tissues and the relationship with oxidative stress in patients with atrial fibrillation].

AIM: To observe the expression of mammalian sirtuin 1 (SIRT1) in right auricle tissues in patients presenting with atrial fibrillation (AF) and make clear the relationship between SIRT1 expression and oxidative stress. METHODS: A total of 38 patients with rheumatic heart disease were divided into 2 groups: AF group (AF lasted more than 6 months, n=25) and SR (sinus rhythm) group (n=13). The expression of SIRT1 in right auricle tissues harvested during heart operations was detected by immunohistochemistry. Oxidative stress was estimated by the amounts of malondialdehyde (MDA) and metallothionein (MT) and the activity of superoxide dismutase (SOD) which were detected using thiobarbituric acid reaction (TBA), enzyme-linked immunosorbent assay (ELISA) and xanthine oxidase assay, respectively. RESULTS: Compared with the SR group, the expression of SIRT1 protein in the atrium significantly increased in AF group [P<0.05, (45.8±4.03)% vs (19.7±2.54)%]. In AF group, MDA was (7.24±1.05) nmol/mg, SOD (1034.25±84.32) U/mg and MT (7.21±1.46) µg/g, all being significantly higher than those in SR group[P<0.05, MDA: (3.01±0.47) nmol/mg; SOD: (723.63±65.23) U/mg; MT: (4.31±1.23) µg/g]. Spearman correlation indicated that the expression of SIRT1 had a significantly negative correlation with the amounts of MDA and MT and the activity of SOD (P<0.05, r=-0.447, -0.521, -0.394, respectively). CONCLUSION: The expression of SIRT1 increased in patients with AF. SIRT1 maybe effects the AF by means of inhibiting the process of oxidative stress.

[Effect of soft tissue crush injury on tensions of thoracic aortic rings in rats].

OBJECTIVE: To observe the effect of soft tissue crush injury on the tensions of thoracic aortic rings (TARs) in rats and to investigate the potential roles of nitric oxide in the change of the tensions. METHODS: Thirty adult SD rats were randomly divided into control group and crush injury (8 h and 16 h after injury) groups. Two kinds of TARs (one with endothelium and the other without endothelium) in vitro were prepared. In the TARs with endothelium, the tensions induced by phenylephrine (PE), acetylcholine (Ach), calcium ionophore A23187 and angiotensin II (AngI) were measured by the vascular tension detective technique. Then the TARs with endothelium were preincubated with nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) for 20 minutes, the tensions induced by PE and Ang II were measured again. In the TARs without endothelium, the tensions induced by PE and Ang II were measured by the same method. RESULTS: In the TARs with endothelium, the tension of relaxation induced by cumulative doses of Ach and A23187 decreased significantly in 8 h and 16h groups. The tension of contraction induced by cumulative doses of PE and Ang II also decreased significantly (P<0.05). The tension of contraction increased after the preincubation with L-NNA. In the TARs without endothelium, the tension of contraction induced by PE and Ang II increased comparing to that of TARs with endothelium. CONCLUSION: The soft tissue crush injury can influence the tensions of TARs in rats and the vascular-derived NO can mediate the effects.

Comparison of virulence-associated traits between a UPEC strain HEC4 and a APEC strain E058.

Since avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar challenges when establishing infection in the extra-intestinal locations of the hosts, they may share a similar content of virulence genes and capacity to cause disease. One APEC and one UPEC isolates were compared by their content of virulence genes and other traits. The two strains showed overlap in terms of their virulence genotypes, including their possession of certain genes associated with a large transmissible plasmid of APEC, and also shared some biochemical activities. Study of the pathogenicity of UPEC in chicks showed the similar symptoms and lesions compare to those caused by APEC. Based on these results, the potential whether APEC might serve as a reservoir of plasmid-linked and virulence genes for UPEC should be considered.