Ubiquitination, a diverse post-translational modification, is carried out by enzymes including E1-activating enzymes, E2-conjugating enzymes, E3 ligases, and deubiquitinating enzymes (DUBs). Ubiquitin itself possesses 7 lysine residues and N-terminal methionine, allowing for the formation of polyubiquitin chains with different lengths and linkages. These chains exhibit various topologies that can be recognized by proteins containing ubiquitin-binding domain, thereby transmitting distinct cellular signals. To unravel the physiological mechanisms associated with ubiquitin, numerous ubiquitin probes have been developed. This review provides an overview of recent advancements in the field of ubiquitin probes, focusing on activity-based and affinity-based probes. Activity-based probes are designed to covalently bind to DUBs, E1s, or E3s, enabling the identification and characterization of these enzymes. Affinity-based probes, on the other hand, selectively bind to ubiquitin-binding domains, facilitating the identification of proteins that interact with ubiquitin. Moreover, this review comprehensively discusses the synthetic methodologies employed for the acquisition of ubiquitin probes. These includes meticulous discussions on the synthesis of individual monomeric modules, the establishment of isopeptide linkages, as well as the incorporation of reactive functional groups. Additionally, the review explores the emerging area of cell-penetrating ubiquitin probes and highlights their latest applications in living cells. These probes incorporate cell-penetrating peptides to enable their internalization into cells, allowing for direct visualization and manipulation of ubiquitin-modified proteins within their native environment. Overall, this review offers insights into the design, synthesis, and applications of ubiquitin probes, highlighting their significance in elucidating ubiquitin-mediated cellular processes.

Objective To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. Methods The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). Results SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. Conclusions The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

ObjectiveAlveolar macrophages (AMs) are critical for maintaining the homeostasis of pulmonary microenvironment. They process surfactants to ensure alveoli patency, and also serve as the first line of immune defense against pathogen invasion. Available studies have shown that monocyte-derived AMs continuously release pro-inflammatory cytokines and chemokines, recruiting other immune cells to the damaged area during pulmonary fibrosis. These monocyte-derived AMs maintains and amplifies inflammation, playing a negative role in pulmonary fibrosis progression. Current researches have predominantly focused on the gene expression levels of AMs in pulmonary fibrosis microenvironment, with less emphasis on the function and regulation of proteins. This study aims to investigate the differentially expressed proteins (DEPs) of AMs under normal physiological conditions and after pulmonary fibrosis, in order to gain a more comprehensive understanding of the role of AMs in the progression of pulmonary fibrosis. MethodsFirstly, the construction of bleomycin-induced pulmonary fibrosis mouse models was evaluated through using measurements such as body mass, lung coefficient, lungwet-to-dry mass ratio, H&E staining and Masson staining. Subsequently, AMs from both the saline controls and the pulmonary fibrosis models (2.5×10⁵ cells per sample) were collected using FACS sorting, and protein expression profiles of these cells were obtained through label-free proteomics approach. The quality of the proteomic data was assessed by comparing our saline control proteomic data with public proteomic data of physiological AMs. Thirdly, DEPs analysis between the saline controls and the bleomycin groups was carried out using R package Prostar. Functional enrichment analyses of significantly upregulated DEPs were performed using R package Clusterprofiler for GO and KEGG pathways. Finally, the STRING database was used to explore the protein-protein interaction networks related to phagocytosis regulation, inflammatory response regulation, andI-κB/NF-κB signaling pathway. The expression levels of Tlr2 and Pycard were detected respectively by FACS and western blotting. ResultsCompared to the saline controls, mice in the bleomycin groups exhibited a lower average body mass, extensive infiltration of inflammatory cells, and deposition of collagen in the lungs. This indicates that bleomycin successfully induced pulmonary fibrosis in mouse models. The proteomic data of AMs obtained from these models was of high quality and fulfilled the research requirements. A comprehensive analysis showed that 778 proteins were upregulated in pulmonary fibrosis groups compared with control groups. Moreover, the signal pathways enriched in up-regulated DEPs were related to the I‑κB/NF‑κB pathway, inflammatory response regulation, phagocytosis regulation, TGF-β signaling, and HIF-1 pathway, indicating that AMs in pulmonary fibrosis microenvironment exerted pro-inflammatory and pro-fibrotic functions. Protein-protein interaction network analysis of the DEPs suggested that the interactions between Tlr2 and Pycard were control nodes for the pro-inflammatory phenotype of AMs, thereby contributing to pulmonary fibrosis progression. Further validation by FACS and Western blotting respectively confirmed that the expression levels of Tlr2 and Pycard in AMs were significantly increased after pulmonary fibrosis. ConclusionThis study investigates the changes in the protein expression profile of AMs in the pulmonary fibrosis microenvironment. The results show that AMs notably enhanced the activity of various pathways associated with inflammation and fibrosis, suggesting that the interaction between Tlr2 and Pycard serves as a key control node for the highly pro-inflammatory behavior of AMs.

The adulteration and counterfeiting of herbal ingredients in medicinal and food homology (MFH) have a serious impact on the quality of herbal materials, thereby endangering human health. Compared to pharmaceutical drugs, health products derived from traditional Chinese medicine (TCM) are more easily accessible and closely integrated into consumers' daily life. However, the authentication of the authenticity of TCM ingredients in MFH has not received sufficient attention. The lack of clear standards emphasizes the necessity of conducting systematic research in this area. This study utilized DNA barcoding technology, combining ITS2, psbA-trnH, matK as universal barcode sequences, and DX, HH, JYH as specific barcode sequences, to identify the authenticity of commercial medicinal and food homology scented herbal teas. The aim was to investigate the authenticity of scented herbal tea products circulating in the market. The research results revealed that among 180 scented herbal tea samples, DNA barcodes were successfully obtained from 164 samples, while the remaining samples either lacked the target components or suffered severe DNA degradation, resulting in failed amplification. Combined with morphological identification studies, it was found that out of the 180 samples, 141 were authentic, accounting for 78.33% of the total samples, while 31 samples showed adulteration and 8 samples lacked the target components, accounting for 21.67% of the total samples. Additionally, water testing revealed that 5 samples of Carthamus tinctorius herbal tea exhibited the phenomenon of weight adulteration. This study exposed the presence of adulteration and weight adulteration in scented herbal tea products, highlighting the need for regulatory authorities to expedite the establishment of quality standards in scented herbal tea industry. These findings provide valuable insights for the rapid development of the scented herbal tea industry.

Objective: To analyze the short-term effect of individual atmospheric PM_2.5 exposure on the diversity, enterotype, and community structure of gut microbiome in healthy elderly people in Jinan, Shandong province. Methods: The present panel study recruited 76 healthy elderly people aged 60-69 years old in Dianliu Street, Lixia District, Jinan, Shandong Province, and followed them up five times from September 2018 to January 2019. The relevant information was collected by questionnaire, physical examination, precise monitoring of individual PM_2.5 exposure, fecal sample collection and gut microbiome 16S rDNA sequencing. The Dirichlet multinomial mixtures (DMM) model was used to analyze the enterotype. Linear mixed effect model and generalized linear mixed effect model were used to analyze the effect of PM_2.5 exposure on gut microbiome α diversity indices (Shannon, Simpson, Chao1, and ACE indices), enterotype and abundance of core species. Results: Each of the 76 subjects participated in at least two follow-up visits, resulting in a total of 352 person-visits. The age of 76 subjects was (65.0±2.8) years old with BMI (25.0±2.4) kg/m². There were 38 males accounting for 50% of the subjects. People with an educational level of primary school or below accounted for 10.5% of the 76 subjects, and those with secondary school and junior college or above accounting for 71.1% and 18.4%. The individual PM_2.5 exposure concentration of 76 subjects during the study period was (58.7±53.7) μg/m³. DMM model showed that the subjects could be divided into four enterotypes, which were mainly driven by Bacteroides, Faecalibacterium, Lachnospiraceae, Prevotellaceae, and Ruminococcaceae. Linear mixed effects model showed that different lag periods of PM_2.5 exposure were significantly associated with a lower gut α diversity index (FDR<0.05 after correction). Further analysis showed that PM_2.5 exposure was significantly associated with changes in the abundances of Firmicutes (Megamonas, Blautia, Streptococcus, etc.) and Bacteroidetes (Alistipes) (FDR<0.05 after correction). Conclusion: Short-term PM_2.5 exposure is significantly associated with a decrease in gut microbiome diversity and changes in the abundance of several species of Firmicutes and Bacteroidetes in the elderly. It is necessary to further explore the underlying mechanisms between PM_2.5 exposure and the gut microbiome, so as to provide a scientific basis for promoting the intestinal health of the elderly.
Aged ; Humans ; Male ; Middle Aged ; Feces/microbiology* ; Gastrointestinal Microbiome ; Particulate Matter ; RNA, Ribosomal, 16S/genetics* ; Female

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.
Rhamnose/pharmacology* ; Plasmids/genetics* ; Pseudomonas aeruginosa ; Escherichia coli/metabolism*

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.
Isatis/genetics* ; Plant Proteins/metabolism* ; Phylogeny ; Arabidopsis/genetics* ; Flavonoids ; Cloning, Molecular

OBJECTIVE: To investigate the correlation of inducible co-stimulatory molecules (ICOS) with mesenteric vascular endothelial- mesenchymal transition (EndMT) and sclerosis in spontaneously hypertensive rats (SHR). METHODS: Twenty 4-week-old WKY rats and 20 SHRs of the same strain were both randomly divided into 4 groups for observation at 4, 6, 10 and 30 weeks of age. ICOS expression frequency in rat spleen CD4⁺T cells was analyzed using flow cytometry, and the expressions of ICOS, VE-cad, α-SMA and Col3 mRNA in rat mesentery were detected by RT-PCR. The distributions of ICOS, IL-17A and TGF-β in rat mesentery were detected by immunohistochemistry. The levels of IL-17A and TGF-β in rat plasma were measured using ELISA. The morphological changes of rat mesenteric vessels were observed with Masson staining. Spearman or Pearson correlation analyses were used to evaluate the correlation between ICOS expression and the expressions of the markers of vascular EndMT and sclerosis. RESULTS: Compared with the control WKY rats, the SHRs began to show significantly increased systolic blood pressure and ICOS expression frequency on CD4⁺T cells at 6 weeks of age (P < 0.05). In the SHRs, the mRNA and protein expressions of ICOS, α-SMA, Col3, IL-17A and TGF-β in the mesentery were significantly higher than those in control group (P < 0.05), while the mRNA and protein expressions of VE-cad started to reduce significantly at 10 weeks of age (P < 0.05). The plasma levels of IL-17A and TGF-β were significantly increased in SHRs since 6 weeks of age (P < 0.05) with progressive worsening of mesenteric vascular sclerosis (P < 0.05). ICOS mRNA and protein expression levels in the mesenteric tissues of SHRs began to show positive correlations with α-SMA and Col3 expression levels and the severity of vascular sclerosis at 6 weeks of age (P < 0.05) and a negative correlation with VE-cad expression level at 10 weeks (P < 0.05). CONCLUSION ICOS play an important pathogenic role in EndMT and sclerosis of mesenteric vessels in essential hypertension by mediating related immune responses.
Rats ; Animals ; Rats, Inbred SHR ; Rats, Inbred WKY ; Hypertension ; Interleukin-17 ; Sclerosis/pathology* ; Transforming Growth Factor beta ; Mesentery/pathology* ; RNA, Messenger/metabolism* ; Blood Pressure

Background Mitochondrial dynamin-related protein 1 (DRP1) regulates mitochondrial division and plays an important role in maintaining hepatocyte function. However, the role of DRP1 in cadmium exposure-induced maternal liver damage in pregnant mice remains unclear. Objective To investigate the role and mechanism of DRP1 in maternal liver damage induced by cadmium exposure during pregnancy. Methods This study consisted of animal experiments and cell experiments. (1) Animal experiments. Mice at 14 days of gestation were randomly divided into three groups: a control group, a low-dose cadmium group (LCd group: 2.5 mg·kg⁻¹), and a high-dose cadmium group (HCd group: 5 mg·kg⁻¹). The pregnant mice were intraperitoneally injected with cadmium chloride (CdCl₂) for 6 and 24 h in the next morning. The weights of pregnant mice, uterus, maternal liver, and fetal mice were recorded after sacrifice. Serum and liver of pregnant mice were collected, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected, and liver tissues were stained with HE to observe changes in liver function and liver tissue structure. The expressions of oxidative phosphorylation-related proteins, hypoxia inducible factor-1α (HIF-1α) and DRP1 proteins in liver of pregnant mice were detected by Western blotting. (2) Cell experiments. AML12 cells were treated with CdCl₂ (10 μmol·L⁻¹) for 0, 2, 6, 12, and 24 h. The expressions of oxidative phosphorylation-related proteins, DRP1, and hypoxia inducible factor-1α (HIF-1α) proteins were detected. AML12 cells were pretreated with DRP1 inhibitor Mdivi-1 for 1 h and then CdCl₂ (10 μmol·L⁻¹) for 12 h to detect the expression of oxidative phosphorylation-related proteins and DRP1 protein. AML12 cells were treated with Hif-1α siRNA for 48 h and CdCl₂ (10 μmol·L⁻¹) for 6 h to detect the expression of HIF-1α and DRP1 proteins. Results The results of animal experiments showed that cadmium exposure in pregnant mice had no effects on maternal liver weight and liver coefficient. However, the histomorphological changes and necrosis in hepatocytes were observed. Compared with the control group, the serum ALT and AST levels of pregnant mice in the LCd group were significantly increased after 6 h (P＜0.05), and the levels in the HCd group were significantly increased after 6 and 24 h (P＜0.05). Cadmium exposure during pregnancy significantly up-regulated HIF-1α and DRP1 expressions and down-regulated the expressions of oxidative phosphorylation-related proteins in maternal livers. In vitro cell experiments showed that the expressions of oxidative phosphorylation-related proteins was significantly decreased and HIF-1α and DRP1 protein expressions were significantly increased in the AML12 cells treated with CdCl₂ for 6 h. Mdivi-1 pretreatment significantly antagonized the inhibitory effect of cadmium on the expressions of oxidative phosphorylation-related proteins in AML12 cells, while Hif-1α siRNA pretreatment significantly antagonized the up-regulative effect of cadmium on DRP1 expression in AML12 cells. Conclusion Cadmium exposure in pregnant mice may up-regulate DRP1 expression by activating HIF-1α signaling, then inhibit oxidative phosphorylation level of hepatic cells, and ultimately lead to maternal liver damage.

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca²⁺, Mn²⁺ and Co²⁺) showed inhibitory effect on the activity of UGT74L2, while Mg²⁺ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the K_m value was 29.84 μmol·L^-1, the k_cat was 0.02 s^-1, and the k_cat·K_m^-1 was 572.6 mol^-1·s^-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.