Development of a new paradigm model for deciphering action mechanism of Danhong injection using a combination of isothermal shift assay and database interrogation.

BACKGROUND: Identification of active components of traditional Chinese Medicine (TCM) and their respective targets is important for understanding the mechanisms underlying TCM efficacy. However, there are still no effective technical methods to achieve this. METHODS: Herein, we have established a method for rapidly identifying targets of a specific TCM and interrogating the targets with their corresponding active components based on Isothermal Shift Assay (iTSA) and database interrogation. RESULTS: We optimized iTSA workflow and identified 110 targets for Danhong injection (DHI) which is used as an effective remedy for cardiovascular and cerebrovascular diseases. Moreover, we identified the targets of the nine major ingredients found in DHI. Database interrogation found that the potential targets for DHI, in which we verified that ADK as the target for salvianolic acid A and ALDH1B1 as the target for protocatechualdehyde in DHI, respectively. CONCLUSION: Overall, we established a novel paradigm model for the identification of targets and their respective ingredients in DHI, which facilitates the discovery of drug candidates and targets for improving disease management and contributes to revealing the underlying mechanisms of TCM and fostering TCM development and modernization.

Thermally Stable and Chemically Recyclable Poly(ketal-ester)s Regulated by Floor Temperature.

Chemical recycling to monomers (CRM) offers a promising closed-loop approach to transition from current linear plastic economy toward a more sustainable circular paradigm. Typically, this approach has focused on modulating the ceiling temperature (Tc) of monomers. Despite considerable advancements, polymers with low Tc often face challenges such as inadequate thermal stability, exemplified by poly(γ-butyrolactone) (PGBL) with a decomposition temperature of ∼200 °C. In contrast, floor temperature (Tf)-regulated polymers, particularly those synthesized via the ring-opening polymerization (ROP) of macrolactones, inherently exhibit enhanced thermodynamic stability as the temperature increases. However, the development of those Tf regulated chemically recyclable polymers remains relatively underexplored. In this context, by judicious design and efficient synthesis of a biobased macrocyclic diester monomer (HOD), we developed a type of Tf -regulated closed-loop chemically recyclable poly(ketal-ester) (PHOD). First, the entropy-driven ROP of HOD generated high-molar mass PHOD with exceptional thermal stability with a Td,5% reaching up to 353 °C. Notably, it maintains a high Td,5% of 345 °C even without removing the polymerization catalyst. This contrasts markedly with PGBL, which spontaneously depolymerizes back to the monomer above its Tc in the presence of catalyst. Second, PHOD displays outstanding closed-loop chemical recyclability at room temperature within just 1 min with tBuOK. Finally, copolymerization of pentadecanolide (PDL) with HOD generated high-performance copolymers (PHOD-co-PPDL) with tunable mechanical properties and chemical recyclability of both components.

A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

Application of panoramic radiographs in the diagnosis of temporomandibular disorders.

To evaluate the feasibility of temporomandibular disorder (TMD) diagnosis with panoramic radiography, and provide standardized data for artificial intelligence-assisted diagnosis by measuring the differences in the condylar and mandibular ramus heights. A total of 500 panoramic radiographs (219 male and 281 female participants) of healthy individuals were examined. The panoramic machine compatible measurement software, SCANORA 5.2.6, was used to measure the bilateral condylar height and mandibular ramus height, and SPSS 27.0 was used to calculate the left- and right-side differences in condylar height and mandibular ramus height of healthy individuals. Magnetic resonance images of the temporomandibular joint region obtained from 46 outpatients in the Stomatology Department were selected along with their corresponding panoramic radiographs. The left- and right-sided differences were measured and compared with the magnetic resonance imaging results. The measurement data are expressed as mean ±â€…standard deviation (mm). t Tests were used to analyze data from healthy male and healthy female groups. The findings revealed that while there was no significant difference (P > .05) in the height of the condyle between men and women, there was a significant difference (P  < .05) in the height of the mandibular ramus. In healthy population, the difference in height between the left and right condyle was 1.09 ±â€…0.99 mm. The difference in height of mandibular ramus in men was 1.26 ±â€…0.85 mm and that in women was 1.19 ±â€…0.87 mm. For the diagnosis of TMD, the sensitivity of panoramic radiographs was 94.74% (36/38), specificity was 75.00% (6/8), and diagnostic accuracy was 91.30% (42/46). The height of the right and left lateral condyles was not identical in healthy individuals, resulting in a discernible height discrepancy. In addition, the height of the mandibular ramus varied. By considering the left-right lateral height differences identified in this study along with clinical examination, it is possible to employ this metric as a preliminary screening tool for patients with TMD. Further, the use of panoramic radiographs for initial TMD screening is both viable and significant.

Novel primers to identify a wider diversity of butyrate-producing bacteria.

Butyrate-producing bacteria are a functionally important part of the intestinal tract flora, and the resulting butyric acid is essential for maintaining host intestinal health, regulating the immune system, and influencing energy metabolism. However, butyrate-producing bacteria have not been defined as a coherent phylogenetic group. They are primarily identified using primers for key genes in the butyrate-producing pathway, and their use has been limited to the Bacillota and Bacteroidetes phyla. To overcome this limitation, we developed functional gene primers able to identify butyrate-producing bacteria through the butyrate kinase gene, which encodes the enzyme involved in the final step of the butyrate-producing pathway. Genomes extracted from human and rat feces were used to amplify the target genes through PCR. The obtained sequences were analyzed using BLASTX to construct a developmental tree using the MEGA software. The newly designed butyrate kinase gene primers allowed to recognize a wider diversity of butyrate-producing bacteria than that recognized using currently available primers. Specifically, butyrate-producing bacteria from the Synergistota and Spirochaetota phyla were identified for the first time using these primers. Thus, the developed primers provide a more accurate method for researchers and doctors to identify potential butyrate-producing bacteria and deepen our understanding of butyrate-producing bacterial species.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment.

Metabolic alterations in the microenvironment significantly modulate tumor immunosensitivity, but the underlying mechanisms remain obscure. Here, we report that tumors depleted of fumarate hydratase (FH) exhibit inhibition of functional CD8+ T cell activation, expansion, and efficacy, with enhanced malignant proliferative capacity. Mechanistically, FH depletion in tumor cells accumulates fumarate in the tumor interstitial fluid, and increased fumarate can directly succinate ZAP70 at C96 and C102 and abrogate its activity in infiltrating CD8+ T cells, resulting in suppressed CD8+ T cell activation and anti-tumor immune responses in vitro and in vivo. Additionally, fumarate depletion by increasing FH expression strongly enhances the anti-tumor efficacy of anti-CD19 CAR T cells. Thus, these findings demonstrate a role for fumarate in controlling TCR signaling and suggest that fumarate accumulation in the tumor microenvironment (TME) is a metabolic barrier to CD8+ T cell anti-tumor function. And potentially, fumarate depletion could be an important strategy for tumor immunotherapy.

Fully Bio-based Poly(ketal-ester)s by Ring-opening Polymerization of a Bicylcic Lactone from Glycerol and Levulinic Acid.

A fully renewable bio-based bicyclic lactone containing a five-membered cyclic ketal moiety, 7-methyl-3,8,10-trioxabicyclo[5.2.1]decan-4-one (TOD), was synthesized through a two-step acid-catalyzed process from glycerol and levulinic acid. The ring-opening polymerization (ROP) of TOD at 30°C with benzyl alcohol (BnOH) as the initiator and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as the catalyst can afford high molar mass PTOD with a cis-2.4-disubstitued 2-methyl 1,3-dioxolane moiety in its repeating unit. PTOD is an amorphous polymer with a glass transition temperature (Tg ) of 13°C. It can be hydrolyzed into structurally defined small molecules under acidic or basic conditions by the selective cleavage of either the cyclic ketal or the ester linkage respectively. The TBD-catalyzed copolymerization of L-lactide (L-LA) and TOD at -20°C was investigated. It was confirmed that L-LA polymerized quickly with racemization to form PLA, followed by a slow incorporation of TOD into the formed PLA chains via transesterification. By varying the feed ratios of L-LA to TOD, a series of random copolymers (PLA-co-PTOD) with different TOD incorporation ratios and tunable Tg s were obtained. Under acidic conditions, PLA-co-PTOD degrades much faster than PLA via the selective cleavage of the cyclic ketal linkages. This work provides insights for the development of more sustainable and acid-accelerated degradable alternatives to aliphatic polyesters.

Assembly of Hydrophobic ZIF-8 on CeO2 Nanorods as High-Efficiency Catalyst for Electrocatalytic Nitrogen Reduction Reaction.

The electrocatalytic nitrogen reduction reaction (NRR) can use renewable electricity to convert water and N2 into NH3 under normal temperature and pressure conditions. However, due to the competitiveness of the hydrogen evolution reaction (HER), the ammonia production rate (RNH3) and Faraday efficiency (FE) of NRR catalysts cannot meet the needs of large-scale industrialization. Herein, by assembling hydrophobic ZIF-8 on a cerium oxide (CeO2) nanorod, we designed an excellent electrocatalyst CeO2-ZIF-8 with intrinsic NRR activity. The hydrophobic ZIF-8 surface was conducive to the efficient three-phase contact point of N2 (gas), CeO2 (solid) and electrolyte (liquid). Therefore, N2 is concentrated and H+ is deconcentrated on the CeO2-ZIF-8 electrocatalyst surface, which improves NRR and suppresses HER and finally CeO2-ZIF-8 exhibits excellent NRR performance with an RNH3 of 2.12 µg h-1 cm-2 and FE of 8.41% at -0.50 V (vs. RHE). It is worth noting that CeO2-ZIF-8 showed excellent stability in the six-cycle test, and the RNH3 and FE variation were negligible. This study paves a route for inhibiting the competitive reaction to improve the NRR catalyst activity and may provide a new strategy for NRR catalyst design.

Flavonoid 4,4'-dimethoxychalcone induced ferroptosis in cancer cells by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH.

Flavonoids are widely distributed in plants as secondary metabolites and have various biological benefits such as anti-tumor, anti-oxidant, anti-inflammatory and anti-aging. We previously reported that 4,4'-dimethoxychalcone (DMC) suppressed cancer cell proliferation by aggravating oxidative stress and inducing G2/M cell cycle arrest. In the present study, we explored the underlying mechanisms by which DMC inhibited cancer cell growth. Given that ferrochelatase (FECH) is a potential target of DMC identified by thermal proteome profiling (TPP) method, herein, we confirmed that DMC inhibited the enzymatic activity of FECH. Furthermore, we proved that DMC induced Keap1 degradation via ubiquitin-proteasome system, which led to the nuclear translocation of Nrf2 and upregulated Nrf2 targeted gene HMOX1. FECH inhibition and HMOX1 upregulation resulted in iron overload and triggered ferroptosis in cancer cells. Collectively, we revealed that DMC induced ferroptosis by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH. Our findings indicate that FECH contributes to the non-canonical ferroptosis induction, shed light on the mechanisms of DMC inhibiting cancer cell growth, and set an example for studying biological functions of flavonoids.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

[Clinical application of disc reduction and anchorage for diacapitular condylar fracture with disc displacement].

Objective: To investigate the effectiveness of disc reduction and anchorage in treatment of diacapitular condylar fracture with disc displacement. Methods: Between June 2019 and June 2021, 20 patients (27 sides) with diacapitular condylar fractures with disc displacement were treated with disc reduction and anchorage combined with internal fixation. There were 15 males and 5 females with a median age of 40 years (range, 8-65 years). The fractures were caused by falling from height in 3 cases, traffic accident in 3 cases, and falling in 14 cases. Among them, there were 13 cases of unilateral fracture and 7 cases of bilateral fractures. Five sides were type A fractures and 22 sides were type B. There were 14 simple diacapitular condylar fractures, 12 diacapitular condylar fractures combined with mandibular chin fractures, and 1 diacapitular condylar fracture combined with mandibular angle fracture. The maximum opening was 5-20 mm (mean, 9.7 mm). The time from injury to operation was 4-20 days, with an average of 11.6 days. The postoperative imaging examination was performed to evaluate the reduction of fracture and disc. The maximum opening at 6 months after operation was recorded, and the clinical dysfunction index (Di) of Helkimo index was used to evaluate the temporomandibular joint function. Results: All incisions healed by first intention. All 20 patients were followed up 6-10 months (mean, 8 months). Postoperative imaging examination showed that 27 fractures were well reduced, of which 26 were anatomically reduced and 1 was basically reduced; the reduction of the temporomandibular joint disc was excellent in 25 sides, good in 1 side, and poor in 1 side, and the effective rate of disc reduction and anchorage was 96.3%. The occlusion relationship of the patient was stable and basically reached the pre-injury level, the incision scar was hidden, and the mouth opening significantly improved when compared with the preoperative level. The maximum mouth opening was 32-40 mm (mean, 36.8 mm) at 6 months after operation. Maximum opening was more than 35 mm in 17 cases. At last follow-up, joint function reached Di 0 grade in 8 sides, DiⅠ grade in 18 sides, and DiⅡ grade in 1 side. After operation, 2 cases of opening deviation, 1 case of joint click, and 2 cases of temporary disappearance of frontal striae on affected side occurred, which recovered to normal after symptomatic treatment. Conclusion: For diacapitular condylar fractures with disc displacement, it is necessary to adopt disc reduction and anchorage at the same time of fracture reduction and internal fixation, which can achieve good clinical results.

Photocaging of Activity-Based Ubiquitin Probes via a C-Terminal Backbone Modification Strategy.

Photocaged, activity-based ubiquitin (Ub) probes (Ub-ABPs) have been developed for the time-resolved probing of deubiquitinating enzyme (DUB) activities, but many Ub-ABPs are still challenging to photocage because their warheads (e.g. propargylamide (PA) or dehydroalanine (Dha)) are difficult to temporally block and activate. Here, we describe a new C-terminal backbone modification strategy for the construction of photocaged Ub-ABPs in which a light-sensitive group is placed at the backbone amide bond of the Ub Gly75. This strategy enabled the facile generation of cell-permeable photocaged Ub-PA and Dha probes that could be activated to capture DUBs after photo-irradiation, and were used to profile DUBs in cells under specially designed conditions (e.g. in cells experiencing oxidative stress) or DUBs with isopeptide linkage selectivity. This backbone modification strategy is anticipated to provide a general solution for the development of photocaged Ub ABPs bearing any warheads for DUB profiling.

Functional vulnerability of liver macrophages to capsules defines virulence of blood-borne bacteria.

Many encapsulated bacteria use capsules to cause invasive diseases. However, it remains largely unknown how the capsules enhance bacterial virulence under in vivo infection conditions. Here we show that the capsules primarily target the liver to enhance bacterial survival at the onset of blood-borne infections. In a mouse sepsis model, the capsules enabled human pathogens Streptococcus pneumoniae and Escherichia coli to circumvent the recognition of liver-resident macrophage Kupffer cells (KCs) in a capsular serotype-dependent manner. In contrast to effective capture of acapsular bacteria by KCs, the encapsulated bacteria are partially (low-virulence types) or completely (high-virulence types) "untouchable" for KCs. We finally identified the asialoglycoprotein receptor (ASGR) as the first known capsule receptor on KCs to recognize the low-virulence serotype-7F and -14 pneumococcal capsules. Our data identify the molecular interplay between the capsules and KCs as a master controller of the fate and virulence of encapsulated bacteria, and suggest that the interplay is targetable for therapeutic control of septic infections.

LATS1 K751 acetylation blocks activation of Hippo signalling and switches LATS1 from a tumor suppressor to an oncoprotein.

Large tumor suppressor 1 (LATS1) is the key kinase controlling activation of Hippo signalling pathway. Post-translational modifications of LATS1 modulate its kinase activity. However, detailed mechanism underlying LATS1 stability and activation remains elusive. Here we report that LATS1 is acetylated by acetyltransferase CBP at K751 and is deacetylated by deacetylases SIRT3 and SIRT4. Acetylation at K751 stabilized LATS1 by decreasing LATS1 ubiquitination and inhibited LATS1 activation by reducing its phosphorylation. Mechanistically, LATS1 acetylation resulted in inhibition of YAP phosphorylation and degradation, leading to increased YAP nucleus translocation and promoted target gene expression. Functionally, LATS1-K751Q, the acetylation mimic mutant potentiated lung cancer cell migration, invasion and tumor growth, whereas LATS1-K751R, the acetylation deficient mutant inhibited these functions. Taken together, we demonstrated a previously unidentified post-translational modification of LATS1 that converts LATS1 from a tumor suppressor to a tumor promoter by suppression of Hippo signalling through acetylation of LATS1.

An integrated strategy for the construction of a species-specific glycan library for mass spectrometry-based intact glycopeptide analyses.

Mass spectrometry (MS)-based strategies and related software tools using glycan mass lists have greatly facilitated the analysis of intact glycopeptides. Most glycan mass lists are derived from normal glycans of mammals and contain limited monosaccharides, which has significantly hindered high throughput studies of unusual glycosylation events observed in other species. In this work, an integrated strategy was developed for the construction of a species-specific glycan mass list from glycan structure databases and published papers. We developed a computational tool called LibGlycan, which could process the different formats of glycans. Then, the software tool generated a glycan library that contained the monoisotope mass, average mass, isotope distribution, and glycan mass list for input into Byonic software. This strategy was applied to analyze the N-glycosylation of rice roots and O-glycosylation of Acinetobacter baumannii ATCC17978, leading to the identification of 296 and 145 intact glycopeptides respectively. Combined, these results show that this strategy is a robust computational approach for the determination of glycan diversity within different complex biological systems.

Induced TRIM21 ISGylation by IFN-ß enhances p62 ubiquitination to prevent its autophagosome targeting.

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-ß also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-ß.

Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility.

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.

Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy.

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.