Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis.

OBJECTIVE: Optimize the extraction process of earthworm fibrinolytic enzyme. METHODS: Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme. RESULTS: Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods. DISCUSSION: The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.

Whole-tumor histogram analysis of synthetic magnetic resonance imaging predicts isocitrate dehydrogenase mutation status in gliomas.

Background: An accurate assessment of isocitrate dehydrogenase (IDH) status in patients with glioma is crucial for treatment planning and is a key factor in predicting patient outcomes. In this study, we investigated the potential value of whole-tumor histogram metrics derived from synthetic magnetic resonance imaging (MRI) in distinguishing IDH mutation status between astrocytoma and glioblastoma. Methods: In this prospective study, 80 glioma patients were enrolled from September 2019 to June 2022. All patients underwent pre- and post-contrast synthetic MRI scan protocol. Immunohistochemistry (IHC) staining or gene sequencing were used to assess IDH mutation status in tumor tissue samples. Whole-tumor histogram metrics, including T1, T2, proton density (PD), etc., were extracted from the quantitative maps, while radiological features were assessed by synthetic contrast-weighted maps. Basic clinical features of the patients were also evaluated. Differences in clinical, radiological, and histogram metrics between IDH-mutant astrocytoma and IDH-wildtype glioblastoma were analyzed using univariate analyses. Variables with statistical significance in univariate analysis were included in multivariate logistic regression analysis to develop the combined model. Receiver operating characteristic (ROC) and area under the curve (AUC) were used to assess the diagnostic performance of metrics and models. Results: The histopathologic analysis revealed that of the 80 cases, 41 were classified as IDH-mutant astrocytoma and 39 as IDH-wildtype glioblastoma. Compared to IDH-wildtype glioblastoma, IDH-mutant astrocytoma showed significantly lower T1 [10th percentile (10th), mean, and median] and post-contrast PD (10th, 90th percentile, mean, median, and maximum) values as well as higher post-contrast T1 (cT1) (10th, mean, median, and minimum) values (all P<0.05). The combined model (T1-10th + cT1-10th + age) was developed by integrating the independent influencing factors of IDH-mutant astrocytoma using the multivariate logistic regression. The diagnostic performance of this model [AUC =0.872 (0.778-0.936), sensitivity =75.61%, and specificity =89.74%] was superior to the clinicoradiological model, which was constructed using age and enhancement degree (AUC =0.822 (0.870-0.898), P=0.035). Conclusions: The combined model constructed using histogram metrics derived from synthetic MRI could be a valuable preoperative tool to distinguish IDH mutation status between astrocytoma and glioblastoma, and subsequently, could assist in the decision-making process of pretreatment.

A nitric oxide-catalytically generating carboxymethyl chitosan/sodium alginate hydrogel coating mimicking endothelium function for improving the biocompatibility.

Thanks to their outstanding mechanical properties and corrosion resistance in physiological environments, titanium and its alloys are broadly explored in the field of intravascular devices. However, the biocompatibility is insufficient, causing thrombus formation and even implantation failure. In this study, inspired by the functions of endothelial glycocalyx and the NO-releasing of endothelial cells (ECs), a biomimetic coating (TNTA-Se) with three-dimensional gel-like structures and NO-catalytically generating ability was constructed on the titanium surface. To this end, the titanium alloy was firstly anodized and then annealed to form nanotube structures imitating the three-dimensional villous of glycocalyx, followed by the preparation of the Cu2+-loaded polydopamine intermediate layer for the immobilization of carboxymethyl chitosan and sodium alginate to form the hydrogel structure. Finally, an organoselenium compound (selenocystamine) as an active catalyst was covalently immobilized on the surface to develop a bioactive coating mimicking endothelial function with NO-generating activity. The surface morphologies and chemical structures of the biomimetic coating were characterized by scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and the results indicated that the NO-catalytically generating hydrogel coating was successfully constructed. The results of water contact angle and protein adsorption suggested that the TNTA-Se coating exhibited excellent hydrophilicity, the promotion of bovine serum albumin (BSA) adsorption while the inhibition of fibrinogen (FIB) adsorption. Upon the addition of NO donor S-nitroso glutathione (GSNO) and reducing agent glutathione (GSH), the surface (TNTA-NO) displayed excellent blood compatibility and cytocompatibility to ECs. Compared with other surfaces, the TNTA-NO coating can not only further promote BSA adsorption and inhibit the adhesion and activation of platelets as well as hemolysis, but also significantly enhance ECs adhesion and proliferation and up-regulate VEGF and NO expression of ECs. The current study demonstrated that the NO-catalytically generating hydrogel coating on the titanium alloy can mimic the glycocalyx structure and endothelium function to catalyze a large number of NO donors in human blood to produce NO, and thus simultaneously enhance the surface hemocompatibility and endothelialization, representing a promising strategy for long-term cardiovascular implants of titanium-based devices.

Recent advances in the pharmacological applications and liver toxicity of triptolide.

Triptolide is a predominant active component of Triptergium wilfordii Hook. F, which has been used for the treatment of cancers and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and diabetic nephropathy. Therefore, triptolide and its derivates are considered to have promising prospects for development into drugs. However, the clinical application of triptolide is limited due to various organ toxicities, especially liver toxicity. The potential mechanism of triptolide-induced hepatotoxicity has attracted increasing attention. Over the past five years, studies have revealed that triptolide-induced liver toxicity is involved in metabolic imbalance, oxidative stress, inflammations, autophagy, apoptosis, and the regulation of cytochrome P450 (CYP450) enzymes, gut microbiota and immune cells. In this review, we summarize the pharmacological applications and hepatotoxicity mechanism of triptolide, which will provide solid theoretical evidence for further research of triptolide.

SARS-CoV-2 spike host cell surface exposure promoted by a COPI sorting inhibitor.

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations.

Fabrication of CO-releasing surface to enhance the blood compatibility and endothelialization of TiO2 nanotubes on titanium surface.

Although the construction of nanotube arrays with the micro-nano structures on the titanium surfaces has demonstrated a great promise in the field of blood-contacting materials and devices, the limited surface hemocompatibility and delayed endothelial healing should be further improved. Carbon monoxide (CO) gas signaling molecule within the physiological concentrations has excellent anticoagulation and the ability to promote endothelial growth, exhibiting the great potential for the blood-contact biomaterials, especially the cardiovascular devices. In this study, the regular titanium dioxide nanotube arrays were firstly prepared in situ on the titanium surface by anodic oxidation, followed by the immobilization of the complex of sodium alginate/carboxymethyl chitosan (SA/CS) on the self-assembled modified nanotube surface, the CO-releasing molecule (CORM-401) was finally grafted onto the surface to create a CO-releasing bioactive surface to enhance the biocompatibility. The results of scanning electron microscopy (SEM), X-ray energy dispersion spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) revealed that the CO-releasing molecules were successfully immobilized on the surface. The modified nanotube arrays not only exhibited excellent hydrophilicity but also could slowly release CO gas molecules, and the amount of CO release increased when cysteine was added. Furthermore, the nanotube array can promote albumin adsorption while inhibit fibrinogen adsorption to some extent, demonstrating its selective albumin adsorption; although this effect was somewhat reduced by the introduction of CORM-401, it can be significantly enhanced by the catalytic release of CO. The results of hemocompatibility and endothelial cell growth behaviors showed that, as compared with the CORM-401 modified sample, although the SA/CS-modified sample had better biocompatibility, in the case of cysteine-catalyzed CO release, the released CO could not only reduce the platelet adhesion and activation as well as hemolysis rate, but also promote endothelial cell adhesion and proliferation as well as vascular endothelial growth factor (VEGF) and nitric oxide (NO) expression. As a result, the research of the present study demonstrated that the releasing CO from TiO2 nanotubes can simultaneously enhance the surface hemocompatibility and endothelialization, which could open a new route to enhance the biocompatibility of the blood-contacting materials and devices, such as the artificial heart valve and cardiovascular stents.

Structural mechanism of a dual-functional enzyme DgpA/B/C as both a C-glycoside cleaving enzyme and an O- to C-glycoside isomerase.

The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature. The knowledge of C-glycoside breakdown and synthesis is very limited. Recently, the enzyme DgpA/B/C cascade from a human intestinal bacterium PUE was identified to specifically cleave the C-glycosidic bond of puerarin (daidzein-8-C-glucoside). Here we investigated how puerarin is recognized and oxidized by DgpA based on crystal structures of DgpA with or without substrate and biochemical characterization. More strikingly, we found that apart from being a C-glycoside cleaving enzyme, DgpA/B/C is capable of efficiently converting O- to C-glycoside showing the activity as a structure isomerase. A possible mechanistic model was proposed dependently of the simulated complex structure of DgpB/C with 3″-oxo-daidzin and structure-based mutagenesis. Our findings not only shed light on understanding the enzyme-mediated C-glycosidic bond breakage and formation, but also may help to facilitate stereospecific C-glycoside synthesis in pharmaceutical industry.

A newly isolated human intestinal strain deglycosylating flavonoid C-glycosides.

Glycosidic bond of C-glycosides is difficult to be broken due to its chemical stability. Screening specific microbe from microbiota is a practical way to deglycosylate these compounds. In this study, a new strain W974-1 which is capable of cleaving C-glycosidic bonds was isolated from human gut microbiota by spread plate method. It deglycosylates flavonoid 8-C-glycosides such as orientin and vitexin to their aglycones with the enzymes secreted outside the bacterial cells. This strain was identified as Enterococcus avium by 16S rDNA sequencing, physiological and biochemical characterization.

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors.

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling-dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.

ER retention is imposed by COPII protein sorting and attenuated by 4-phenylbutyrate.

Native cargo proteins exit the endoplasmic reticulum (ER) in COPII-coated vesicles, whereas resident and misfolded proteins are substantially excluded from vesicles by a retention mechanism that remains unresolved. We probed the ER retention process using the proteostasis regulator 4-phenylbutyrate (4-PBA), which we show targets COPII protein to reduce the stringency of retention. 4-PBA competes with p24 proteins to bind COPII. When p24 protein uptake is blocked, COPII vesicles package resident proteins and an ER-trapped mutant LDL receptor. We further show that 4-PBA triggers the secretion of a KDEL-tagged luminal resident, implying that a compromised retention mechanism causes saturation of the KDEL retrieval system. The results indicate that stringent ER retention requires the COPII coat machinery to actively sort biosynthetic cargo from diffusible misfolded and resident ER proteins.

TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats.

The supramolecular cargo procollagen is loaded into coat protein complex II (COPII)-coated carriers at endoplasmic reticulum (ER) exit sites by the receptor molecule TANGO1/cTAGE5. Electron microscopy studies have identified a tubular carrier of suitable dimensions that is molded by a distinctive helical array of the COPII inner coat protein Sec23/24•Sar1; the helical arrangement is absent from canonical COPII-coated small vesicles. In this study, we combined X-ray crystallographic and biochemical analysis to characterize the association of TANGO1/cTAGE5 with COPII proteins. The affinity for Sec23 is concentrated in the proline-rich domains (PRDs) of TANGO1 and cTAGE5, but Sec23 recognizes merely a PPP motif. The PRDs contain repeated PPP motifs separated by proline-rich linkers, so a single TANGO1/cTAGE5 receptor can bind multiple copies of coat protein in a close-packed array. We propose that TANGO1/cTAGE5 promotes the accretion of inner coat proteins to the helical lattice. Furthermore, we show that PPP motifs in the outer coat protein Sec31 also bind to Sec23, suggesting that stepwise COPII coat assembly will ultimately displace TANGO1/cTAGE5 and compartmentalize its operation to the base of the growing COPII tubule.

Rules for the recognition of dilysine retrieval motifs by coatomer.

Cytoplasmic dilysine motifs on transmembrane proteins are captured by coatomer α-COP and ß'-COP subunits and packaged into COPI-coated vesicles for Golgi-to-ER retrieval. Numerous ER/Golgi proteins contain K(x)Kxx motifs, but the rules for their recognition are unclear. We present crystal structures of α-COP and ß'-COP bound to a series of naturally occurring retrieval motifs-encompassing KKxx, KxKxx and non-canonical RKxx and viral KxHxx sequences. Binding experiments show that α-COP and ß'-COP have generally the same specificity for KKxx and KxKxx, but only ß'-COP recognizes the RKxx signal. Dilysine motif recognition involves lysine side-chain interactions with two acidic patches. Surprisingly, however, KKxx and KxKxx motifs bind differently, with their lysine residues transposed at the binding patches. We derive rules for retrieval motif recognition from key structural features: the reversed binding modes, the recognition of the C-terminal carboxylate group which enforces lysine positional context, and the tolerance of the acidic patches for non-lysine residues.

Phosphorylation of DCC by ERK2 is facilitated by direct docking of the receptor P1 domain to the kinase.

Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades.