Biochimica et biophysica acta. General subjects最新文献

Background

Sonodynamic antimicrobial chemotherapy (SACT) is an effective antimicrobial treatment that can avoid the production of drug-resistant bacteria. Design and development of new high-efficiency sonosensitizers play a key role in the practical application of SACT.

Methods

The bacteriostatic effects of two phenothiazine compounds, toluidine blue (TB) and azure A (AA) combined with ultrasonic (US) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were studied, and the sonodynamic antibacterial activities of TB and AA were compared. The reactive oxygen species (ROS) and the types of ROS produced in the sonodynamic system were detected and the sonodynamic mechanisms of TB and AA were proposed.

Results

The sonodynamic bacteriostasis mediated by TB and AA increased with the increasing concentration of sonosensitizer, the extension of sonication time and the increase of reaction temperature. The production of ROS was the main reason that TB and AA had excellent sonodynamic antibacterial performance. Singlet oxygen (¹O₂) and hydroxyl radical (•OH) were the main ROS types in the sonodynamic antibacterial system. The ROS produced by the combined action of AA and US was higher than that of TB.

Conclusion

Both TB and AA displayed excellent sonodynamic antibacterial activities. Moreover, AA had a higher sonodynamic activity than TB. The electron donation effect and steric hindrance effect of the methyl group of phenothiazine parent nucleus of TB might be the cause of the decrease of its sonodynamic activity. These results would provide a valuable reference for the further study of phenothiazines sonosensitizers and their clinical application in SACT.

Increased iron level is detected in rat kidney and human urine in diabetic condition and implicated in associated nephropathy. However, the biological cue and mechanism of the iron accumulation remain unclear. Here we reveal that glucose increases iron uptake by promoting transferrin receptor 1 (TFRC) in kidney cells by a translational mechanism but does not alter expression of endosomal iron transporter DMT1. Glucose decreases iron exporter ferroportin (FPN) by a protein degradation mechanism. Hepcidin is known to bind at Cys-326 residue in promoting degradation of human ferroportin. When Cys-326 was mutated to Ser in human-FPN-FLAG and expressed in kidney cells, glucose still could degrade FPN-FLAG implicating involvement of hepcidin independent mechanism in glucose induced ferroportin degradation. Chronic hyperglycemia was generated in rats by administering streptozotocin (STZ) with periodic insulin injection to determine the level of iron homeostasis components. Increased TFRC and decreased ferroportin levels were detected in hyperglycemic rat kidney by Western blot and immunohistochemistry analyses. Hepcidin mRNA was not significantly altered in kidney but was marginally decreased in liver. Perls' staining and non-heme iron estimation showed an elevated iron level in hyperglycemic rat kidney. These results suggest that high glucose dysregulates iron transport components resulting iron accumulation in diabetic kidney.

A prokaryotic resistance-based directed evolution system leveraging protein-fragment complementation assay (PCA) was devised, and its proficiency in detecting protein-protein interactions and discriminating varying degrees of binding affinity was demonstrated by two well-characterized protein pairs. Furthermore, we constructed a random mutant library based on the GBP^R36K/E45K mutant, characterized by almost no affinity towards EGFP. This library was subjected to PCA-based prokaryotic directed evolution, resulting in the isolation of back-mutated variants. In summary, we have established an expedited, cost-effective, and structural information-independent PCA-based prokaryotic directed evolution platform for nanobody affinity maturation, featuring tunable screening stringency via modulation of antibiotic concentrations.

Background

The β1,6-GlcNAc branch in N-glycans, produced by a glycosyltransferase N-acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases.

Scope

Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the β1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5-deficient mice, focusing on cancer and the immune system.

Major conclusions

GnT-V has a unique structure for substrate recognition, and its activity and function are regulated by shedding. The glycans produced by GnT-V play pivotal roles in the differentiation of neural cells, cancer malignancy and immunotherapy, and the development of autoimmune diseases by regulating the functions and cell surface residency of glycoproteins.

General significance

Controlling the expression or activity of GnT-V could be a therapeutic option against cancer and autoimmune diseases. Future work should clarify how GnT-V selectively modifies the specific glycoproteins or N-glycosylation sites in vivo.

Heme is an essential prosthetic molecule for life activities and is well known to act as the active center of many proteins, however, labile heme (LH) released from proteins is a harmful molecule that produces reactive oxygen species and must be strictly controlled. Recently, LH has been suggested to function as an important molecule for diverse physiological responses. Quantitative analysis of the intracellular dynamics of LH is essential for understanding its physiological functions, a substantially practical method has not been established. Here, we successfully developed an alternative method that can be used to complement quantification of the dynamics of intracellular LH using H-FluNox, an activity-based specific fluorescent probe recently constructed. Our newly established method should be effective in elucidating the physiological functions of LH.

Background

Ferroptosis, a type of autophagy-dependent cell death, has been implicated in the pathogenesis of lung adenocarcinoma (LUAD). This study aimed to investigate the involvement of coatomer protein complex I subunit zeta 1 (COPZ1) in ferroptosis and ferritinophagy in LUAD.

Methods

Publicly available human LUAD sample data were obtained from the TCGA database to analyze the association of COPZ1 expression with LUAD grade and patient survival. Clinical samples of LUAD and para-carcinoma tissues were collected. COPZ1-deficient LUAD cell model and xenograft model were established. These models were analyzed to evaluate tumor growth, lipid peroxidation levels, mitochondrial structure, autophagy activation, and iron metabolism.

Results

High expression of COPZ1 was indicative of malignancy and poor overall survival. Clinical LUAD tissues showed increased COPZ1 expression and decreased nuclear receptor coactivator 4 (NCOA4) expression. COPZ1 knockdown inhibited xenograft tumor growth and induced apoptosis. COPZ1 knockdown elevated the levels of ROS, Fe²⁺ and lipid peroxidation. COPZ1 knockdown also caused mitochondrial shrinkage. Liproxstatin-1, deferoxamine, and z-VAD-FMK reversed the effects of COPZ1 knockdown on LUAD cell proliferation and ferroptosis. Furthermore, COPZ1 was directly bound to NCOA4. COPZ1 knockdown restricted FTH1 expression and promoted NCOA4 and LC3 expression. NCOA4 knockdown reversed the regulation of iron metabolism, lipid peroxidation, and mitochondrial structure induced by COPZ1 knockdown. COPZ1 knockdown induced the translocation of ferritin to lysosomes for degradation, whereas NCOA4 knockdown disrupted this process.

Conclusion

This study provides novel evidence that COPZ1 regulates NCOA4-mediated ferritinophagy and ferroptosis. These findings provide new insights into the pathogenesis and potential treatment of LUAD.

Background

Cationic liposomes represent a promising non-viral carrier platform for gene delivery. The successful intracellular delivery of genes to the target cell is highly influenced by lipid compositions in the liposomal formulation. In the present study, a Box-Behnken design was applied to investigate the optimal lipid composition for the liposome-based transfection agent.

Methods

The concentrations of DOTAP, DSPE-PEG, and cholesterol were set as independent factors. A total of 15 lipid compositions were generated and tested for specific responses, including particle size, encapsulation efficiency, cell viability, and cell transfection. The data were then analyzed to predict the optimal composition using response surface methodology (RSM).

Results

The results for particle size, encapsulation efficiency, cell viability and fluorescence intensity ranged from 158.7 to 2064 nm, 48.19–95.72%, 81.50–122.67%, and 0.0–9.08, respectively. Compositions of liposome-based transfection agent without DOTAP, those without cholesterol, and those containing DSPE-PEG2000 with a molar ratio equal to or greater than that of cholesterol tended to exhibit low encapsulation efficiency. The ability of the liposome to complex DNA, as determined through electrophoresis gel retardation assay, showed that the composition without DOTAP produced DNA bands, indicating that the prepared liposomes had a less ability to complex DNA. The cytotoxicity test results indicated that all lipid compositions were considered non-toxic, as they exhibited >80% cell viability. The cell transfection assay demonstrated that the lipid composition containing a combination of DOTAP and cholesterol was able to transfect DNA into cells. According to response analysis, RSM predicted that the optimal lipid composition consisted of 2.75 μmol DOTAP and 0.91 μmol cholesterol, with a desirability value of 0.85.

Conclusions

Although the equation model is still acceptable for predicting the optimal lipid composition, further study is needed to obtain a model with higher desirability, such as by using more lipid compositions, increased replications, and different variable responses.

The clinical efficacy of tissue plasminogen activator (tPA) is limited by its lack of specific delivery, requiring large therapeutic doses that increase the risk of intracerebral hemorrhage, bleeding at the surgical site, and patient mortality after angioplasty. To address these limitations, this study aimed to develop a chitosan polysulfate (CsPs)-coated liposomal formulation for the sustained release of tPA. The CsPs-coated liposomes containing tPA (Liposome-tPA/CsPs) were fabricated using the thin-film hydration technique and their properties were compared to tPA-encapsulated nanoliposomes without a coating layer (Liposome-tPA). Liposome-tPA/CsPs showed a quasi-spherical morphology with a hydrodynamic diameter of 110 nm, while Liposome-tPA had a diameter of 80 nm. The thermal analysis showed that the degradation temperature and glass transition temperature (Tg) of Liposome-tPA/CsPs were higher than that of tPA alone, indicating improved temperature stability. The in vitro release study demonstrated a slow and sustained release of tPA from the Liposome-tPA/CsPs, with a concentration of 0.02 mg/ml at 1 h and 0.23 mg/ml at 180 h. The CsPs coating layer enhanced the antibacterial and antioxidant activity of the nanoliposomes. Liposome-tPA/CsPs exhibited higher cell viability compared to Liposome-tPA. It also achieved a higher percentage of thrombolysis, with complete clot dissolution observed after 3 h of treatment. These findings suggest that the Liposome-tPA/CsPs can be a promising approach to overcome the limitations associated with the systemic administration of tPA, potentially enhancing its clinical efficacy while reducing the risk of adverse events.