LC-MS/MS methods for simultaneous determination of youkenafil and its metabolite M1 in human seminal plasma and plasma: Application to evaluate the acute effect of youkenafil on semen quality and its pharmacokinetics in human.

Youkenafil is a novel Phosphodiesterase type 5 inhibitor used for treating erectile dysfunction. N-desethyl compound of youkenafil (M1) is its main active metabolite. In this study, two methods were developed and validated for the simultaneous determination of youkenafil and M1 by HPLC-MS/MS in human matrices including seminal plasma and plasma, in which the multiple reaction monitoring and electrospray ionization in positive mode were adopted, and the deuterated youkenafil (youkenafil-d5) was selected as the internal standard. The collected semen sample was kept at room temperature for approximately 30 min until fully liquefied. The volume of the liquefied semen was measured and then divided into two parts. One part was centrifuged to obtain the seminal plasma for the content detection of youkenafil and M1, while the other part was used for routine semen analysis. The chromatographic separation was accomplished with the column of Poroshell 120 EC-C18 (5 × 2.1 mm, 2.7 µm, Agilent). Protein precipitation with methanol was used for the pretreatment of seminal plasma and plasma. The intra-run and inter-run precisions were less than 6.4 % (relative standard deviation) and accuracies were all within -4.7 %-6.8 % (relative error) in both matrices. All other validated bioanalytical parameters were within the acceptance criteria set by the FDA. The methods were successfully applied to different clinical studies of youkenafil. In the clinical study of the acute effect of youkenafil on semen quality in healthy males, the content of youkenafil in seminal plasma was extremely low. Concentrations of youkenafil and M1 in seminal plasma were lower than those in plasma, at 20.7 % and 4.49 % of the plasma concentration, respectively. There was no significant acute effect of youkenafil on semen quality. In the pharmacokinetic study of youkenafil after single dose-escalation administration, the exposure to youkenafil and M1 was non-linear with the dose in the range of 100-400 mg.

A DNA tetrahedron-based ferroptosis-suppressing nanoparticle: superior delivery of curcumin and alleviation of diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a significant complication that poses continuous threat to the bone health of patients with diabetes; however, currently, there are no effective treatment strategies. In patients with diabetes, the increased levels of ferroptosis affect the osteogenic commitment and differentiation of bone mesenchymal stem cells (BMSCs), leading to significant skeletal changes. To address this issue, we aimed to target ferroptosis and propose a novel therapeutic approach for the treatment of DOP. We synthesized ferroptosis-suppressing nanoparticles, which could deliver curcumin, a natural compound, to the bone marrow using tetrahedral framework nucleic acid (tFNA). This delivery system demonstrated excellent curcumin bioavailability and stability, as well as synergistic properties with tFNA. Both in vitro and in vivo experiments revealed that nanoparticles could enhance mitochondrial function by activating the nuclear factor E2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) pathway, inhibiting ferroptosis, promoting the osteogenic differentiation of BMSCs in the diabetic microenvironment, reducing trabecular loss, and increasing bone formation. These findings suggest that curcumin-containing DNA tetrahedron-based ferroptosis-suppressing nanoparticles have a promising potential for the treatment of DOP and other ferroptosis-related diseases.

Cucurbitacin B induces apoptosis in colorectal cells through reactive oxygen species generation and endoplasmic reticulum stress pathways.

Cucurbitacin B (CuB) is a member of the cucurbitacin family, which has shown potent anticancer pharmacological activity. Prolonged or severe endoplasmic reticulum stress (ERS) induces apoptosis; therefore, the present study investigated whether CuB may activate the ERS pathway to induce apoptosis. HT-29 and SW620 colorectal cancer (CRC) cells were treated with a range of concentrations of CuB for 48 h, and the viability and proliferation of cells were determined using Cell Counting Kit 8 (CCK8) and colony formation assays. Subsequently, the appropriate CuB concentration (5 µM) was selected for treatment of CRC cells for 48 h. Western blot analysis was used to measure the expression levels of ERS-related proteins, flow cytometry was used to evaluate apoptosis, the dichlorodihydrofluorescein diacetate fluorescent probe was used to detect reactive oxygen species (ROS) production, and the relationship between ROS and ERS was determined by western blot analysis. Furthermore, flow cytometry was used to evaluate apoptosis after treatment with the ERS inhibitor 4-phenylbutyric acid, the ROS inhibitor N-acetylcysteine and following knockdown of CHOP expression. In addition, western blot analysis was performed to measure Bax and Bcl2 protein expression levels, and a CCK8 assay was performed to evaluate the viability of cells following knockdown of CHOP. Notably, CuB treatment increased apoptosis and inhibited cell proliferation in CRC cell lines, and these effects were mediated by ROS and ROS-regulated activation of the PERK and XBP1 ERS pathways. In conclusion, CuB may induce apoptosis in HT-29 and SW620 CRC cells via ROS and ERS.

Tetrahedral Framework Nucleic Acids Based Small Interfering RNA Targeting Receptor for Advanced Glycation End Products for Diabetic Complications Treatment.

Complications arising from diabetes can threaten multiple organs. Advanced glycation end products (AGEs) play a significant role in inducing these complications. Highly processed diets and hyperglycemia facilitate the accumulation of AGEs in the body. Interaction between AGEs and their main receptor (RAGE) initiates the transmission of intracellular inflammatory and cell death signals, which ultimately lead to complications. To counter AGEs-induced damage, we developed an siRNA-binding tetrahedral framework nucleic acids (TDN) system, termed Tsi, which combines the potent cell membrane penetrability and serum stability of TDN with the gene-targeting specificity of siRNA-RAGE. Tsi effectively and persistently downregulates the expression of RAGE, thereby suppressing inflammation by blocking the NF-κB pathway as well as exhibiting antioxidant functions. Furthermore, Tsi regulates the pyroptosis state of macrophages via the NLRP3/caspase-1 axis, which inhibits the spread of cell death signals and maintains homeostasis. This is of great significance for the synergistic treatment strategy for systemic complications in patients with refractory hyperglycemia. In summary, this study describes a nanomedicine that targets the RAGE and suppresses AGE-induced inflammation. This nucleic acid drug holds long-lasting efficacy and is independent of lowering hyperglycemia, which provides a strategy for the treatment of diabetic complications and age-related diseases.

Functional micro-RNA drugs acting as a fate manipulator in the regulation of osteoblastic death.

Bone loss is prevalent in clinical pathological phenomena such as osteoporosis, which is characterized by decreased osteoblast function and number, increased osteoclast activity, and imbalanced bone homeostasis. However, current treatment strategies for bone diseases are limited. Regulated cell death (RCD) is a programmed cell death pattern activated by the expression of specific genes in response to environmental changes. Various studies have shown that RCD is closely associated with bone diseases, and manipulating the death fate of osteoblasts could contribute to effective bone treatment. Recently, microRNA-targeting therapy drugs have emerged as a potential solution because of their precise targeting, powerful curative effect, and limited side effects. Nevertheless, their clinical application is limited by their inherent instability, easy enzymatic degradation, and poor membrane penetrability. To address this challenge, a self-assembling tetrahedral DNA nanostructure (TDN)-based microRNA (Tmi) delivery system has been proposed. TDN features excellent biocompatibility, cell membrane penetrability, serum stability, and modification versatility, making it an ideal nucleic acid carrier for miRNA protection and intracellular transport. Once inside cells, Tmi can dissociate and release miRNAs to manipulate key molecules in the RCD signaling pathway, thereby regulating bone homeostasis and curing diseases caused by abnormal RCD activation. In this paper, we discuss the impact of the miRNA network on the initiation and termination of four critical RCD programs in bone tissues: apoptosis, autophagy, pyroptosis, and ferroptosis. Furthermore, we present the Tmi delivery system as a miRNA drug vector. This provides insight into the clinical translation of miRNA nucleic acid drugs and the application of miRNA drugs in bone diseases.

Simultaneous determination of maltol and maltol glucuronide in human plasma and urine by HPLC-MS/MS: Application in clinical study in patients with iron deficiency.

Ferric maltol has been used as an oral drug for iron deficiency. This study developed and fully validated the novel HPLC-MS/MS methods to determine maltol and maltol glucuronide simultaneously in plasma and urine. The protein precipitation was performed by addition of acetonitrile in the plasma samples. The dilution was performed for the urine samples to reach the suitable concentrations for injection. The multiple reaction monitoring (MRM) with an electrospray ionization (ESI) positive ion detection mode was used for the quantification. The maltol concentration linear ranges were 6.00-150 ng/mL and 0.100-10.0 µg/mL for the plasma and urine samples, respectively. The maltol glucuronide concentration linear ranges were 50.0-15000 ng/mL and 2.00-2000 µg/mL for the plasma and urine samples, respectively. These methods were applied to a single dose clinical study at a dose of 60 mg ferric maltol capsule in the patients with iron deficiency. The half-lives of maltol and maltol glucuronide were 0.90 ± 0.40 h and 1.02 ± 0.25 h in the iron deficiency patients, respectively. 39.52 ± 7.11 % maltol were excreted in urine in the form of maltol glucuronide.

LPCAT1 enhances the invasion and migration in gastric cancer: Based on computational biology methods and in vitro experiments.

BACKGROUND AND AIM: The biological functions and clinical implications of lysophosphatidylcholine acyltransferase 1 (LPCAT1) remain unclarified in gastric cancer (GC). The aim of the current study was to explore the possible clinicopathological significance of LPCAT1 and its perspective mechanism in GC tissues. MATERIALS AND METHODS: The protein expression and mRNA levels of LPCAT1 were detected from in-house immunohistochemistry and public high-throughput RNA arrays and RNA sequencing. To have a comprehensive understanding of the clinical value of LPCAT1 in GC, all enrolled data were integrated to calculate the expression difference and standard mean difference (SMD). The biological mechanism of LPCAT1 in GC was confirmed by computational biology and in vitro experiments. Migration and invasion assays were also conducted to confirm the effect of LPCAT1 in GC. RESULTS: Both protein and mRNA expression levels of LPCAT1 in GC were remarkably higher than those in noncancerous controls. Comprehensively, the SMD of LPCAT1 mRNA was 1.11 (95% CI = 0.86-1.36) in GC, and the summarized AUC was 0.85 based on 15 datasets containing 1727 cases of GC and 940 cases of non-GC controls. Moreover, LPCAT1 could accelerate the invasion and migration of GC by boosting the neutrophil degranulation pathway and disturbing the immune microenvironment. CONCLUSION: An increased level of LPCAT1 may promote the progression of GC.

Corrigendum: Machine learning in non-small cell lung cancer radiotherapy: a bibliometric analysis.

[This corrects the article DOI: 10.3389/fonc.2023.1082423.].

Machine learning in non-small cell lung cancer radiotherapy: A bibliometric analysis.

Background: Machine learning is now well-developed in non-small cell lung cancer (NSCLC) radiotherapy. But the research trend and hotspots are still unclear. To investigate the progress in machine learning in radiotherapy NSCLC, we performed a bibliometric analysis of associated research and discuss the current research hotspots and potential hot areas in the future. Methods: The involved researches were obtained from the Web of Science Core Collection database (WoSCC). We used R-studio software, the Bibliometrix package and VOSviewer (Version 1.6.18) software to perform bibliometric analysis. Results: We found 197 publications about machine learning in radiotherapy for NSCLC in the WoSCC, and the journal Medical Physics contributed the most articles. The University of Texas MD Anderson Cancer Center was the most frequent publishing institution, and the United States contributed most of the publications. In our bibliometric analysis, "radiomics" was the most frequent keyword, and we found that machine learning is mainly applied to analyze medical images in the radiotherapy of NSCLC. Conclusion: The research we identified about machine learning in NSCLC radiotherapy was mainly related to the radiotherapy planning of NSCLC and the prediction of treatment effects and adverse events in NSCLC patients who were under radiotherapy. Our research has added new insights into machine learning in NSCLC radiotherapy and could help researchers better identify hot research areas in the future.

Oxymatrine suppresses colorectal cancer progression by inhibiting NLRP3 inflammasome activation through mitophagy induction in vitro and in vivo.

Chinese herb Radix sophorae tonkinensis extract oxymatrine shows anticancer effects. This study evaluated the role of oxymatrine in colorectal cancer (CRC) and the underlying molecular events in vitro and in vivo. CRC cells were treated with different doses of oxymatrine to assess cell viability, reactive oxygen species production, gene expression, and gene alterations. Meanwhile, mouse xenograft and liver metastasis models were used to assess the effects of oxymatrine using histology examination, transmission electron microscopy, and Western blot, respectively. Our results showed that oxymatrine treatment triggered CRC cell mitophagy to inhibit CRC cell growth, migration, invasion, and metastasis in vitro and in vivo. At the gene level, oxymatrine inhibited LRPPRC to promote Parkin translocation into the mitochondria and reduce the mitophagy-activated NLRP3 inflammasome. Thus, oxymatrine had an anticancer activity through LRPPRC inhibition, mitophagy induction, and NLRP3 inflammasome suppression in the CRC cell xenograft and liver metastasis models. In conclusion, the study demonstrates the oxymatrine anti- CRC activity through its unique role in regulating CRC cell mitophagy and NLRP3 inflammasome levels in vitro and in vivo.

Ambra1 regulates apoptosis and chemosensitivity in breast cancer cells through the Akt-FoxO1-Bim pathway.

The sensitivity of cells to chemotherapeutic agents has a major effect on disease outcome in breast cancer patients. Unfortunately, there are numerous factors involved in the regulation of chemosensitivity, and the mechanisms need to be further investigated. Autophagy/Beclin 1 regulator 1 (Ambra1) is a key protein in the crosstalk between autophagy and apoptosis. It controls the switch between these two processes, which determines whether cells survive or die. Induction of apoptosis is the primary mechanism by which most chemotherapeutic drugs eliminate cancer cells. Recently, Ambra1 has been shown to modulate paclitaxel-induced apoptosis in breast cancer cells via the Bim/mitochondrial pathway, thereby modifying the sensitivity of cells to paclitaxel. However, how Ambra1 regulates Bim expression remains unclear. Here, we further confirmed that Bim plays an indispensable role in Ambra1's regulation of apoptosis and chemosensitivity in breast cancer cells. Furthermore, Ambra1 was found to regulate Bim expression at the transcriptional level through the Akt-FoxO1 pathway. Therefore, we propose a novel pathway, Ambra1-Akt-FoxO1-Bim, which regulates apoptosis and chemosensitivity in breast cancer cells. Thus, Ambra1 may represent a potential target for breast cancer treatment.

Co-pathogens in Periodontitis and Inflammatory Bowel Disease.

Localized inflammatory lesions in one area of the body may affect other distant organs through various modes of transmission thus initiating secondary inflammatory infections. Periodontal disease (PD) and inflammatory bowel disease (IBD) have been shown to coexist. Periodontitis is a multifactorial inflammatory disease, and dental plaque is considered to be the initial risk factor. Individuals with genetic susceptibility are more likely to develop periodontitis when exposed to external stimuli. IBD is affected by host genetics, immunoregulation, daily diet, and the gut microbiota, and its risk factors appear to be shared with those of PD. However, the key etiologies of both diseases remain unclear, thus hindering the exploration of possible links between IBD and PD. Recent studies and systematic reviews have focused on evidence-based statistics of the prevalence and clinical manifestations of both diseases, but discussions of the microbial etiological correlation between periodontitis and intestinal inflammation are scarce. Here, we summarize the potential common pathogenic microorganisms that may serve as bridges between the two diseases. Studies have shown that invasive microorganisms such as Porphyromonas gingivalis, Fusobacterium nucleatum, Klebsiella spp. and Campylobacter spp. play key roles in the comorbidity of PD and IBD.

Structure and Function of Oral Microbial Community in Periodontitis Based on Integrated Data.

Objective: Microorganisms play a key role in the initiation and progression of periodontal disease. Research studies have focused on seeking specific microorganisms for diagnosing and monitoring the outcome of periodontitis treatment. Large samples may help to discover novel potential biomarkers and capture the common characteristics among different periodontitis patients. This study examines how to screen and merge high-quality periodontitis-related sequence datasets from several similar projects to analyze and mine the potential information comprehensively. Methods: In all, 943 subgingival samples from nine publications were included based on predetermined screening criteria. A uniform pipeline (QIIME2) was applied to clean the raw sequence datasets and merge them together. Microbial structure, biomarkers, and correlation network were explored between periodontitis and healthy individuals. The microbiota patterns at different periodontal pocket depths were described. Additionally, potential microbial functions and metabolic pathways were predicted using PICRUSt to assess the differences between health and periodontitis. Results: The subgingival microbial communities and functions in subjects with periodontitis were significantly different from those in healthy subjects. Treponema, TG5, Desulfobulbus, Catonella, Bacteroides, Aggregatibacter, Peptostreptococcus, and Eikenella were periodontitis biomarkers, while Veillonella, Corynebacterium, Neisseria, Rothia, Paludibacter, Capnocytophaga, and Kingella were signature of healthy periodontium. With the variation of pocket depth from shallow to deep pocket, the proportion of Spirochaetes, Bacteroidetes, TM7, and Fusobacteria increased, whereas that of Proteobacteria and Actinobacteria decreased. Synergistic relationships were observed among different pathobionts and negative relationships were noted between periodontal pathobionts and healthy microbiota. Conclusion: This study shows significant differences in the oral microbial community and potential metabolic pathways between the periodontitis and healthy groups. Our integrated analysis provides potential biomarkers and directions for in-depth research. Moreover, a new method for integrating similar sequence data is shown here that can be applied to other microbial-related areas.

Identification and Characterization of a Novel Thermostable and Salt-Tolerant ß-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1.

ß-1,3 xylanase is an important enzyme in the biorefinery process for some algae. The discovery and characterization of new ß-1,3 xylanase is a hot research topic. In this paper, a novel ß-1,3 xylanase (Xyl88) is revealed from the annotated genome of Flammeovirga pacifica strain WPAGA1. Bioinformatic analysis shows that Xyl88 belongs to the glycoside hydrolase 26 (GH26) with a suspected CBM (carbohydrate-binding module) sequence. The activity of rXyl88 is 75% of the highest enzyme activity (1.5 mol/L NaCl) in 3 mol/L NaCl buffer, which suggests good salt tolerance of rXy188. The optimum reaction temperature in the buffer without NaCl and with 1.5 mol/L NaCl is 45 °C and 55 °C, respectively. Notably, the catalytic efficiency of rXyl88 (kcat/Km) is approximately 20 higher than that of the thermophilic ß-1,3 xylanase that has the highest catalytic efficiency. Xyl88 in this study becomes the most efficient enzyme ever found, and it is also the first reported moderately thermophilic and salt-tolerant ß-1,3 xylanase. Results of molecular dynamics simulation further prove the excellent thermal stability of Xyl88. Moreover, according to the predicted 3D structure of the Xyl88, the surface of the enzyme is distributed with more negative charges, which is related to its salt tolerance, and significantly more hydrogen bonds and Van der Waals force between the intramolecular residues, which is related to its thermal stability.

Erratum: Exogenous glutathione contributes to cisplatin resistance in lung cancer A549 cells.

[This corrects the article on p. 1295 in vol. 10, PMID: 29887946.].

Erratum: Exogenous glutathione contributes to cisplatin resistance in lung cancer A549 cells.

[This corrects the article on p. 1295 in vol. 10, PMID: 29887946.].

Ambra1 modulates the sensitivity of breast cancer cells to epirubicin by regulating autophagy via ATG12.

The sensitivity of breast cancer cells to epirubicin (EPI) is closely related to the efficacy of the drug and the prognosis of patients. A growing body of research has suggested that autophagy is involved in the treatment of a variety of cancers, including breast cancer, and modifies the sensitivity of anticancer drugs. However, the mechanism by which autophagy participates in cancer therapy and modulates drug sensitivity has not been fully elucidated. In this study, we showed that the expression of Autophagy/Beclin 1 regulator 1 (Ambra1), a key protein of autophagy, was negatively correlated with EPI sensitivity in breast cancer cells. In addition, it altered the sensitivity of breast cancer cells to EPI by regulating EPI-induced autophagy. As a potential mechanism, we demonstrated that autophagy-related protein 12 (ATG12) was a downstream protein that Ambra1-regulated EPI-induced autophagy. Therefore, Ambra1 plays an important role in regulating the sensitivity of breast cancer cells to EPI. And the regulatory effect of Ambra1 on EPI sensitivity is achieved through the regulation of autophagy by targeting ATG12. Overall, we propose a novel mechanism by which autophagy modulates the sensitivity of breast cancer cells to EPI. ATG12 is a novel targeting protein of Ambra1 in regulating EPI-induced autophagy. In addition, the important role of Ambra1 in modulating the sensitivity of breast cancer cells to EPI is confirmed in vivo. This finding indicates that Ambra1 might be a target for developing breast cancer treatments.

Characterization of a novel psychrophilic and halophilic ß-1, 3-xylanase from deep-sea bacterium, Flammeovirga pacifica strain WPAGA1.

ß-1, 3-Xylanase is one of the most important hydrolytic enzymes to prepare oligosaccharides as functional foods in seaweed industry. However, less than five ß-1, 3-xylanases have been experimentally expressed and characterized; moreover, none of them is psychrophilic and salt tolerant. Here, we mined a novel ß-1, 3-xylanase (Xyl512) from the genome of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1 and biochemically characterized it in detail. The Xyl512 did not contain any carbohydrate-binding module; the catalytic domain of it belonged to the glycoside hydrolase family 26. The optimum temperature and pH of the purified ß-1, 3-xylanase was 20 °C and pH 7.0 in the condition of no NaCl. However, they shifted to 30 °C and 7.5 with 1.5 mol/L NaCl, respectively. In this condition (1.5 mol/L NaCl), the overall activity was 2-fold as high as that without NaCl. Based on the residue interactions and the electrostatic surfaces, we addressed the possible mechanism of its adaption to low temperature and relative high NaCl concentration. The Xyl512 showed significantly reduced numbers of hydrogen bonds leading to a more flexible structure, which is likely to be responsible for its cold adaptation. While the negatively charged surface may contribute to its salt tolerance. The ß-1, 3-xylanase we identified here was the first reported psychrophilic and halophilic one with functionally characterized. It could make new contributions to exploring and studying the ß-1, 3-xylanase for further associated investigations.

Exogenous glutathione contributes to cisplatin resistance in lung cancer A549 cells.

BACKGROUND: Recent studies have reported that an elevated intracellular glutathione (GSH) level is associated with resistance of non-small cell lung cancer (NSCLC) cell lines to cisplatin (CDDP). It is well-known that GSH is widely used in the clinic as a hepatoprotective agent. However, whether exogenous GSH can affect the sensitivity of NSCLC cells to CDDP remains unclear. The aim of this study is to evaluate the role of exogenous GSH in the resistance of A549 cells to CDDP. METHODS: The effect of GSH and CDDP on the proliferation of A549 cells was analyzed by MTT assay. Subsequent experiments were conducted in A549 cells divided into four groups: control group (untreated cells), GSH group (treated with 120 µg/ml GSH for 48 h), CDDP group (treated with 10 µg/ml CDDP for 48 h) and CDDP+GSH group (treated with 10 µg/ml CDDP+120 µg/ml GSH for 48 h). Apoptosis was detected by flow cytometry. Light microscopy, fluorescence microscopy and electron microscopy were performed to study morphologic and ultrastructural differences among the four groups of cells. Intracellular GSH level and γ-GCS expression were determined by immunohistochemistry (IHC). Cellular platinum uptake was assessed by inductively coupled plasma mass spectrometry (ICP-MS). Quantitative RT-PCR analysis was performed to measure the expression of caspase3, caspase9, bax, bcl-2 and MDR-1. Western blot analysis was conducted to examine the protein levels of GST-π, MRP-1 and P-gp. RESULTS: Growth inhibition and apoptosis were reduced in A549 cells in the CDDP+GSH group compared to those in the CDDP group 48 h post-treatment. Alterations in cellular morphology and ultrastructure, as well as typical characteristics of apoptosis, were observed. Intracellular GSH and γ-GCS levels were elevated by exogenous administration of GSH; in contrast, cellular platinum concentration fell rapidly. Relative to the CDDP group, the CDDP+GSH group exhibited 47.92%, 47.82% and 63.75% downregulation in caspase3, caspase9 and bax mRNA expression, respectively, and a 2.17-fold increase in bcl-2 mRNA level. In addition, there were 1.58-fold and 2.67-fold increases in the level of GST-π and MRP-1, respectively; however, the changes in MDR-1 and P-gp levels were not statistically significant. CONCLUSIONS: Our data demonstrated that exogenous GSH used as hepatinica in the clinic could induce resistance of A549 cells to CDDP by inhibiting apoptosis, elevating cellular GSH levels, inactivating the mitochondria-mediated signaling pathway, and increasing the expression of GST-π, γ-GCS and MRP1 to increase CDDP efflux.

A novel method for simultaneous purification and immobilization of a xylanase-lichenase chimera via SpyTag/SpyCatcher spontaneous reaction.

We generated a bifunctional enzyme chimera containing the xylanase and lichenase coupled with SpyTag between them. Meanwhile, we generated another chimera containing SpyCatcher and elastin-like polypeptides (ELPs). As ELPs could bond to the xylanase-lichenase chimera through SpyTag/SpyCatcher spontaneous reaction in mild condition, which would lead to the formation of a 3-arm star multifunctional chimera. We purified the xylanase-lichenase by the non-chromatographic purification tag of ELPs. Interestingly, 57.5% of the xylanase and 47.2% of the lichenase in chimera self-assembled into insoluble active particles during the process of purification, which could serve as immobilized bifunctional enzymes. Notably, the immobilized chimera xylanase-lichenase showed a remarkable stability even after 10 reaction cycles, which retained around 56% (lichenase) and 44% (xylanase) of their initial activities, respectively. Moreover, the enhanced thermostability of the immobilized enzymes was also achieved. After incubating at 60 °C for 60 min, the residual activity of the immobilized lichenase was 35%, while the free one was only 24%. Unexpectedly, the free xylanase almost lost its activity when incubated at 55 °C for 60 min, whereas the immobilized xylanase retained 10% of its activity. However, the catalytic efficiency (kcat/Km) of the free xylanase was 1.7-fold higher than the immobilized one, while the free lichenase was 1.1-fold higher than the immobilized one. This is among the first known reports that two enzymes are purified and immobilized in one-step. This novel strategy is easy to scale up and may meet the demands of biofuel industry. It would have great potentials in other biotechnological fields, such as the multifunctional biomaterials systems.