Discovery and characterization of two novel polyethylene terephthalate hydrolases: One from a bacterium identified in human feces and one from the Streptomyces genus.

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Production and Functional Analysis of Collagen Hexapeptide Repeat Sequences in Pichia pastoris.

In the development of biomaterials with specific structural domains associated with various cellular activities, the limited integrin specificity of commonly used adhesion sequences, such as the RGD tripeptide, has resulted in an inability to precisely control cellular responses. To overcome this limitation, we conducted multiple replications of the integrin α2ß1-specific ligand, the collagen hexapeptide Gly-Phe-Pro-Gly-Glu-Arg (GFPGER) in Pichia pastoris. This enabled the development of recombinant collagen with high biological activity, which was subsequently expressed, isolated, and purified for structural and functional analysis. The proteins carrying the multiple replications GFPGER sequence demonstrated significant bioactivity in cells, leading to enhanced cell adhesion, osteoblast differentiation, and mineralization when compared to control groups. Importantly, these effects were mediated by integrin α2ß1. The new collagen constructed in this study is expected to be a biomaterial for regulating specific cell functions and fates.

Ginsenoside Rh4 alleviates gastrointestinal mucositis and enhances chemotherapy efficacy through modulating gut microbiota.

BACKGROUND: Gastrointestinal mucositis stands as one of the most severe side effects of irinotecan (CPT-11). however, only palliative treatment is available at present. Therefore, there is an urgent need for adjunctive medications to alleviate the side effects of CPT-11. PURPOSE: In this study, our objective was to explore whether ginsenoside Rh4 could serve as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, thereby alleviating the side effects of CPT-11 and augmenting its anti-tumor efficacy. STUDY DESIGN: A CPT-11-induced gastrointestinal mucositis model was used to investigate whether ginsenoside Rh4 alleviated CPT-11-induced gastrointestinal mucositis and enhanced the anti-tumor activity of CPT-11. METHODS: In this study, we utilized CT26 cells to establish a xenograft tumor model, employing transcriptomics, genomics, and metabolomics techniques to investigate the impact of ginsenoside Rh4 on CPT-11-induced gastrointestinal mucositis and the effect on the anti-tumor activity of CPT-11. Furthermore, we explored the pivotal role of gut microbiota and their metabolites through fecal microbiota transplantation (FMT) experiments and supplementation of the key differential metabolite, hyodeoxycholic acid (HDCA). RESULTS: The results showed that ginsenoside Rh4 repaired the impairment of intestinal barrier function and restored intestinal mucosal homeostasis in a gut microbiota-dependent manner. Ginsenoside Rh4 treatment modulated gut microbiota diversity and upregulated the abundance of beneficial bacteria, especially Lactobacillus_reuteri and Akkermansia_muciniphila, which further regulated bile acid biosynthesis, significantly promoted the production of the beneficial secondary bile acid hyodeoxycholic acid (HDCA), thereby alleviating CPT-11-induced gut microbiota dysbiosis. Subsequently, ginsenoside Rh4 further alleviated gastrointestinal mucositis through the TGR5-TLR4-NF-κB signaling pathway. On the other hand, ginsenoside Rh4 combination therapy could further reduce the weight and volume of colon tumors, promote tumor cell apoptosis, and enhance the anti-tumor activity of CPT-11 by inhibiting the PI3K-Akt signaling pathway, thus exerting a synergistic anti-tumor effect. CONCLUSION: In summary, our findings confirm that ginsenoside Rh4 can alleviate CPT-11-induced gastrointestinal mucositis and enhance the anti-tumor activity of CPT-11 by modulating gut microbiota and its related metabolites. Our study validates the potential of ginsenoside Rh4 as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, offering new therapeutic strategies for addressing chemotherapy side effects and improving chemotherapy efficacy.

Synergistic anticancer effects of ginsenoside CK and gefitinib against gefitinib-resistant NSCLC by regulating the balance of angiogenic factors through HIF-1α/VEGF.

Drug resistance is a serious problem for gefitinib in the treatment of lung cancer. Ginsenoside CK, a metabolite of diol ginsenosides, have many excellent pharmacological activities, but whether ginsenoside CK can overcome gefitinib resistance remains unclear. In our study, the sensitizing activity of ginsenoside CK on gefitinib-resistant non-small cell lung cancer (NSCLC) in vitro and in vivo was investigated. Ginsenoside CK was confirmed to enhance the anti-proliferation, pro-apoptotic and anti-migration effects of gefitinib in primary and acquired resistant NSCLC. Furthermore, the combined administration of CK and gefitinib effectively promoted the sensitivity of lung cancer xenograft to gefitinib in vivo, and the tumor inhibition rate reached 70.97% (vs. gefitinib monotherapy 32.65%). Subsequently, tubule formation experiment and western blot results showed that co-treatment of ginsenoside CK inhibited the angiogenesis ability of HUVEC cells, and inhibited the expression of HIF-1α, VEGF, FGF and MMP2/9. More interestingly, ginsenoside CK co-treatment enhanced the expression of anti-angiogenic factor PF4, increased pericellular envelope, and promoted the normalization of vascular structure. In conclusion, ginsenoside CK improved the resistance of gefitinib by regulating the balance of angiogenic factors through down-regulating the HIF-1α/VEGF signaling pathway, providing a theoretical basis for improving the clinical efficacy of gefitinib and applying combined strategies to overcome drug resistance.

Ginsenoside CK Alleviates DSS-Induced IBD in Mice by Regulating Tryptophan Metabolism and Activating Aryl Hydrocarbon Receptor via Gut Microbiota Modulation.

Dysbiosis of gut microbiota is believed to be associated with inflammatory bowel disease (IBD). Ginsenoside compound K (CK), the main metabolite of Panax ginseng ginsenoside, has proven effective as an anti-inflammatory agent in IBD. However, the mechanisms by which CK modulates gut microbiota to ameliorate IBD remain poorly understood. Herein, CK demonstrated the potential to suppress the release of proinflammatory cytokines by gut microbiota modulation. Notably, supplementation with CK promoted the restoration of a harmonious balance in gut microbiota, primarily by enhancing the populations of Lactobacillus and Akkermansia. Furthermore, CK considerably elevated the concentrations of tryptophan metabolites derived from Lactobacillus that could activate the aryl hydrocarbon receptor. Overall, the promising alleviative efficacy of CK primarily stemmed from the promotion of Lactobacillus growth and production of tryptophan metabolites, suggesting that CK should be regarded as a prospective prebiotic agent for IBD in the future.

Expression, characterization and antivascular activity of amino acid sequence repeating collagen hexadecapeptide.

Type V collagen is an essential component of the extracellular matrix (ECM), and its remodeling releases specific protein fragments that can specifically inhibit endothelial cell responses such as proliferation, migration, and invasion. In this study, we have successfully constructed two engineered strains of Pichia pastoris capable of producing recombinant collagen through a new genetic engineering approach. Through high-density fermentation, the expression of 1605 protein and 1610 protein could reach 2.72 g/L and 4.36 g/L. With the increase of repetition times, the yield also increased. Bioactivity analysis showed that recombinant collagen could block the angiogenic effect of FGF-2 on endothelial cells by eliminating FGF-2-induced endothelial cell migration and invasion. Collectively, the recombinant proteins we successfully expressed have a wide range of potential for inhibiting angiogenesis in the biomaterials and biomedical fields.

Ginsenoside Rk3 Regulates Tryptophan Metabolism along the Brain-Gut Axis by Targeting Tryptophan Hydroxylase and Remodeling the Intestinal Microenvironment to Alleviate Depressive-Like Behavior in Mice.

Depression is a neuropsychiatric disease that significantly impacts the physical and mental health of >300 million people worldwide and places a major burden on society. Ginsenosides are the main active ingredient in ginseng and have been proven to have various pharmacological effects on the nervous system. Herein, we investigated the antidepressant effect of ginsenoside Rk3 and its underlying mechanism in a murine model of depression. Rk3 significantly improved depression-like behavior in mice, ameliorated the disturbance of the hypothalamus-pituitary-adrenal axis, and alleviated neuronal damage in the hippocampus and prefrontal cortex of mice. Additionally, Rk3 improved the abnormal metabolism of tryptophan in brain tissue by targeting tryptophan hydroxylase, thereby reducing neuronal apoptosis and synaptic structural damage in the mouse hippocampus and prefrontal cortex. Furthermore, Rk3 reshaped the composition of the gut microbiota of mice and regulated intestinal tryptophan metabolism, which alleviated intestinal barrier damage. Thus, this study provides valuable insights into the role of Rk3 in the tryptophan metabolic cycle along the brain-gut axis, suggesting that Rk3 may have the potential for treating depression.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.

Ginsenoside Rk1 induces autophagy-dependent apoptosis in hepatocellular carcinoma by AMPK/mTOR signaling pathway.

Hepatocellular carcinoma (HCC) is the third most lethal cancer in the world. Recent studies have shown that suppression of autophagy plays an important role in the development of HCC. Ginsenoside Rk1 is a protopanaxadiol saponin isolated from ginseng and has a significant anti-tumor effect, but its role and mechanism in HCC are still unclear. In this study, a mouse liver cancer model induced by diethylnitrosamine and carbon tetrachloride (DEN + CCl4) was employed to investigate the inhibitory effect of Rk1 on HCC. The results demonstrate that ginsenoside Rk1 effectively inhibits liver injury, liver fibrosis, and cirrhosis during HCC progression. Transcriptome data analysis of mouse liver tissue reveals that ginsenoside Rk1 significantly regulates the AMPK/mTOR signaling pathway, autophagy pathway, and apoptosis pathway. Subsequent studies show that ginsenoside Rk1 induces AMPK protein activation, upregulates the expression of autophagy marker LC3-II protein to promote autophagy, and then downregulates the expression of Bcl2 protein to trigger a caspase cascade reaction, activating AMPK/mTOR-induced toxic autophagy to promote cells death. Importantly, co-treatment of ginsenoside Rk1 with autophagy inhibitors can inhibit apoptosis of HCC cells, once again demonstrating the ability of ginsenoside Rk1 to promote autophagy-dependent apoptosis. In conclusion, our study demonstrates that ginsenoside Rk1 inhibits the development of primary HCC by activating toxic autophagy to promote apoptosis through the AMPK/mTOR pathway. These findings confirm that ginsenoside Rk1 is a promising new strategy for the treatment of HCC.

One-Step Purification and Immobilization of Glycosyltransferase with Zn-Ni MOF for the Synthesis of Rare Ginsenoside Rh2.

Uridine diphosphate (UDP)-glucosyltransferases (UGTs) have received increasing attention in the field of ginsenoside Rh2 conversion. By harnessing the metal chelation between transition metal ions and imidazole groups present on His-tagged enzymes, a specific immobilization of the enzyme within metal-organic frameworks (MOFs) is achieved. This innovative approach not only enhances the stability and reusability of the enzyme but also enables one-step purification and immobilization. Consequently, the need for purifying crude enzyme solutions is effectively circumvented, resulting in significant cost savings during experimentation. The use of immobilized enzymes in catalytic reactions has shown great potential for achieving higher conversion rates of ginsenoside Rh2. In this study, highly stable mesoporous Zn-Ni MOF materials were synthesized at 150 °C by a solvothermal method. The UGT immobilized on the Zn-Ni MOF (referred to as UGT@Zn-Ni MOF) exhibited superior pH adaptability and thermal stability, retaining approximately 76% of its initial activity even after undergoing 7 cycles. Furthermore, the relative activity of the immobilized enzyme remained at an impressive 80.22% even after 45 days of storage. The strong specific adsorption property of Zn-Ni MOF on His-tagged UGT was confirmed through analysis using polyacrylamide gel electrophoresis. UGT@Zn-Ni MOF was used to catalyze the conversion reaction, and the concentration of rare ginsenoside Rh2 was generated at 3.15 µg/mL. The results showed that Zn-Ni MOF is a material that can efficiently purify and immobilize His-tagged enzyme in one step and has great potential for industrial applications in enzyme purification and ginsenoside synthesis.

Janus hydrogels: merging boundaries in tissue engineering for enhanced biomaterials and regenerative therapies.

In recent years, the design and synthesis of Janus hydrogels have witnessed a thriving development, overcoming the limitations of single-performance materials and expanding their potential applications in tissue engineering and regenerative medicine. Janus hydrogels, with their exceptional mechanical properties and excellent biocompatibility, have emerged as promising candidates for various biomedical applications, including tissue engineering and regenerative therapies. In this review, we present the latest progress in the synthesis of Janus hydrogels using commonly employed preparation methods. We elucidate the surface and interface interactions of these hydrogels and discuss the enhanced properties bestowed by the unique "Janus" structure in biomaterials. Additionally, we explore the applications of Janus hydrogels in facilitating regenerative therapies, such as drug delivery, wound healing, tissue engineering, and biosensing. Furthermore, we analyze the challenges and future trends associated with the utilization of Janus hydrogels in biomedical applications.

Ginsenoside Rk3 modulates gut microbiota and regulates immune response of group 3 innate lymphoid cells to against colorectal tumorigenesis.

The gut microbiota plays a pivotal role in the immunomodulatory and protumorigenic microenvironment of colorectal cancer (CRC). However, the effect of ginsenoside Rk3 (Rk3) on CRC and gut microbiota remains unclear. Therefore, the purpose of this study is to explore the potential effect of Rk3 on CRC from the perspective of gut microbiota and immune regulation. Our results reveal that treatment with Rk3 significantly suppresses the formation of colon tumors, repairs intestinal barrier damage, and regulates the gut microbiota imbalance caused by CRC, including enrichment of probiotics such as Akkermansia muciniphila and Barnesiella intestinihominis, and clearance of pathogenic Desulfovibrio. Subsequent metabolomics data demonstrate that Rk3 can modulate the metabolism of amino acids and bile acids, particularly by upregulating glutamine, which has the potential to regulate the immune response. Furthermore, we elucidate the regulatory effects of Rk3 on chemokines and inflammatory factors associated with group 3 innate lymphoid cells (ILC3s) and T helper 17 (Th17) signaling pathways, which inhibits the hyperactivation of the Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway. These results indicate that Rk3 modulates gut microbiota, regulates ILC3s immune response, and inhibits the JAK-STAT3 signaling pathway to suppress the development of colon tumors. More importantly, the results of fecal microbiota transplantation suggest that the inhibitory effect of Rk3 on colon tumors and its regulation of ILC3 immune responses are mediated by the gut microbiota. In summary, these findings emphasize that Rk3 can be utilized as a regulator of the gut microbiota for the prevention and treatment of CRC.

A fingerprint-like supramolecular-assembled Ag3PO4/polydopamine/g-C3N4 heterojunction nanocomposite for enhanced solar-driven oxygen evolution in vivo.

Biocompatible photocatalytic water-splitting systems are promising for tissue self-oxygenation. Herein, a structure-function dual biomimetic fingerprint-like silver phosphate/polydopamine/graphitic carbon nitride (Ag3PO4/PDA/g-C3N4) heterojunction nanocomposite is proposed for enhanced solar-driven oxygen (O2) evolution in vivo in situ. Briefly, a porous nitrogen-defected g-C3N4 nanovoile (CN) is synthesized as the base. Dopamine molecules are controllably inserted into the CN interlayer, forming PDA spacers (4.28 nm) through self-polymerization-induced supramolecular-assembly. Ag3PO4 nanoparticles are then in situ deposited to create Ag3PO4/PDA/CN. The fingerprint-like structure of PDA/CN enlarges the layer spacing, thereby accelerating mass transfer and increasing reaction sites. The PDA spacer roles as excellent light harvester, electronic-ionic conductor, and redox pair through conformational changes, resulting in tailored electronic band structure, optimized carrier behavior, and reduced electrochemical impedance. In physiological conditions, Ag3PO4/PDA/CN exhibits O2 evolution rate of 45.35 µmol⋅g-1⋅h-1, 9-fold of bulk g-C3N4. The biocompatibility and in vivo oxygen supply effectiveness for biomedical applications have been verified in animal models.

Ginsenoside Rk3 Ameliorates Obesity-Induced Colitis by Modulating Lipid Metabolism in C57BL/6 Mice.

Lipid metabolism is closely related to obesity and its complications. Our previous study found that ginsenoside Rk3 (Rk3), a natural bioactive substance derived from ginseng, can effectively alleviate obesity-induced colitis, while its impact on the improvement of the lipid metabolism disorder remains unclear. Here, we demonstrated that Rk3 significantly alleviated inflammation, oxidative stress, and lipid dysregulation in high-fat diet-induced colitis C57BL/6 mice. The potential mechanism by which Rk3 mitigated colon inflammation in the context of obesity may involve the modulation of polyunsaturated fatty acid metabolism with specific attention to n-6 fatty acids, linoleic acid, and arachidonic acid. Rk3 intervention markedly reduced the production of pro-inflammatory factors (PGE2, PGD2, TXB2, HETE, and HODE) by inhibiting cyclooxygenase and lipoxygenase pathways, while enhancing the production of anti-inflammatory factors (EET and diHOME) via cytochrome P450 pathways. Our findings suggest that Rk3 is a potential anti-inflammatory natural drug that can improve obesity-induced intestinal inflammation by regulating lipid metabolism.

Ginsenoside Rg5 inhibits lipid accumulation and hepatocyte apoptosis via the Notch1 signaling pathway in NASH mice.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a prevalent chronic liver disease that lacks an FDA-approved treatment medicine. Despite the known antitumor and hypoglycemic properties of Ginsenoside Rg5, its effects and underlying mechanisms in the context of NASH remain largely unexplored. PURPOSE: This study aims to investigate the effect of Rg5 on NASH mice induced by a high-fat diet and CCl4. STUDY DESIGN: In vivo experiments, a mouse NASH model was established by a HFHC diet plus intraperitoneal injection of low-dose CCl4. In vitro experiments, a cellular steatosis model was established using free fatty acids (FFA) induced HepG2 cells. In addition, a fibrogenesis model was established using HSC-LX2 cells. METHODS: The effects of Ginsenoside Rg5 on lipid accumulation and oxidative damage were analyzed by ELISA kit, H&E staining, Oil Red O staining, flow cytometry and Western blot. The effects of Ginsenoside Rg5 on liver fibrosis were analyzed by Masson staining, Sirus Red staining, immunohistochemistry and Western blot. The effect of Ginsenoside Rg5 on Notch1 signaling pathway in liver was studied by protein Oil Red staining, protein immunoblotting and immunofluorescence. RESULTS: In terms of lipid accumulation, Rg5 has the ability to regulate key proteins related to lipogenesis, thereby inhibiting hepatic lipid accumulation and oxidative stress. Additionally, Rg5 can reduce the occurrence of hepatocyte apoptosis by regulating the p53 protein. Moreover, after Rg5 intervention, the presence of fibrotic proteins (α-SMA, Collagen 1, TGF-ß) in the liver is significantly suppressed, thus inhibiting liver fibrosis. Lastly, Rg5 leads to a decrease in the expression levels of Notch1 and its ligand Jagged-1 in the liver. CONCLUSION: In summary, the regulatory effects of Rg5 on the Notch1 signaling pathway play a crucial role in modulating hepatic lipid metabolism and preventing hepatocyte apoptosis, thereby impeding the progression of NASH. These findings highlight the potential of Rg5 as a promising natural product for interventions targeting NASH.

Status and developmental trends in recombinant collagen preparation technology.

Recombinant collagen is a pivotal topic in foundational biological research and epitomizes the application of critical bioengineering technologies. These technological advancements have profound implications across diverse areas such as regenerative medicine, organ replacement, tissue engineering, cosmetics and more. Thus, recombinant collagen and its preparation methodologies rooted in genetically engineered cells mark pivotal milestones in medical product research. This article provides a comprehensive overview of the current genetic engineering technologies and methods used in the production of recombinant collagen, as well as the conventional production process and quality control detection methods for this material. Furthermore, the discussion extends to foresee the strides in physical transfection and magnetic control sorting studies, envisioning an enhanced preparation of recombinant collagen-seeded cells to further fuel recombinant collagen production.

Rapid dissolution microneedle based on polyvinyl alcohol/chitosan for local oral anesthesia.

At present, the main clinical methods of oral local anesthesia are direct injection of anesthetic and surface ointment. However, the pain and fear caused by the injection, the discomfort of topical anesthetic creams, and the scour and moist oral environment during the procedure pose great challenges to oral anesthesia. Herein, we designed a Lido-PVP/PVA DMNP microneedle (MN) for oral local anesthesia. The microneedle tip was consisted of Polyvinylpyrrolidone/Polyvinyl alcohol (PVP/PVA), which can quickly dissolve and release the lidocaine hydrochloride (Lido) drug within 5 min to achieve rapid anesthesia. The backing was composed of polyvinyl alcohol/chitosan (PVA/CS), and its excellent adhesion can overcome saliva erosion and anchor firmly to the oral mucosa, significantly improving the utilization rate of drugs, as well as the patient compliance. MNs have good mechanical properties for tissue insertion while possessing high drug loading (3 mg/MNs). Von Frey tests proved that MNs showed a faster and more effective local anesthetic effect (anesthesia takes effect at 5 min) compared to cream (anesthesia takes effect at 30 min). In addition, the excellent biocompatibility and no skin irritation endowed Lido-PVP/PVA DMNP MNs a great potential for oral local anesthesia in the oral cavity.

Molecularly imprinted solid-phase extraction combined with non-ionic hydrophobic deep eutectic solvents dispersed liquid-liquid microextraction for efficient enrichment and determination of the estrogens in serum samples.

Hormonal drugs in biological samples are usually in low concentration and highly intrusive. It is of great significance to enhance the sensitivity and specificity of the detection process of hormone drugs in biological samples by utilizing appropriate sample pretreatment methods for the detection of hormone drugs. In this study, a sample pretreatment method was developed to effectively enrich estrogens in serum samples by combining molecularly imprinted solid-phase extraction, which has high specificity, and non-ionic hydrophobic deep eutectic solvent-dispersive liquid-liquid microextraction, which has a high enrichment ability. The theoretical basis for the effective enrichment of estrogens by non-ionic hydrophobic deep eutectic solvent was also computed by simulation. The results showed that the combination of molecularly imprinted solid-phase extraction and deep eutectic solvent-dispersive liquid-liquid microextraction could improve the sensitivity of HPLC by 33∼125 folds, and at the same time effectively reduce the interference. In addition, the non-ionic hydrophobic deep eutectic solvent has a relatively low solvation energy for estrogen and possesses a surface charge similar to that of estrogen, and thus can effectively enrich estrogen. The study provides ideas and methods for the extraction and determination of low-concentration drugs in biological samples and also provides a theoretical basis for the application of non-ionic hydrophobic deep eutectic solvent extraction.

Selumetinib overcomes gefitinib primary and acquired resistance by regulating MIG6/STAT3 in NSCLC.

Gefitinib, as the first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has achieved great advances in the treatment of non-small cell lung cancer (NSCLC), but drug resistance will inevitably occur. Therefore, exploring the resistance mechanism of gefitinib and developing new combination treatment strategies are of great importance. In our study, the results showed that selumetinib (AZD6244) synergistically inhibited the proliferation of NSCLC with gefitinib. Selumetinib also enhanced gefitinib-induced apoptosis and migration inhibition ability in gefitinib-resistant lung cancer cell lines. Subsequently, the negative regulation between MIG6 and STAT3 was observed and verified through the STRING database and western blotting assays. Sustained activation of STAT3 was significantly downregulated when co-treatment with selumetinib in gefitinib-resistant cells. However, the downregulation of p-STAT3, resulting from the combination of selumetinib and gefitinib was counteracted by the deletion of MIG6, suggesting that selumetinib enhanced gefitinib sensitivity by regulating MIG6/STAT3 in NSCLC. In contrast, p-STAT3 was further inhibited after treatment with gefitinib and selumetinib when MIG6 was overexpressed. Furthermore, the combined administration of selumetinib and gefitinib effectively promoted the sensitivity of lung cancer xenografts to gefitinib in vivo, and the tumor inhibition rate reached 81.49%, while the tumor inhibition rate of the gefitinib monotherapy group was only 31.95%. Overall, MIG6/STAT3 negative regulation plays an important role in the sustained activation of STAT3 and the resistance to EGFR-TKIs. Our study also suggests that EGFR-TKIs combined with MEK1/2 inhibitors, such as selumetinib, may be beneficial to those NSCLC patients who develop a primary or acquired resistance to EGFR-TKIs, providing theoretical support for combining TKIs and selumetinib in clinical cancer treatment.

Protein engineering of multi-enzyme virus-like particle nanoreactors for enhanced chiral alcohol synthesis.

In the past decade, virus-like particles (VLPs) that can encapsulate single or multiple enzymes have been studied extensively as typical nanoreactors for biocatalysis in vitro, yet their catalytic efficiencies are usually inadequate for real applications. These biocatalytic nanoreactors should be engineered like their free-enzyme counterparts to improve their catalytic performance for potential applications. Herein we engineer biocatalytic VLPs for the enhanced synthesis of chiral alcohols. Different methods including directed evolution were applied to the entire bacteriophage P22 VLPs (except the coat protein), which encapsulated a carbonyl reductase from Scheffersomyces stipitis (SsCR) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) in their capsids. The best variant, namely M5, showed an enhanced turnover frequency (TOF, min-1) up to 15-fold toward the majority of tested aromatic prochiral ketones, and gave up to 99% enantiomeric excess in the synthesis of chiral alcohol pharmaceutical intermediates. A comparison with the mutations of the free-enzyme counterparts showed that the same amino acid mutations led to different changes in the catalytic efficiencies of free and confined enzymes. Finally, the engineered M5 nanoreactor showed improved efficiency in the scale-up synthesis of chiral alcohols. The conversions of three substrates catalyzed by M5 were all higher than those catalyzed by the wild-type nanoreactor, demonstrating that enzyme-encapsulating VLPs can evolve to enhance their catalytic performance for potential applications.