Adaptive Responses of a Peroxidase-like Polyoxometalate-Based Tri-Assembly to Bacterial Microenvironment (BME) Significantly Improved the Anti-Bacterial Effects.

The present study presents the tertiary assembly of a POM, peptide, and biogenic amine, which is a concept to construct new hybrid bio-inorganic materials for antibacterial applications and will help to promote the development of antivirus agents in the future. To achieve this, a Eu-containing polyoxometalate (EuW10) was first co-assembled with a biogenic amine of spermine (Spm), which improved both the luminescence and antibacterial effect of EuW10. Further introduction of a basic peptide from HPV E6, GL-22, induced more extensive enhancements, both of them being attributed to the cooperation and synergistic effects between the constituents, particularly the adaptive responses of assembly to the bacterial microenvironment (BME). Further intrinsic mechanism investigations revealed in detail that the encapsulation of EuW10 in Spm and further GL-22 enhanced the uptake abilities of EuW10 in bacteria, which further improved the ROS generation in BME via the abundant H2O2 involved there and significantly promoted the antibacterial effects.

Amelioration of alcohol-induced acute liver injury in C57BL/6 mice by a mixture of TCM phytochemicals and probiotics with antioxidative and anti-inflammatory effects.

Background: There are many causes of acute liver injury (ALI), such as alcohol, drugs, infection, and toxic materials, which have caused major health problems around the world. Among these causes, alcohol consumption induced liver injury is a common alcoholic liver disease, which can further lead to liver failure even liver cancer. A number of traditional Chinese medicine (TCM) and TCM derived compounds have been used in treating the liver-associated diseases and combination use of probiotics with TCM phytochemicals has attracted interests for enhanced biological effects. Methods: This study investigated the hepatoprotective effect of TCM-probiotics complex (TCMPC) and its underlying mechanism for the treatment of ALI in mice. The TCMPC is composed of TCM phytochemicals puerarin, curcumin, ginsenosides, and 5 lactobacteria strains. We first established a mouse model of alcohol-induced ALI, then the therapeutic effects of TCMPC on alcohol-induced ALI were monitored. A series of measurements have been performed on antioxidation, anti-inflammation, and lipid metabolism regulation. Results: The results showed that TCMPC can reduce the level of liver injury biomarkers and regulate oxidative stress. Histopathological results indicated that TCMPC could ameliorate ALI in mice. In addition, it can also significantly reduce the production of inflammatory cytokines caused by ALI. Conclusion: Our research has proved the therapeutic effect of TCMPC on alcohol-induced ALI. The potential mechanism of hepatoprotective effects of TCMPC may be related to its antioxidative and anti-inflammatory effects. Our research might provide a new way for liver disease treatment.

A tetrapeptide from maize combined with probiotics exerted strong anti-inflammatory effects and modulated gut microbiota in DSS-induced colitis mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent gastrointestinal inflammation caused by abnormal immune response, and patients usually have intestinal flora imbalance. At present, the pathogenesis of UC is not well understood, and it appears that there is chronic activation of the immune and inflammatory cascade in genetically susceptible individuals. Some food supplements such as specific peptides and probiotics have been investigated and shown the potential for the treatment of UC. The purpose of this study is to investigate the therapeutic effect and potential mechanism of tetrapeptide from maize (TPM) and probiotic treatment on dextran sulfate sodium (DSS)-induced UC in C57BL/6J mice. Our results indicated that the therapeutic effects of TPM and probiotics are positively associated with a reduction in pro-inflammatory cytokine levels and restoration of the gut microbiota. Treatment with TPM or probiotics effectively alleviated the adverse effects of UC, including weight loss, shortened colon length, and colon and kidney tissue damage in mice. Additionally, both TPM and probiotics significantly reduced pro-inflammatory cytokine levels and oxidative stress in UC mice, and the effect was more pronounced when both were used together. Moreover, co-treatment with TPM and probiotics increased the diversity of gut microbes in UC mice, reduced the ratio of Firmicutes to Bacteroidetes (F/B) and increased the abundance of bacterial species, including Muribaculaceae, Alistipes, Ligilactobacillus and Lactobacillus, and has been shown to be beneficial for a variety of pathological conditions.

In Silico Screening of Novel TMPRSS2 Inhibitors for Treatment of COVID-19.

COVID-19, a pandemic caused by the virus SARS-CoV-2, has spread globally, necessitating the search for antiviral compounds. Transmembrane protease serine 2 (TMPRSS2) is a cell surface protease that plays an essential role in SARS-CoV-2 infection. Therefore, researchers are searching for TMPRSS2 inhibitors that can be used for the treatment of COVID-19. As such, in this study, based on the crystal structure, we targeted the active site of TMPRSS2 for virtual screening of compounds in the FDA database. Then, we screened lumacaftor and ergotamine, which showed strong binding ability, using 100 ns molecular dynamics simulations to study the stability of the protein-ligand binding process, the flexibility of amino acid residues, and the formation of hydrogen bonds. Subsequently, we calculated the binding free energy of the protein-ligand complex by the MM-PBSA method. The results show that lumacaftor and ergotamine interact with residues around the TMPRSS2 active site, and reached equilibrium in the 100 ns molecular dynamics simulations. We think that lumacaftor and ergotamine, which we screened through in silico studies, can effectively inhibit the activity of TMPRSS2. Our findings provide a basis for subsequent in vitro experiments, having important implications for the development of effective anti-COVID-19 drugs.

Synergistically enhanced photothermal transition of a polyoxometalate/peptide assembly improved the antibiofilm and antibacterial activities.

We successfully developed an antimicrobial assembly (Mo154/TK-14) using molybdenum-polyoxometalate and a positively charged peptide of TK-14. It was characterized and assayed using zeta-potential, dynamic light scattering (DLS), and TEM measurements. The Mo154/TK-14 assembly showed an enhanced 808 nm absorption and, therefore, improved the photothermal conversion efficiency of Mo154 (30.3%) to 38.6%. Consequently, in comparison to 5 µM Mo154 without irradiation, both the biofilm formation and bacterial viability of S. aureus were 24.6% and 20.2%, respectively, for the Mo154/TK-14 assembly; the biofilm formation and bacterial viability were further decreased to 7.7% and 4.4% under 808 nm irradiation, respectively. Therefore, the Mo154/TK-14 assembly reflects convincing antibacterial properties compared to Mo154. This is due to the synergistic effect between the peptide-binding enhanced 808 nm absorption and the improved PTT properties. The antimicrobial assembly offers a novel strategy for the rational design of light-responsive antibacterial materials.

Implications of Gut Microbiota in Complex Human Diseases.

Humans, throughout the life cycle, from birth to death, are accompanied by the presence of gut microbes. Environmental factors, lifestyle, age and other factors can affect the balance of intestinal microbiota and their impact on human health. A large amount of data show that dietary, prebiotics, antibiotics can regulate various diseases through gut microbes. In this review, we focus on the role of gut microbes in the development of metabolic, gastrointestinal, neurological, immune diseases and, cancer. We also discuss the interaction between gut microbes and the host with respect to their beneficial and harmful effects, including their metabolites, microbial enzymes, small molecules and inflammatory molecules. More specifically, we evaluate the potential ability of gut microbes to cure diseases through Fecal Microbial Transplantation (FMT), which is expected to become a new type of clinical strategy for the treatment of various diseases.

Transdermal Drug Delivery Systems and Their Use in Obesity Treatment.

Transdermal drug delivery (TDD) has recently emerged as an effective alternative to oral and injection administration because of its less invasiveness, low rejection rate, and excellent ease of administration. TDD has made an important contribution to medical practice such as diabetes, hemorrhoids, arthritis, migraine, and schizophrenia treatment, but has yet to fully achieve its potential in the treatment of obesity. Obesity has reached epidemic proportions globally and posed a significant threat to human health. Various approaches, including oral and injection administration have widely been used in clinical setting for obesity treatment. However, these traditional options remain ineffective and inconvenient, and carry risks of adverse effects. Therefore, alternative and advanced drug delivery strategies with higher efficacy and less toxicity such as TDD are urgently required for obesity treatment. This review summarizes current TDD technology, and the main anti-obesity drug delivery system. This review also provides insights into various anti-obesity drugs under study with a focus on the recent developments of TDD system for enhanced anti-obesity drug delivery. Although most of presented studies stay in animal stage, the application of TDD in anti-obesity drugs would have a significant impact on bringing safe and effective therapies to obese patients in the future.

Correction: Wu, H.; et al. A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2. Int. J. Mol. Sci. 2020, 21, 72.

The authors wish to make the following corrections to this paper [...].

Boletus aereus protects against acute alcohol-induced liver damage in the C57BL/6 mouse via regulating the oxidative stress-mediated NF-κB pathway.

CONTEXT: Alcoholic liver disease, caused by abuse and consumption of alcohol, exhibits high morbidity and mortality. Boletus aereus Bull. (Boletaceae) (BA) shows antioxidant, anti-inflammatory and antimicrobial effects. OBJECTIVES: To investigate the hepatoprotective effects of BA using an acute alcohol-induced hepatotoxicity mice model. MATERIALS AND METHODS: The composition of BA fruit body was first systematically analyzed. Subsequently, a C57BL/6 mice model of acute alcohol-induced liver injury was established by intragastrically administration of alcohol, which was intragastrically received with BA powder at 200 mg/kg and 800 mg/kg for 2 weeks, 60 mg/kg silybin treatment was used as positive control group. By employing the pathological examination, ELISA, RT-PCR and western blot, the regulation of BA on oxidative stress signals was investigated. RESULTS: The LD50 of BA was much higher than 4 g/kg/p.o. In acute alcohol-damaged mice, BA reduced the levels of alanine aminotransferase (>18.3%) and aspartate aminotransferase (>27.6%) in liver, increased the activity of liver alcohol dehydrogenase (>35.0%) and serum acetaldehyde dehydrogenase (>18.9%). BA increased the activity of superoxide dismutase (>13.4%), glutathione peroxidase (>11.0%) and 800 mg/kg BA strongly reduced chemokine (C-X-C motif) ligand 13 (14.9%) and chitinase-3 like-1 protein (13.4%) in serum. BA reversed mRNA over-expression (>70%) and phosphor-stimulated expression (>45.0%) of an inhibitor of nuclear factor κ-B kinase (NF-κB, an inhibitor of nuclear factor κ-B α and nuclear factor κ-B in the liver. CONCLUSIONS: BA is effective in ameliorating alcohol-induced liver injury through regulating oxidative stress-mediated NF-κB signalling, which provides a scientific basis for further research on its clinical applications.

Akkermansia muciniphila Aspartic Protease Amuc_1434* Inhibits Human Colorectal Cancer LS174T Cell Viability via TRAIL-Mediated Apoptosis Pathway.

Mucin2 (Muc2) is the main component of the intestinal mucosal layer and is highly expressed in mucous colorectal cancer. Previous studies conducted by our lab found that the recombinant protein Amuc_1434 (expressed in Escherichia coli prokaryote cell system, hereinafter termed Amuc_1434*), derived from Akkermansia muciniphila, can degrade Muc2. Thus, the main objective of this study was to explore the effects of Amuc_1434* on LS174T in colorectal cancer cells expressing Muc2. Results from this study demonstrated that Amuc_1434* inhibited the proliferation of LS174T cells, which was related to its ability to degrade Muc2. Amuc_1434* also blocked the G0/G1 phase of the cell cycle of LS174T cells and upregulated the expression of tumor protein 53 (p53), which is a cell cycle-related protein. In addition, Amuc_1434* promoted apoptosis of LS174T cells and increased mitochondrial ROS levels in LS174T cells. The mitochondrial membrane potential of LS174T cells was also downregulated by Amuc_1434*. Amuc_1434* can activate the death receptor pathway and mitochondrial pathway of apoptosis by upregulating tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL). In conclusion, our study was the first to demonstrate that the protein Amuc_1434* derived from Akkermansia muciniphila suppresses LS174T cell viability via TRAIL-mediated apoptosis pathway.

A Purified Aspartic Protease from Akkermansia Muciniphila Plays an Important Role in Degrading Muc2.

Akkermansia muciniphila can produce various mucin-degrading proteins. However, the functional characteristics of these proteins and their role in mucin degradation are unclear. Of the predicted protein-coding genes, Amuc_1434, which encodes for a hypothetical protein, is the focus in this study. A recombinant enzyme Amuc_1434 containing the 6× His-tag produced in Escherichia coli (hereinafter termed Amuc_1434*) was isolated to homogeneity and biochemically characterised. Results showed that the enzyme can hydrolyse hemoglobin with an activity of 17.21 U/µg. The optimal pH and temperature for hemoglobin hydrolysis of Amuc_1434* were found to be around 8.0 and 40 °C, respectively. Amuc_1434* is identified as a member of the aspartic protease family through the action of inhibitor pepstatin A. Amuc_1434* promotes the adhesion of colon cancer cell line LS174T, which can highly express Muc2. Significantly Amuc_1434* can degrade Muc2 of colon cancer cells. Amuc_1434 is mainly located in the colon of BALB/c mice. These results suggest that the presence of Amuc_1434 from Akkermansia muciniphila may be correlated with the restoration of gut barrier function by decreasing mucus layer thickness.

Screening, Identification, and Characterization of an Affinity Peptide Specific to MT1-MMP and Its Application in Tumor Imaging.

Membrane type-1 matrix metalloproteinase (MT1-MMP) plays a crucial role in many physiological and pathological processes, especially in tumor invasion and metastasis. Bioimaging of this key molecule may find wide usage in various applications. MT-loop is a unique sequence of MT1-MMP and locates in the surface of the protein. In our previous studies, AF7p, an affinity peptide that targeting the MT-loop domain of MT1-MMP, was identified by screening a phage display (Ph.D.) peptide library. However, the target of AF7p is a synthetic sequence which lacked native conformation of the MT-loop region; thus, the binding affinity and specificity in reality may not be optimal. In this study, we considered the 3-dimensional (3-D) conformation of the MT-loop area in the MT1-MMP molecule and designed a novel strategy to screen the Ph.D. peptide library. The peptide we obtained showed a better binding affinity to WT-MT1-MMP than AF7p as observed through enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). The new peptide labeled and attached MT1-MMP expression cell lines HT1080 and did not show any toxicity to cells. Furthermore, for in vivo imaging, HT1080 tumor-bearing mice with higher MT1-MMP expression accumulated more Cy5.5-HS7 than mice with MT1-MMP low-expression cell lines A549 at tumor sites, and the half-life of HS7 was longer than that of AF7p, as confirmed by ex vivo imaging of the main organs. These results suggest the feasibility of using the subtraction biopanning strategy to screen the affinity peptide targeting MT-loop regions and HS7 is a superior probe for noninvasively imaging MT1-MMP expression in MT1-MMP-positive tumor models. It provides impetus for further studies to use HS7 in early diagnosis of tumors and in peptide-mediated drugs.

Identifying a Membrane-Type 2 Matrix Metalloproteinase-Targeting Peptide for Human Lung Cancer Detection and Targeting Chemotherapy with Functionalized Mesoporous Silica.

Membrane-type 2 matrix metalloproteinase (MT2-MMP) is critical for the aggressive lung tumor growth, progression, and metastasis. Here, to obtain the peptides in binding specifically to MT2-MMP, a phage-displayed 12 peptide library was used and the affinity of peptides toward MT2-MMP was identified by multitest methods. The results showed that a specific MT2-MMP-targeting peptide with the sequence of HHRLHSAPPPQA (MT2-AF5p) exhibited a high specificity and strong affinity against lung tumors. To further achieve specific targeting and precise therapeutic effects, MT2-AF5p was conjugated onto fluorescent mesoporous silica nanoparticles (FMSN-NH2) and loaded with doxorubicin (DOX) to construct a chemotherapeutic drug-targeting delivery system (DOX-loaded FMSN@MT2-AF5p). The DOX-loaded FMSN@MT2-AF5p achieved a boost in DOX release in an acidic environment. Most importantly, FMSN@MT2-AF5p efficiently targeted the tumor area, as seen in the fluorescent imaging ex vivo. The novel peptide-functionalized nanoparticles with a good biocompatibility are promising for clinical use as a precise targeting nanodrug for lung cancer diagnosis and therapy.

Identification of the endoplasmic reticulum localization sequence and N-glycosylation of matrix metalloproteinase 26.

Matrix metalloproteinase 26 (MMP-26), also called endometase and matrilysin-2, belongs to the MMP superfamily. Previous studies have focused on its role in tumor invasion and migration but detailed subcellular localization of MMP-26 remains poorly understood. In this study, sequence deletion mutants of MMP-26 revealed that residues 88-123 function to localize MMP-26 to the endoplasmic reticulum (ER). Moreover, using homologous recombination, we show that exchanging residues 88-123 of secretory MMP-7 with the same region in MMP-26 causes localization of this MMP-7 construct to the ER. Moreover, two (N64, N221) of the three possible N-glycosylation sites in MMP-26 were shown to be N-glycosylated, and N-glycosylation is not required for ER localization. These results demonstrate that the 88-123 region of MMP-26 is a noncanonical ER retention signal and MMP-26 is an N-glycosylated protein, thereby providing novel insights into the properties of MMP-26 within the cell.

In silico study on identification of novel MALT1 allosteric inhibitors.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which plays a crucial role in the nuclear factor-kappa B (NF-κB) activation signaling pathway as a paracaspase, is a new target for immunomodulatory and antitumor drugs. Here, novel inhibitors that target MALT1 allosteric sites were identified by virtual screening FDA-approved drug databases. Paliperidone, a compound that binds to the allosteric site of MALT1, is investigated. An in vitro study found that the proteolytic activity of MALT1 substrate cleavage was blocked by paliperidone. Meanwhile, the MALT1 proteolytic activity was reversible, as demonstrated by the partial recovery of the MALT1 substrate cleavage following compound wash out. The docking analysis of the interaction of MALT1 and paliperidone suggested that two hydrogen bonds formed in the allosteric pocket of MALT1. MALT1 and paliperidone achieved a good equilibrium, as demonstrated by 100 ns molecular dynamic (MD) simulations conducted with the program Gromacs. However, the catalytically active site of the MALT1 complex with paliperidone remained in an inactive conformation. Thus, paliperidone, a noncompetitive and allosteric inhibitor, was screened through in silico and in vitro methods. This study will be of significance for the development of effective and selective drugs that can treat MALT1-driven cancer or autoimmune diseases.

Two new secolignans from the roots of Urtica fissa.

Two new secolignans, 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyrolactone (1) and 3,4-trans-3-hydroxymethyl-4- [bis(3,4-dimethoxyphenyl)methyl]butyrolactone (2) have been isolated from the roots of Urtica fissa E.Pritz. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS. The inhibitory effects on N1 and N2, two subtypes of neuraminidases (NAs), of these two compounds were assayed.

Optimization of a MT1-MMP-targeting Peptide and Its Application in Near-infrared Fluorescence Tumor Imaging.

Membrane type 1 metalloproteinase (MT1-MMP) is an important regulator of cancer invasion, growth and angiogenesis, thus making it an attractive target for cancer imaging and therapy. A non-substrate peptide (MT1-AF7p) that bonded to the "MT-Loop" region of MT1-MMP was identified by using a phage-displayed peptide library and was used to image the MT1-MMP expression in vivo through optical imaging. However, the substrate in the screening did not have a 3D structure, thus resulting in a loose bonding of MT1-AF7p. To simulate the real conformation of the "MT-Loop" and improve the performance of MT1-AF7p, molecular simulations were performed, because this strategy provides multiple methods for predicting the conformation and interaction of proteinase in 3D. In view of the binding site of the receptor-ligand interactions, histidine 4 was selected for mutation to achieve an increased affinity effect. The optimized peptides were further identified and conformed by atomic force microscopy, isothermal titration calorimetry, cell fluorescence imaging in vitro, and near-infrared fluorescence tumor optical imaging in vivo. The results revealed that the optimized peptide with a mutation of histidine 4 to arginine has the highest affinity and specificity, and exhibited an increased fluorescence intensity in the tumor site in optical imaging.

Inhibiting effects of common trivalent metal ions on transmembrane-type 2 matrix metalloproteinase.

Transmembrane-type 2 matrix metalloproteinase (MT2-MMP) degrades connective extracellular matrix between cells and enables tumor cells to migrate and metastasize, making this substance a potential therapeutic target in various diseases. In this work, the interactions between MT2-MMP and common trivalent metal ions, including aluminum (Al3+) and ferrum (Fe3+) ions, were investigated. Enzymatic detection revealed that Al3+ and Fe3+ strongly inhibited the MT2-MMP. Fluorescence spectrography elucidated a static quenching interaction between the negatively charged amino acids on MT2-MMP and the inhibitory trivalent metal ions, indicating that a stable complex was formed between MT2-MMP and metal ions. In addition, fluorescence data and molecular modeling analysis of the binding characteristics revealed that one trivalent metal ion bound with a protein in the stable complex formation process. The potential inhibitory effect of Al3+ on MT2-MMP was further examined in an MT2-MMP-overexpressed cell line, HT1080, by using flow cytometry. As a result, Al3+ can promote HT1080 cell apoptosis in a micromolar concentration-dependent manner. This work illustrated that common trivalent metal ions can potentially inhibit MT2-MMP-related tumors.

Quantum dots tethered membrane type 3 matrix metalloproteinase-targeting peptide for tumor optical imaging.

Membrane type matrix metalloproteinases (MT-MMPs) play important roles in malignant tumor progression through the degradation of the extracellular matrix and signal transduction. However, a member of the family, MT3-MMP, has attracted the least concern compared with other MT-MMPs. Here, a novel MT3-MMP-targeting peptide with high affinity and specificity has been developed by a phage-display peptide screening technology and multiple biophysics measurements, including single-molecule recognition force spectroscopy and isothermal titration calorimetry. The binding peptides are conjugated on the surface of CdSe/ZnS quantum dots (QDs) and consequently acted as a ligand that specifically targets MT3-MMP overexpressed tumor cells. The imaging nanoprobes used QDs as the photographic developer for optical imaging in vivo. The nanoprobes exhibited a desirable targeting effect and generated good biodistribution profiles for visualization and imaging of MT3-MMP overexpressed tumor. The peptide could be useful to evaluate the distribution and expression of MT3-MMP. Furthermore, the peptide-functionalized QDs show potential application for cancer diagnosis.