The Content and Principle of the Rare Ginsenosides Produced from Gynostemma pentaphyllum after Heat Treatment.

Ginsenoside Rg3, Rk1, and Rg5, rare ginsenosides from Panax ginseng, have many pharmacological effects, which have attracted extensive attention. They can be obtained through the heat treatment of Gynostemma pentaphyllum. In this study, scanning electron microscopy (SEM) and thermal gravity-differential thermal gravity (TG-DTG) were employed to investigate this process and the content change in ginsenosides was analyzed using liquid chromatography-mass spectrometry (LC-MS). SEM and TG-DTG were used to compare the changes in the ginsenosides before and after treatment. In SEM, the presence of hydrogen bond rearrangement was indicated by the observed deformation of vascular bundles and ducts. The before-and-after changes in the peak patterns and peaks values in TG-DTG indicated that the content of different kinds of compounds produced changes, which all revealed that the formation of new saponins before and after the heat treatment was due to the breakage or rearrangement of chemical bonds. Additionally, the deformation of vascular bundles and vessels indicated the presence of hydrogen bond rearrangement. The glycosidic bond at the 20 positions could be cleaved by ginsenoside Rb3 to form ginsenoside Rd, which, in turn, gave rise to ginsenoside Rg3(S) and Rg3(R). They were further dehydrated to form ginsenoside Rk1 and Rg5. This transformation process occurs in a weak acidic environment provided by G. pentaphyllum itself, without the involvement of endogenous enzymes. In addition, the LC-MS analysis results showed that the content of ginsenoside Rb3 decreased from 2.25 mg/g to 1.80 mg/g, while the contents of ginsenoside Rk1 and Rg5 increased from 0.08 and 0.01 mg/g to 3.36 and 3.35 mg/g, respectively. Ginsenoside Rg3(S) and Rg3(R) were almost not detected in G. pentaphyllum, and the contents of them increased to 0.035 and 0.23 mg/g after heat treatment. Therefore, the rare ginsenosides Rg3(S), Rg3(R), Rk1, and Rg5 can be obtained from G. pentaphyllum via heat treatment.

The role of multiple metabolic genes in predicting the overall survival of colorectal cancer: A study based on TCGA and GEO databases.

The recent advances in gene chip technology have led to the identification of multiple metabolism-related genes that are closely associated with colorectal cancer (CRC). Nevertheless, none of these genes could accurately diagnose or predict CRC. The prognosis of CRC has been made by previous prognostic models constructed by using multiple genes, however, the predictive function of multi-gene prognostic models using metabolic genes for the CRC prognosis remains unexplored. In this study, we used the TCGA-CRC cohort as the test dataset and the GSE39582 cohort as the experimental dataset. Firstly, we constructed a prognostic model using metabolic genes from the TCGA-CRC cohort, which were also associated with CRC prognosis. We analyzed the advantages of the prognostic model in the prognosis of CRC and its regulatory mechanism of the genes associated with the model. Secondly, the outcome of the TCGA-CRC cohort analysis was validated using the GSE39582 cohort. We found that the prognostic model can be employed as an independent prognostic risk factor for estimating the CRC survival rate. Besides, compared with traditional clinical pathology, it can precisely predict CRC prognosis as well. The high-risk group of the prognostic model showed a substantially lower survival rate as compared to the low-risk group. In addition, gene enrichment analysis of metabolic genes showed that genes in the prognostic model are enriched in metabolism and cancer-related pathways, which may explain its underlying mechanism. Our study identified a novel metabolic profile containing 11 genes for prognostic prediction of CRC. The prognostic model may unravel the imbalanced metabolic microenvironment, and it might promote the development of biomarkers for predicting treatment response and streamlining metabolic therapy in CRC.

Novel Cyclic Peptides from Lethal Amanita Mushrooms through a Genome-Guided Approach.

Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the "MSDIN" family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms.

Synthesis and evaluation of disulfide-rich cyclic α-conotoxin [S9A]TxID analogues as novel α3ß4 nAChR antagonists.

Head-to-tail cyclization is an effective strategy to improve the biological stability of peptides. The α-conotoxin [S9A]TxID is a peptide that inhibits α3ß4 nAChR with high activity and selectivity. Herein, we established a method for cyclizing and oxidative folding of [S9A]TxID, and six cyclic analogues of [S9A]TxID were chemically synthesized with various linker lengths. We used the electrophysiology assay to measure activity values of these cyclic analogues, and obtained the most potent analogue c[S9A]TxID-6, which was more stable than native [S9A]TxID against proteinase K.

Effects of Cyclization on Activity and Stability of α-Conotoxin TxIB.

α-Conotoxin TxIB specifically blocked α6/α3ß2ß3 acetylcholine receptors (nAChRs), and it could be a potential probe for studying addiction and other diseases related to α6/α3ß2ß3 nAChRs. However, as a peptide, TxIB may suffer from low stability, short half-life, and poor bioavailability. In this study, cyclization of TxIB was used to improve its stability. Four cyclic mutants of TxIB (cTxIB) were synthesized, and the inhibition of these analogues on α6/α3ß2ß3 nAChRs as well as their stability in human serum were measured. All cyclized analogues had similar activity compared to wild-type TxIB, which indicated that backbone cyclization of TxIB had no significant effect on its activity. Cyclization of TxIB with a seven-residue linker improved its stability significantly in human serum. Besides this, the results showed that cyclization maintained the activity of α-conotoxin TxIB, which is conducive to its future application.

Structure and Activity Studies of Disulfide-Deficient Analogues of αO-Conotoxin GeXIVA.

αO-conotoxin GeXIVA from Conus generalis is a potent antagonist of the α9α10 nAChR and analgesic in animal models of pain. This peptide has two disulfide bond cross-links, and the bead and ribbon isomers have similar inhibitory activity against α9α10 nAChRs. We synthesized 12 disulfide-deficient analogues of bead GeXIVA, and all remained potent inhibitors of α9α10 nAChR. The most potent disulfide-deficient analogue displayed IC50 values of 6 and 33 nM at rat and human α9α10 nAChRs, respectively, representing less than a 2-fold increase compared with bead GeXIVA. The disulfide-deficient analogs and parent peptides also do not have a well-defined structure according to NMR spectroscopy. Molecular simulations suggest that the disulfide bonds and termini of GeXIVA do not establish stable interactions with the receptor. Overall, this study proposes that the structure of the analgesic peptide GeXIVA could be simplified through disulfide bond deletions and potentially termini truncations.

Interaction of rat α9α10 nicotinic acetylcholine receptor with α-conotoxin RgIA and Vc1.1: Insights from docking, molecular dynamics and binding free energy contributions.

The α9α10 nicotinic acetylcholine receptor (nAChR) is an effective therapeutic target for neuropathic pain. α-Conotoxin RgIA and Vc1.1 are two well-known peptides blocking α9α10 nAChR potently and selectively, which have been extensively investigated as drug candidates. Several key residues were established in previous experimental research. However, the mechanism of the specific interaction still needs to be elucidated in more detail. In this work, we explored the interaction mechanism between RgIA/Vc1.1 and rat α9α10 nAChR using docking and molecular dynamics (MD) simulations. Energy and network analysis programs were used to reveal key residues responsible for their interaction. Our results indicated that the most critical residues were in accord with previous studies. Importantly, several novel residues, including Tyr95, Trp151 in α9 (+)α10 (-) interface as well as Tyr196, Arg59in α10 (+)α9 (-) interface, were found in our models. Furthermore, we analyzed noncovalent interaction energies between RgIA/Vc1.1 and rat α9α10 nAChR. The results showed that three negatively charged residues (Glu197 in α10 subunit, Asp168 in α9 subunit and Asp205 in α10 subunit) were involved in the interaction between RgIA and rat α9α10 nAChR. In contrast, the interaction between Vc1.1 and rat α9α10 nAChR was mediated by the positively charged residues Arg59, Arg81 in α9 (-) subunit. These findings provided further insights into the molecular mechanisms of interaction between RgIA and Vc1.1 and rat α9α10 nAChR.