Cooperative effects of three preservatives on physiological quality, endophytic bacterial community and volatile organic compounds of postharvest Codonopsis pilosula var. modesta roots.

BACKGROUND: Codonopsis pilosula var. modesta (CPVM) is a famous medicinal and edible plant of Campanulaceae. However, fresh CPVM roots (FCPVR) are prone to softening, browning and spoilage after concentrated harvesting in the main production area of Gansu Province, China in autumn, which poses great challenges to their large-scale storage and modern processing. In this study, effects of chitosan (CS), natamycin (NA) and modified atmosphere agent (MA) on the postharvest quality of FCPVR were first investigated. The roots after different treatments were stored at 4 °C and relative humidity of 75 ± 5% for 100 days. Their overall quality changes were evaluated from three perspectives: physiological quality, endophytic bacterial community and volatile organic compounds. RESULTS: The clustering heatmap and principal component analysis results indicated that CS (2 g kg-1), NA (0.5 g kg-1) and MA (5 g) had a synergistic effect on physiological quality. The roots in the CS + NA + MA group maintained better physiological state, effective components and antioxidant capacity throughout the storage process. On this basis, compared with room temperature storage, the relative abundance of the main spoilage bacterium Pseudomonas in the CS + NA + MA group roots decreased by 44% on the 100th day of storage. Furthermore, after CS + NA + MA composite treatment, the roots produced richer esters with fruit aroma during low-temperature storage. CONCLUSIONS: The CS + NA + MA composite treatment could maintain the physiological quality and flavor of FCPVR, inhibit spoilage by microbial contamination and maintain the optimal quality during low-temperature storage for up to 100 days. © 2024 Society of Chemical Industry.

Microglial Piezo1 senses Aß fibril stiffness to restrict Alzheimer's disease.

The pathology of Alzheimer's disease (AD) is featured with extracellular amyloid-ß (Aß) plaques, whose impact on the mechanical properties of the surrounding brain tissues is unclear. Microglia sense and integrate biochemical cues of the microenvironment. However, whether the microglial mechanosensing pathways influence AD pathogenesis is unknown. Here, we surveyed the elevated stiffness of Aß-plaque-associated tissues and observed the selective upregulation of the mechanosensitive ion channel Piezo1 in Aß-plaque-associated microglia. Piezo1 sensed the stiffness stimuli of Aß fibrils and subsequently induced Ca2+ influx for microglial clustering, phagocytosis, and compacting of Aß plaques. Microglia lacking Piezo1 led to the exacerbation of Aß pathology and cognitive decline, whereas pharmacological activation of microglial Piezo1 ameliorated brain Aß burden and cognitive impairment in 5 × FAD mice. Together, our results reveal that Piezo1, a mechanosensor of Aß fibril stiffness in microglia, represents a potential therapeutic target for AD.

Discrimination of three varieties of Codonopsis Radix based on fingerprint profiles of oligosaccharides by high performance liquid chromatography- evaporative light scattering detector combined with multivariate analysis.

Codonopsis Radix (CR) is a plant that is important in the practice of traditional Chinese medicine (TCM). The Chinese Pharmacopoeia 2020 records dried roots prepared from three varieties of Campanulaceae plants under the designation CR ("Dang-shen" in Chinese), including Codonopsis pilosula (Franch.) Nannf (C. pilosula), Codonopsis pilosula Nannf.var.modesta (Nannf.) L81. T. Shen (C. pilosula var. modesta) and Codonopsis tangshen Oliv (C. tangshen). As major constituents of CR, oligosaccharides might contribute to its clinical efficacy except for other known active compounds, yet the differences in the oligosaccharide profiles of these three varieties of CR remain incompletely understood. In the present study, 135 samples from these different CR varieties were harvested, and oligosaccharide fingerprints for these samples were characterized via HPLC-ELSD, with 19 common peaks being matched. Oligosaccharides were further identified through the combination of electrospray ionization MS/MS (ESI-MS/MS) with nuclear magnetic resonance (NMR). Principal component analysis and linear discriminant analysis (LDA) approaches were then used to compare the oligosaccharide profiles of these three CR varieties. These analyses ultimately revealed that CR was compared with principal component analysis (PCA) and linear discriminant (LDA) methods. The analyses ultimately revealed that these CR samples contained high levels of inulin- and levan-type fructooligosaccharides (FOS), with variations in the relative levels of these FOS compounds among the three analyzed CR varieties. Through the combined analysis of oligosaccharide fingerprints and LDA results, it was possible to differentiate among these CR varieties, with an accurate classification rate of 96.3% and a cross-validation rate of 95.6%. Together, these results highlight a valuable approach to the classification and identification of different CR varieties.

Mammalian target of rapamycin inhibitor abrogates abnormal osteoclastogenesis in neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is the most common genetic syndrome predisposing patients to various tumors due to dysregulation of the Ras signaling pathway. Recent research has shown NF1 patients also suffer a spectrum of bone pathologies. The pathogenesis of NF1 bone diseases is largely unknown. There is no current treatment. By Nf1 heterozygote (Nf1+/-) mice and Nf1 conditional knockout mice, we and other groups demonstrated abnormal osteoblast and osteoclast function due to dysregulation of Ras signaling. However, the specific downstream effector pathways linked to NF1 abnormal osteoblastogenesis and osteoclastogenesis have not been defined. In this study, we investigated the Ras downstream effector related with NF1 bone disease. METHODS: We used Nf1+/+ and Nf1+/- mice as normal and NF1 models. Bone stromal cells extracted from Nf1+/+ and Nf1+/- mice were induced osteoclasts. The osteoclast cell was stained by tartrate resistant acid phosphatase staining. The osteoclast cell number was counted and the surface area of osteoclast cells was calculated under the microscope. The mRNA of mammalian target of rapamycin (mTOR) was determined by quantitative reverse-transcription-polymerase chain reaction. The presence of ribosomal protein S6 kinase was determined by Western blotting. RESULTS: Compared with Nf1+/+ mice, Nf1+/- mice had about 20% more of osteoclast cells. These osteoclast cells were larger in size with more nuclei. Hyperactive mTOR was detected in Nf1+/- osteoclast cells. Inhibition of mTOR signaling by rapamycin in Nf1+/- osteoclasts abrogated abnormalities in cellular size and number. CONCLUSION: mTOR pathway inhibition may represent a viable therapy for NF1 bone diseases.

Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. METHODS: Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B. RESULTS: HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. CONCLUSIONS: DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.

MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1.

MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting neurofibromin and RAS signaling.

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells.

5-Aza-2'-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [(3)H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as gamma-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.

An ATM- and Rad3-related (ATR) signaling pathway and a phosphorylation-acetylation cascade are involved in activation of p53/p21Waf1/Cip1 in response to 5-aza-2'-deoxycytidine treatment.

Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2'-deoxycytidine (5-aza-CdR) or cytarabine treatment. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (ATR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of ATR to chromatin. Upon treatment with 5-aza-CdR, ATR activation is clearly associated with p53 phosphorylation at Ser(15), but not at Thr(18), Ser(20), or Ser(37). This specific p53 phosphorylation at Ser(15) in turn results in acetylation of p53 at Lys(320) and Lys(373)/Lys(382) through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21(Waf1/Cip1) expression because the binding activity of acetylated p53 at Lys(320)/Lys(373)/Lys(382) to the p21(Waf1/Cip1) promoter, as well as p21(Waf1/Cip1) expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser(15) and acetylations at Lys(320)/Lys(373)/Lys(382) mutually interact in the 5-aza-CdR induced p21(Waf1/Cip1) expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21(Waf1/Cip1) expression.

Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1).

Generally, histone deacetylase (HDAC) inhibitor-induced p21(Waf1/Cip1) expression is thought to be p53 independent. Here we found that an inhibitor of HDAC, depsipeptide (FR901228), but not trichostatin A (TSA), induces p21(Waf1/Cip1) expression through both p53 and Sp1/Sp3 pathways in A549 cells (which retain wild-type p53). This is demonstrated by measuring relative luciferase activities of p21 promoter constructs with p53 or Sp1 binding site mutagenesis and was further confirmed by transfection of wild-type p53 into H1299 cells (p53 null). That p53 was acetylated after depsipeptide treatment was tested by sequential immunoprecipitation/Western immunoblot analysis with anti-acetylated lysines and anti-p53 antibodies. The acetylated p53 has a longer half-life due to a significant decrease in p53 ubiquitination. Further study using site-specific antiacetyllysine antibodies and transfection of mutated p53 vectors (K319/K320/K321R mutated and K373R/K382R mutations) into H1299 cells revealed that depsipeptide specifically induces p53 acetylation at K373/K382, but not at K320. As assayed by coimmunoprecipitation, the K373/K382 acetylation is accompanied by a recruitment of p300, but neither CREB-binding protein (CBP) nor p300/CBP-associated factor (PCAF), to the p53 C terminus. Furthermore, activity associated with the binding of the acetylated p53 at K373/K382 to the p21 promoter as well as p21(Waf1/Cip1) expression is significantly increased after depsipeptide treatment, as tested by chromatin immunoprecipitations and Western blotting, respectively. In addition, p53 acetylation at K373/K382 is confirmed to be required for recruitment of p300 to the p21 promoter, and the depsipeptide-induced p53 acetylation at K373/K382 is unlikely to be dependent on p53 phosphorylation at Ser15, Ser20, and Ser392 sites. Our data suggest that p53 acetylation at K373/K382 plays an important role in depsipeptide-induced p21(Waf1/Cip1) expression.