The involvement of calcium in the toxic effect of 4-methylethcathinone on SH-SY5Y cells.

Neurotoxicity induced by psychoactive substances is often accompanied by an imbalance of intracellular calcium ions. It is unclear whether calcium ions play a role in the toxicity induced by psychoactive substances. In the present study, we aimed to evaluate the occurrence of calcium dysregulation and its contribution to cytotoxicity in human neurotypic SH-SY5Y cells challenged with a recently developed psychoactive substance 4-methylethcathinone (4-MEC). An increase in the intracellular calcium was detected by inductively coupled plasma atomic emission spectrometry and Fluo-3 AM dye in SH-SY5Y cells after being treated with 4-MEC. The increase of intracellular Ca2+ level mediated G0/G1 cell cycle arrest and ROS/endoplasmic reticulum stress-autophagy signaling pathways to achieve the toxicity of 4-MEC. In particular, N-acetyl-L-cysteine, a classical antioxidant, was found to be a potential treatment for 4-MEC-induced toxicity. Taken together, our results demonstrate that an increase in intracellular calcium content is one of the mechanisms of 4-MEC-induced toxicity. This study provides a molecular basis for the toxicity mechanism and therapeutic intervention of psychoactive substances.

The correlation between CpG island methylation of hTERT promoter and human age prediction.

DNA methylation is an epigenetic modification that occurs during the life cycle of individuals. Its degree is closely associated with the methylation status of CpG sites in its promoter region. Based on the previous screening that the hTERT methylation is both related to tumors and age, we suspected that the age inference based on hTERT methylation would be disturbed by the disease of the tested person. Herein, eight CpG sites in the hTERT promoter region were analyzed by real-time methylation-specific PCR, and we found that CpG2, CpG5, and CpG8 were closely related to the tumor (P < 0.05). The remaining five CpG sites had a large error in predicting age alone. Combining them to establish a model yielded better results, with an average age error of 4.35 years. This study provides a reliable and accurate detection method for the DNA methylation status of multiple CpG sites on the hTERT gene promoter, which can be used for the prediction of forensic age and assistant diagnosis of clinical diseases.

Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells.

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Identification of three novel new psychoactive substances 4F-AB-BUTINACA, AB-PHETINACA, and 2F-NENDCK.

The identification of new psychoactive substances (NPS) is an active and cutting-edge topic in forensic science. With the emergence of a large number of NPS, their timely identification to prevent spread can pose a challenge to clinical and forensic toxicology laboratories. Three emerging NPS had been identified in recently seized materials, including two synthetic cannabinoids [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-AB-BUTINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-phenethyl-1H-indazole-3-carboxamide (AB-PHETINACA)] and a ketamine-like substance [2-(2-fluorophenyl)-2-(ethylamino) cyclohexan-1-one(2F-NENDCK)]. The three compounds were first identified by Fourier transform infrared spectrometry (FT-IR), gas chromatography-mass spectrometry (GC-MS), ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS), and nuclear magnetic resonance (NMR). These data may assist forensic analysts in analyzing the same substances or their homologous compounds.

Antimicrobial and anticancer activities of Hainan dry noni fruit alcoholic extracts and their novel compounds identification using UPLC-Q-Exactive Obitrap-MS/MS.

Morinda citrifolia Linn (noni) is an important plant in the Pacific Asian region. The fruit has been used as a food source and has shown therapeutical benefits for health. Recently, it has become a source for bioactive compounds. In this study, we investigated the antimicrobial and anticancer activities of alcoholic extracts of Hainan dry noni fruit with machinery assistance and identified their novel compounds by UPLC-Q-Exactive Obitrap-MS/MS. By IE extractor aided method, the extraction of both NFE (Noni Fruit Ethanol) and NFM (Noni Fruit Methanol) solvent crude sample extracts were obtained with recovery yields of 98.48% and 71.65%, respectively. The antimicrobial effect of the crude extracts was subjected to disc diffusion test screening against two microbial strains bacterium SA (Staphylococcus aureus) and, fungal CA (Candida albicans). The MIC values of SA and CA were 35.34 and 47.80 mg/mL for NFE, 117.40 and 108.01 mg/mL for NFM, respectively. Further on, cell viability assay showed that IC50 values of extract NFE and NFM on human UMUC-3 bladder carcinogenic cells were 865.1 and 789.1 µg/mL with less effect to human SVHUC-1 normal cell line for 72hr incubation. Using UPLC-Q-exactive Orbitrap-MS/MS, ten compounds were identified in the noni extracts and confirmed from the HMDB and FooDB. Five known bioactive compounds had been used for treatments in anti-cancer, anti-obesity, and Covid-19 patients. The remaining five compounds were found novel in noni fruit. They were Cyanidin 3-(2 G-xylosylrutinoside), Inulobiose, Clausarinol, Pectachol, and 4,7-Megastigmadien-9-ol. The potential bioactivities of these novel compounds will be studied in the near future. These findings form a basis on screening natural medicinal plant extracts for beneficial use as a food and health source.

Effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella.

Berberine, a traditional Chinese medicine, was found to exhibit anticoccidial activity. However, its mechanism is unclear. Trace metals such as copper and zinc are extremely low (less than 0.01% of the total weight of the body) but play a vital role in organisms. In the present study, we investigated the effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella. Firstly, our data confirmed that infected chickens with E. tenella exhibited classic impairment on the 8th day of post infection, such as weight loss and increased feed conversion. Further study showed that E. tenella infection decreased the contents of copper and zinc in the liver and serum of chickens. Berberine was similar to amprolium and significantly improved the pathogenic conditions. Berberine could restore copper and zinc imbalance caused by E. tenella in chickens to a large extent. Studies on the development of cecum lesions demonstrated that the protective effect of berberine on the intestinal cecum was similar to that of the Cu/Zn mixture. Additionally, the mRNA expression of several metal transport related genes of the chick small intestine, including zinc transporter 1, copper transporter 1 and divalent metal ion transporter 1, was elevated by the treatment with berberine. Taken together, we speculate that the anticoccidial activity of berberine may be related to the maintenance of certain metals (Cu/Zn) homeostasis by affecting mRNA expression of their transport genes. However, the mode of action of BBR on these vital metals in the chicks infected with E. tenella still needs to be further studied.

Rapid quantitation of three synthetic cathinones in urine by magnetic dispersive solid-phase extraction combined with DART-HRMS.

For the rapid quantitation of three synthetic cathinones, namely 1-(4-chlorophenyl)-2-(1-pyrrolidinyl)pentan-1-one (4-Cl-α-PVP), 1-(4-methylphenyl)-2-(methylamino)pentan-1-one (4-MPD), and 1-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-(1-pyrrolidinyl)pentan-1-one (ß-TH-naphyrone), in urine, a new method was established using magnetic dispersive solid-phase extraction (MDSPE) combined with direct analysis in real time and high-resolution mass spectrometry (DART-HRMS). Methcathinone-D3 and proadifen (SKF525A) were used as the internal standards. Hydrophobic magnetic adsorbents were used and consisted of hydrophobic functional group (divinylbenzene) and hydrophilic functional group (vinylpyrrolidone) at a ratio of 3 : 1, and NaH2PO4//NaOH buffer (0.2 M, pH 7) was used in MDSPE. Detection was conducted by DART-HRMS in less than 1 min. For 4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone, the limits of detection were 0.1 ng mL-1, 0.05 ng mL-1 and 0.1 ng mL-1, and the linear ranges were 0.5-100 ng mL-1, 0.2-100 ng mL-1 and 0.2-100 ng mL-1, respectively. The correlation coefficients were all greater than 0.99. The precision and deviation of accuracy were all within ±15%, and the stability of the samples was high under various conditions. The method was successfully applied to detect 4-Cl-α-PVP, 4-MPD and ß-TH-naphyrone in rat urine after subcutaneous administration. In summary, a fast and convenient detection method was established, providing new and effective technical support for the rapid quantitation of three synthetic cathinones (4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone) for forensic purposes.

BSC2 enhances cell resistance to AmB by inhibiting oxidative damage in Saccharomyces cerevisiae.

Amphotericin B has been the gold standard for the treatment of invasive mycosis for many years. Its resistance mechanisms are reported to be mainly related to the decrease of ergosterol content or the changes of cell wall. Previous study has shown that Saccharomyces cerevisiae strain lack of BSC2 was sensitive significantly to Amphotericin B. In the present study, the role of BSC2 on Amphotericin B resistance were investigated. We found that BSC2 enhanced the resistance of yeast cells to Amphotericin B, which was not related to cellular ergosterol content. BSC2 can maintain the permeability of mitochondrial membrane and cell membrane integrity by inhibiting the accumulation of intercellular reactive oxygen species and alleviating the production of lipid peroxidation and superoxide radical. These alterations were attributed to the enhancement of the activities of superoxide dismutase, catalase and glutathione peroxidase, and the increased glutathione content. Taken together, BSC2 inhibits oxidative damage induced by Amphotericin B through increasing activities of antioxidant enzymes and levels of GSH to alleviate the accumulation of reactive oxygen species, lipid peroxidation and superoxide radical, resulting in the maintenance of mitochondrial membrane potential and cell membrane integrity. However, Amphotericin B resistance mediated by BSC2 is independent of Yap1p, GSH1 and Hog1p. The results demonstrate for the first time that BSC2 enhances cell resistance to Amphotericin B by inhibiting oxidative damage in yeast. Our findings improve current understanding of the mechanism of Amphotericin B resistance and provide potential strategy for reducing Amphotericin B resistance.

Effect of cadmium on mRNA mistranslation in Saccharomyces cerevisiae.

Although highly accurate molecular processes and various messenger RNA (mRNA) quality control and ribosome proofreading mechanisms are used by organisms to transcribe their genes and maintain the fidelity of genetic information, errors are inherent in all biological systems. Low-level translation errors caused by an imbalance of homologous and nonhomologous amino acids caused by stress conditions are particularly common. Paradoxically, advantageous phenotypic diversity can be generated by such errors in eukaryotes through unknown molecular processes. Here, we found that the significant cadmium-resistant phenotype was correlated with an increased mistranslation rate of the mRNA in Saccharomyces cerevisiae. This phenotypic change was also related to endogenous sulfur amino acid starvation. Compared with the control, the mistranslation rate caused by cadmium was significantly increased (p < .01). With the increase of cysteine contents in medium, the mistranslation rate of WT(BY4742a) decreased significantly (p < .01). This demonstrates that cadmium treatment and sulfur amino acid starvation both can induce translation errors. Although cadmium uptake is independent of the Sul1 transporter, cadmium-induced mRNA mistranslation is dependent on the sulfate uptake of the Sul1p transporter. Furthermore, cadmium-induced translation errors depend on methionine biosynthesis. Taken together, cadmium causes endogenous sulfur starvation, leading to an increase in the mRNA mistranslation, which contributes to the resistance of yeast cells to cadmium. We provide a new pathway mediating the toxicity of cadmium, and we propose that altering mRNA mistranslation may portray a different form of environmental adaptation.

Succession of oral microbiota community as a tool to estimate postmortem interval.

The establishment of postmortem interval is one of the most important aspects of forensic expertise. Microbes may provide a novel way to estimate the postmortem intervals in order to avoid many of these limitations. The oral cavity harbors one of the most diverse microbiomes that play a key role in the decomposition of corpses. In this study, the oral bacterial community showed obvious changes in relative abundance during the process of mice decomposition. Meanwhile, at different taxonomic levels, specific bacteria were found to be significantly correlated with the postmortem interval. Linear regression models between relative abundance and the postmortem interval were constructed. Among these species, Gamma-proteobacteria and Proteus were the best ones that can be used to infer the postmortem interval, especially late postmortem interval. Therefore, we suggest that succession of oral microbial community can be developed as a forensic tool for estimating the postmortem interval.