Ultrasensitive upconverting nanoprobes for in situ imaging of drug-induced liver injury using miR-122 as the biomarker.

Drug-induced liver injury (DILI) is a frequent adverse drug reaction. The current clinical diagnostic methods are inadequate for accurate and early detection of DILI due to the lack of effective diagnostic biomarkers. Hepatocyte-specific miR-122 is released from injured hepatocytes promptly and its efflux is significantly correlated with the progression of DILI. Therefore, achieving precise in situ detection of miR-122 with high sensitivity is vital for early visualization of DILI. Herein, a new nanoprobe, consisting of miR-122 aptamer, upconversion nanoparticles (UCNPs) and Prussian blue nanoparticles (PBNPs) was introduced for the early and sensitive detection of DILI in situ. As the nanoprobes reached in the liver, miR-122 aptamer-based entropy-driven strand displacement (ESDR) signal amplification reaction was triggered and luminescence resonance energy transfer (LRET) between UCNPs and PBNPs was responded to achieve the high-fidelity detection of DILI. A negative correlation was observed between the intensity of upconversion luminescence (UCL) and the concentration of miR-122. UCL imaging conducted both in vivo and ex vivo indicated that a reduction in miR-122 concentration led to an increase in UCL intensity, revealing a precise state of DILI. The detection technique demonstrated a positive correlation between signal intensity and severity, offering a more straightforward and intuitive method of visualizing DILI.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.

Comprehensive analysis of JAZ family members in Ginkgo biloba reveals the regulatory role of the GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis.

Ginkgo biloba L., an ancient relict plant known as a 'living fossil', has a high medicinal and nutritional value in its kernels and leaves. Ginkgolides are unique diterpene lactone compounds in G. biloba, with favorable therapeutic effects on cardiovascular and cerebrovascular diseases. Thus, it is essential to study the biosynthesis and regulatory mechanism of ginkgolide, which will contribute to quality improvement and medication requirements. In this study, the regulatory roles of the JAZ gene family and GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis were explored based on genome and methyl jasmonate-induced transcriptome. Firstly, 18 JAZ proteins were identified from G. biloba, and the gene characteristics and expansion patterns along with evolutionary relationships of these GbJAZs were analyzed systematically. Expression patterns analysis indicated that most GbJAZs expressed highly in the fibrous root and were induced significantly by methyl jasmonate. Mechanistically, yeast two-hybrid assays suggested that GbJAZ3/11 interacted with both GbMYC2 and GbCOI1, and several GbJAZ proteins could form homodimers or heterodimers between the GbJAZ family. Moreover, GbMYC2 is directly bound to the G-box element in the promoter of GbLPS, to regulate the biosynthesis of ginkgolide. Collectively, these results systematically characterized the JAZ gene family in G. biloba and demonstrated that the GbCOI1/GbJAZs/GbMYC2 module could regulate ginkgolides biosynthesis, which provides a novel insight for studying the mechanism of JA regulating ginkgolide biosynthesis.

An integrated strategy combining network toxicology and feature-based molecular networking for exploring hepatotoxic constituents and mechanism of Epimedii Folium-induced hepatotoxicity in vitro.

Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.

Baohuoside I inhibits FXR signaling pathway to interfere with bile acid homeostasis via targeting ER α degradation.

Epimedii folium (EF) is an effective herbal medicine in osteoporosis treatment, but the clinical utilization of EF has been limited due to potential hepatotoxicity. The previous studies identified that baohuoside I (BI), the main active component of EF, was relevant to EF-induced liver injury. However, the mechanisms of BI causing direct injury to hepatocytes remain unclear. Here, we reveal that BI inhibits FXR-mediated signaling pathway via targeting estrogen receptor α (ER α), leading to the accumulation of bile acids (BAs). Targeted bile acid analyses show BI alters the BA composition and distribution, resulting in impaired BA homeostasis. Mechanistically, BI induces FXR-dependent hepatotoxicity at transcriptional level. Additionally, ER α is predicted to bind to the FXR promoter region based on transcription factor binding sites databases and we further demonstrate that ER α positively regulates FXR promoter activity and affects the expression of target genes involved in BA metabolism. Importantly, we discover that ER α and its mediated FXR transcription regulation might be involved in BI-induced liver injury via ligand-dependent ER α degradation. Collectively, our findings indicate that FXR is a newly discovered target gene of ER α mediated BI-induced liver injury, and suggest BI may be responsible for EF-induced liver injury.

[Advance in biosynthesis and metabolic regulation of ginkgolides].

Ginkgolides,the unique terpenoids in Ginkgo biloba,have a significant effect on the prevention and treatment of cardiovascular and cerebrovascular diseases. Metabolic regulation and synthetic biology strategies are efficient methods to obtain high-quality ginkgolides. The present study reviewed the cloning and functions of genes related to the biosynthetic pathway of ginkgolides,as well as relevant studies of omics,genetic transformation,and metabolic regulation in recent years,and predicted the research trends and prospects,aiming to provide a reference for discovering the key genes related to the biosynthetic pathway and the biosynthesis of ginkgolides.

A novel method for ginkgolide biosynthesis elucidation based on MeJA induction and differential metabolomics.

Ginkgolides from Ginkgo Biloba have significantly therapeutic effect to cardiovascular and cerebrovascular diseases. However, the biosynthetic pathway of ginkgolides has not been fully elucidated until now. As ginkgolides are synthesized in the ginkgo roots, the accumulation of ginkgolides intermediate metabolites varies greatly between roots and leaves. As Methyl jasmonate (MeJA) can effectively enhance the biosynthesis of ginkgolides, a novel method based on MeJA induction and differential metabolomics was used to screen the differentially intermediate metabolites among ginkgo leaves, roots and roots-MJ-3. Two differential intermediate metabolites (dehydroabietadienal and 1, 2, 3, 4, 4a, 9, 10, 10a-Octahydro-6-hydroxy-7-isopropyl-1, 4a-dimethyl-1-phenanthrenemethanol) were identified in ginkgo roots by UPLC-QTOF-MS. Then, a new ginkgolides biosynthetic pathway was proposed based on differential metabolomics. This study provides a novel method for the elucidation of nature product precursor and is helpful to promote the clarification of ginkgolides biosynthetic pathway.

Comparative analysis and natural evolution of squalene epoxidase in three Fritillaria species.

Fritillariae Bulbus are the most commonly used antitussive and edible herbs in China. Based on UPLC-QTOF-MS and UPLC-QQQ-MS, the validated MRM-based non-targeted quantitative method was applied to determinate the contents of 48 Fritillaria alkaloids (FAs) in three Fritillaria species (F. thunbergii Miq., F. unibracteata and F. ussuriensis). The RNA-Seq results showed that gene transcript levels have different expression patterns in three Fritillaria species. Based on transcriptome data, the full-length cDNA sequences of squalene epoxidase gene were cloned and characterized. Natural evolution of squalene epoxidase genes resulted in four mutations (C236R, M489L, G510A and K517R) in three Fritillaria species. Molecular docking analysis showed that the 236 residue is located inside the pocket and the binding center while other three residues are located on the surface of the protein. Functional verification indicated the mutations of SQE (C236R) could effectively increase the activity of SQE and obtain higher yield of 2,3-oxidosqualene in recombinant yeast. And the mutations of SQE (M489L and G510A), which increased the hydrophobicity of the protein surface, could also enhance the activity of SQE. This study provides major insights into the metabolites differentiation of FAs biosynthesis, and a firm foundation for the quality control and metabolic engineering of Fritillariae bulbus.

[Sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis].

OBJECTIVE: To improve the sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis. METHODS: Filter paper blood samples were collected from 30 non-malaria fever patients and 20 infectious disease patients (common cold, influenza, typhoid, hepatitis, etc.). Four ml blood each taken from one falciparum malaria patient and one vivax malaria patient was serially diluted. Healthy blood sample was used as negative control. Improved direct heating method was used to prepare DNA template. The cytochrome oxidase gene (coxI) located in mitochondrion was selected as target gene. Relevant web resources and software (PUBMED, NCBI-BLAST, Mfold server and Primer Premier 5.0) were employed to design and optimize Tag-primer nested/multiplex PCR (UT-PCR) which was used to test all blood samples. RESULTS: A 611 bp band and a 255 bp band were seen in serially diluted infected blood samples (1,000, 100, 10 and 1 parasite/microl) from P.f and P.v patient tested by UT-PCR. The detection limit of either P. falciparum or P. vivax reached 1 parasite/microl, and the tested blood samples of non-malaria fever patients, patients with other infectious diseases and healthy persons were all negative. Consistent results of each sample in more than 3 duplicated tests were obtained. CONCLUSION: The optimized Tag-primer nested/multiplex PCR shows high sensitivity, specificity and stability in malaria diagnosis.

[Experimental infection of Sarcocystis suihominis in pig and human volunteer in Guangxi].

OBJECTIVE: To confirm existence of Sarcocystis suihominis and possible transmission cycle between human and pigs. METHODS: Based on the human-pig-human infection cycle of Sarcocystis suihominis, feces of naturally infected pigs were collected and over 10,000 sporocysts were received by flotation technique, which were mixed with fodder to infect a normal pig. Fresh pork meat containing mature sarcocysts was chopped into pieces and swallowed by a volunteer (the first author of this paper) with about 71,000 sporocysts. Symptoms and development of the parasites after infection were observed. RESULTS: The volunteer showed abdominal distension in about 5 hours after infection, with watery diarrhea 13 times from the 8th to 36th hour, vomiting 4 times, chilling and fever with a temperature of 38.5 degrees C, dizziness, headache, joint and muscle ache, epigastralgia, and anorexia. Un-sporized sporocysts were found in the faces 10 days after infection and sporocysts appeared on the 12th day. The average size of sporocysts was 11.9 (8.8-14.5) microm x 9.2 (7.5-12.5) microm. The infected pig showed a slight anorexia, fatigue, constipation, hair loosen in 5-8 days after infection, and returned normal on the 17th day. The average size of the sarcocysts was 299.2 (175-575 ) microm x 62.3 (30-102.5) microm. Size of bradyzoites was 11.5 (9.5-13.5) microm x 4.1 (2.8-5.0) microm. The volunteer was treated with acetylspiramycin for 15 days (0.2 g/time, 4 times/d) after 46 days of infection, and fecal examination turned negative 30 days later. CONCLUSION: There is a man-pig cycle for Sarcocystis suihominis in Guangxi.

[A discussion on the CSP genotyping of Plasmodium vivax and malaria control in five southern provinces of China].

OBJECTIVE: To explore circumsporozoite protein (CSP) genotype structure of Plasmodium vivax in southern China and evaluate its epidemiological significance. METHODS: Filter paper blood samples were collected from 346 vivax malaria patients in 5 provinces (Autonomous Region) including Hainan, Yunnan, Guangxi, Guangdong and Guizhou for identifying CSP genotypes, by using the method of single-tube nested/multiplex PCR. The findings combined with relevant data were statistically analyzed. RESULTS: In Guangdong, Guangxi and Guizhou Provinces (Autonomous Region), the temperate zone family strains accounted for more than 90%, with only a few tropical zone family strains and no PV-type II each strain. In Yunnan Province, temperate strains and tropical strains accounted for 71.4% and 28.6% respectively, with occasional PV-type II strain. In Hainan Province, strains of temperate zone, tropical zone and PV-type II accounted for about one-third. CONCLUSIONS: The temperate zone family strains were the predominant ones in the Provinces (Autonomous Region) of Guangdong, Guangxi and Guizhou where malaria control was carried out effectively; while in Hainan and Yunnan Provinces the difficulties in malaria control may probably be related to the complex structure of P. vivax population and multiple infections of different genotypes. The findings indicate that the complexity of the P. vivax genotype structure might be an indicative epidemiological feature for malaria control and surveillance.

Enantiomeric Synthesis of L-(or 1'R,2'S)-Carbocyclic Cyclopropyl Nucleosides.

The enantiomeric synthesis of carbocyclic cyclopropyl L-nucleosides has been accomplished from L-gulonic gamma-lactone. The key intermediate 3 was stereoselectively synthesized by DIBAL-H reduction and silyl protection followed by cyclopropanation from ester 2, which was in turn prepared from L-gulonic gamma-lactone (1) in five steps. Desilylation of cyclopropyl intermediate 3 gave alcohol 4, which was then converted to the urea derivative 5 and cyclopropylamine 7 by Curtius rearrangement of an acyl azide. The urea intermediate 5 was utilized to prepare thymine 10, uracil 11, and cytosine 14 derivatives. The hypoxanthine, adenine, and guanine nucleosides 21, 22, and 24 were synthesized from the amino intermediate 7.