Novel Mycoviruses Discovered from a Metatranscriptomics Survey of the Phytopathogenic Alternaria Fungus.

Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (-ssRNA) viruses. In these viruses, five +ssRNA and four -ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel -ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses.

Expression profiling of circular RNAs and their potential role in early­stage diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a severe cardiovascular complication of diabetes mellitus (DM). Detecting DCM during the early stages of the disease remains a challenge, as the molecular mechanisms underlying early­stage DCM are not clearly understood. Circular RNA (circRNA), a type of non­coding RNA, has been confirmed to be associated with numerous diseases. However, it is still unclear how circRNAs are involved in early­stage DCM. In the present study, heart tissues harvested from BKS­db/db knock­out mice were identified through high­throughput RNA sequencing technology. A total of 58 significantly differentially expressed circRNAs were identified in the db/db sample. Among these, six upregulated circRNAs and seven downregulated circRNAs were detected by reverse transcription­quantitative PCR and analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Furthermore, based on the predicted binding site with microRNAs (miRNAs) involved in DCM, five circRNAs (mmu_circ_0000652, mmu_circ_0000547, mmu_circ_0001058, mmu_circ_0000680 and novel_circ_0004285) were shown to serve as competing endogenous (ce)RNAs. The corresponding miRNAs and mRNAs of the ceRNAs were also verified, and two promising circRNA­miRNA­mRNA regulatory networks were determined. Finally, internal ribosome entry site prediction combined with open reading frame prediction indicated that it was highly possible that mmu_circ_0001160 encoded a protein. In the present study, a comprehensive analysis of the circRNA expression profile during the early phase of DCM was performed, and two promising circRNA­miRNA­mRNA regulatory networks were identified. These results lay the foundation for unravelling the underlying pathogenesis of DCM, and highlight potential biomarkers and therapeutic targets for the treatment of DCM at an early stage.

Computational and experimental identification of strong synergy of the Fe/ZnO catalyst in promoting acetic acid synthesis from CH4 and CO2.

DFT calculations have identified reaction pathways for acetic acid synthesis from CO2 and CH4 on ZnO, Cu/ZnO and Fe/ZnO surfaces. Fe/ZnO exhibits strong synergy in facilitating CH4 activation, dissociation and C-C coupling. Thus, the surface acetate formation is significantly enhanced. The DFT predictions have been confirmed by in situ DRIFTS experiments.

A simple fluorescent strategy based on triple-helix molecular switch for sensitive detection of chloramphenicol.

A simple fluorescent strategy based on the formation of triple-helix molecular switch (THMS) between a signal transduction probe (STP) and an aptamer (Apt) was constructed for the determination of chloramphenicol (CAP). A weak fluorescence intensity was observed for STP solution due to the proximity of fluorophore and quencher through intramolecular DNA hybridization, causing the fluorescence quenching. The fluorescence intensity of the system was significantly enhanced after the addition of Apt. It was attributed to the formation of THMS between the Apt and STP through the Watson-Crick and Hoogsteen base pairing, resulting in the restoration of fluorescence because of the long distance between the fluorophore and quencher of STP. The fluorescence intensity of the system decreased due to the release of STP caused by the specific binding between Apt and CAP. The quantitative analysis of CAP could be achieved based on the decreased fluorescence intensity. The parameters affecting the performance of THMS including the Apt arm length, pH of buffer solution, Mg2+ concentration and the formation time of THMS were investigated in detail. Under the optimal conditions (Apt arm length of 9 bases, pH of 6.5, 2.5 × 103 µmol L-1 Mg2+, THMS formation time of 30 min), the decreased fluorescence intensity and the concentration of chloramphenicol were linear in the range of 5.0 × 10-3-2.0 × 10-1 µmol L-1 with the correlation coefficient of 0.9963. The limit of detection was 1.2 nmol L-1. Subsequently, the developed method was applied to the analysis of chloramphenicol in honey sample, and the recovery was between 84.5% and 103.0% with relative standard deviation less than 4.6%.

Determination of Chloramphenicol in Honey and Milk by HPLC Coupled with Aptamer-Functionalized Fe3 O4 /Graphene Oxide Magnetic Solid-Phase Extraction.

An aptamer-functionalized Fe3 O4 /graphene oxide was synthesized by chemical co-precipitation method and then employed in the magnetic solid-phase extraction for selective enrichment of chloramphenicol before HPLC. The aptamer was covalently bonded to the Fe3 O4 /graphene oxide complex by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Parameters affecting extraction efficiency including solution pH, extraction time and temperature, types and volume of elution solvent, and elution time were investigated in detail. Under the optimal conditions, good linearity was obtained between the peak area and analyte concentration in the range of 7.0 to 1.0 × 103  µg/L with the correlation coefficient of 0.9994. The limit of detection and quantitation were 0.24 µg/L and 0.79 µg/L, respectively. The developed method was employed to the analysis of chloramphenicol in honey and milk samples. The recoveries ranged from 80.5% to 105.0% with relative standard deviations less than 8.9%. PRACTICAL APPLICATION: An aptamer-functionalized Fe3 O4 /graphene oxide was synthesized and employed in magnetic solid phase extraction for the enrichment of chloramphenicol before HPLC. The presented assay was employed for the determination of chloramphenicol in honey and milk with satisfactory results.

Using miRNAs and circRNAs to estimate PMI in advanced stage.

In our previous study, we evaluated the stability of multi-RNA markers in heart, liver and skeletal muscle tissues of mice within 8 days after death and concluded that microRNAs (miRNAs) and circular (circRNAs) were more stable as reference genes in dead bodies than other kinds of RNAs. Based on their tissue-specific expression, we obtained reference genes for three kinds of tissues: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 in skeletal muscle tissues. For the estimation of post mortem interval (PMI), we also selected suitable biomarkers, which exhibited the best correlation coefficient with PMI. In our stability analysis of multi-RNA markers, Gapdh, Rps18, U6 and ß-actin were unstable and selected as candidate target biomarkers. By analyzing the correlation between the expression levels of candidate target biomarkers and PMI, we obtained suitable target biomarkers for the three kinds of tissues, respectively. Finally, we established mathematical models of PMI estimation using the above selected reference genes and target biomarkers. The low estimated error in the validated samples demonstrated that PMI in advanced stage could be accurately predicted by real-time quantitative polymerase chain reaction (qPCR) through systematically selected effective reference genes and target biomarkers. Besides, combining the estimated results of various tissues and multi-biomarkers could improve the accuracy of PMI estimation.

Opposite effects of cannabinoid CB1 and CB2 receptors on antipsychotic clozapine-induced cardiotoxicity.

BACKGROUND AND PURPOSE: Clozapine is an atypical antipsychotic drug that is very efficacious in treating psychosis, but the risk of severe cardiotoxicity limits its clinical use. The present study investigated the harmful effects of clozapine on myocardium and assessed the involvement of cannabinoid receptors in its cardiotoxicity. EXPERIMENTAL APPROACH: Clozapine alone or in combination with selective cannabinoid receptor antagonists or agonists were used to treat mice and cardiomyocytes. KEY RESULTS: Clozapine induced myocardial inflammation and infiltration 7 days after i.p. injection. Mice survival rate and myocardial infiltration, and fibrotic lesions were dose-dependently worsened by clozapine. Clozapine decreased major endocannabinoid levels in sera and cultured cardiomyocytes. Cannabinoid CB1 receptors decreased in clozapine-treated hearts and were translocated from cytomembranes to cytoplasm and nuclei, whereas CB2 receptors increased in clozapine-treated hearts and inversely translocated from nuclei to the cytomembrane. Selective antagonists of CB1 receptors, rimonabant and AM281, but not its selective agonist arachidonyl-2'-chloroethylamide, ameliorated clozapine-induced myocardial inflammatory infiltration and fibrotic lesions. In contrast, selective agonists of CB2 receptors, AM1241 and JWH-133, but not its selective antagonist AM630, blunted clozapine-mediated cardiotoxicity in mice. In cultured cardiomyocytes, clozapine increased the pro-inflammatory factor IL-1ß and the concentrations of myocardial injury markers (LDH and aspartate aminotransferase); these effects were reversed by either a CB1 antagonist or CB2 agonist and further prevented by combined pretreatments. CONCLUSIONS AND IMPLICATIONS: Our data provide evidence that cannabinoid CB1 and CB2 receptors have opposite effects and selective antagonists of CB1 or agonists of CB2 receptors might confer protective effects against clozapine in myocardium.

Metabolic profiling of femoral muscle from rats at different periods of time after death.

Clarification of postmortem metabolite changes can help characterize the process of biological degradation and facilitate investigations of forensic casework, especially in the estimation of postmortem interval (PMI). Metabolomics can provide information on the molecular profiles of tissues, which can aid in investigating postmortem metabolite changes. In this study, liquid chromatography-mass spectrometric (LC-MS) analysis was performed to identify the metabolic profiles of rat femoral muscle at ten periods of time after death within 168 h. The results obtained by reversed-phase liquid chromatography (RPLC)- and hydrophilic interaction liquid chromatography (HILIC)- electrospray ionization (ESI±) have revealed more than 16,000 features from all four datasets. Furthermore, 915 of these features were identified using an in-house database. Principal component analysis (PCA) demonstrated the time-specific features of molecular profiling at each period of time after death. Moreover, results from partial least squares projection to latent structures-discriminant analysis (PLS-DA) disclosed a strong association of metabolic alterations of at least 59 metabolites with the time since death, especially within 48 h after death, which expounds these metabolites as potential indicators in PMI estimation. Altogether, our results illustrate the potentiality of metabolic profiling in the evaluation of PMI and provide candidate metabolite markers with strong correlation with time since death for forensic purpose.

Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI.

The precise estimation of postmortem interval (PMI) is a critical step in death investigation of forensic cases. Detecting the degradation of RNA in tissues by real time quantitative polymerase chain reaction (RT-qPCR) technology provides a new theoretical basis for estimation of PMI. However, most commonly used reference genes degrade over time, while previous studies seldom consider this when selecting suitable reference genes for the estimation of PMI. Studies have shown microRNAs (miRNAs) are very stable and circular RNAs (circRNAs) have recently emerged as a novel class of RNAs with high stability. We aimed to evaluate the stability of the two kinds of RNAs and normal reference genes using geNorm and NormFinder algorithms to identify tissue-specific reference genes for PMI estimation. The content of candidate RNAs from mouse heart, liver and skeletal muscle tissues were dynamically examined in 8 consecutive days after death. Among the 11 candidate genes (ß-actin, Gapdh, Rps18, 5S, 18S, U6, miR-133a, miR-122, circ-AFF1, LC-Ogdh and LC-LRP6), the following genes showed prioritized stability: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 and LC-LRP6 in skeletal muscle tissues. Our results suggested that miRNAs and circRNAs were more stable as reference genes than other kinds of RNAs regarding PMI estimation. The appropriate internal control genes were not completely the same across tissue types.

Aberrant endoplasmic reticulum stress mediates coronary artery spasm through regulating MLCK/MLC2 pathway.

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.

[Analysis of monosaccharides in the saffron corm glycoconjugate by capillary electrophoresis].

The monosaccharides in the saffron corm glycoconjugate were separated by capillary electrophoresis (CE) coupled with pre-column derivatization. 4-Methoxyaniline was used as derivatization reagent. The derivatization and CE separation conditions were investigated. The ultraviolet detection wavelength was 234 nm. The maximum yield of this derivatization reaction was obtained under the presence of 9.5% (v/v) acetic acid at 80 degrees C for 2 h. An uncoated fused-silica capillary of 50 microm i.d. and 50/60 cm length (effective length/total length) was employed, and a pressure injection (3.4475 kPa, 5 s) was applied. The baseline separation of 11 monosaccharides and disaccharides (lyxose, xylose, ribose, glucose, mannose, galactose, rhamnose, cellobiose, maltose, lactose, fructose) was reached at 25 degrees C, 20 kV of separation voltage and with 350 mmol/L boric acid (pH 10.21) as running buffer. The developed method has been successfully applied to quantitatively determine the components of saffron corm glycoconjugate, and the results showed that the recovery of each monosaccharide was in the range of 94.3% - 105.4%, the relative standard deviation was 3.3% - 4.6%.