Serum miR-195-5p Exhibits Clinical Significance in the Diagnosis of Essential Hypertension with Type 2 Diabetes Mellitus by Targeting DRD1

OBJECTIVES: Diagnosis and management of essential hypertension (EH) or type 2 diabetes mellitus (T2DM) by combining comprehensive treatment and classificatory diagnosis have been continuously improved. However, understanding the pathogenesis of EH patients with concomitant T2DM and subsequent treatment remain the major challenges owing to the lack of non-invasive biomarkers and information regarding the underlying mechanisms. METHODS: Herein, we collected 200 serum samples from EH and/or T2DM patients and healthy donors (N). Gene-expression profiling was conducted to identify candidate microRNAs with clinical significance. Then, a larger cohort of the aforementioned patients and 50 N were used to identify the correlation between the tumor suppressor miR-195-5p and EH and/or T2DM. The dual-luciferase reporter assay was used to explore the target genes of miR-195-5p. The suppressive effects of miR-195-5p on the 3′-UTR of the dopamine receptor D1 (DRD1) transcript in EH patients with concomitant T2DM were verified as well. RESULTS: Compared with that in other groups, serum miR-195-5p was highly downregulated in EH patients with concomitant T2DM. miR-195-5p overexpression efficiently suppressed DRD1 expression by binding to the two 3′-UTRs. Additionally, two single nucleotide polymorphisms, including 231T-A and 233C-G, in the miR-195-5p binding sites of the DRD1 3′-UTR were further identified. Collectively, we identified the potential clinical significance of DRD1 regulation by miR-195-5p in EH patients with concomitant T2DM. CONCLUSIONS: Our data suggested that miR-195-5p circulating in the peripheral blood served as a novel biomarker and therapeutic target for EH and T2DM, which could eventually help address major challenges during the diagnosis and treatment of EH and T2DM

Inhibition of ERK-DLP1 signaling and mitochondrial division alleviates mitochondrial dysfunction in Alzheimer's disease cybrid cell

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease (AD). The underlying mechanisms and strategies to repair it remain unclear. Here, we demonstrate for the first time the direct consequences and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in AD. Using cytoplasmic hybrid (cybrid) neurons with incorporated platelet mitochondria from AD and age-matched non-AD human subjects into mitochondrial DNA (mtDNA)-depleted neuronal cells, we observed that AD cybrid cells had significant changes in morphology and function; such changes associate with altered expression and distribution of dynamin-like protein (DLP1) and mitofusin 2 (Mfn2). Treatment with antioxidant protects against AD mitochondria-induced extracellular signal-regulated kinase (ERK) activation and mitochondrial fission-fusion imbalances. Notably, inhibition of ERK activation not only attenuates aberrant mitochondrial morphology and function but also restores the mitochondrial fission and fusion balance. These effects suggest a role of oxidative stress-mediated ERK signal transduction in modulation of mitochondrial fission and fusion events. Further, blockade of the mitochondrial fission protein DLP1 by a genetic manipulation with a dominant negative DLP1 (DLP1K38A), its expression with siRNA-DLP1, or inhibition of mitochondrial division with mdivi-1 attenuates mitochondrial functional defects observed in AD cybrid cells. Our results provide new insights into mitochondrial dysfunction resulting from changes in the ERK-fission/fusion (DLP1) machinery and signaling pathway. The protective effect of mdivi-1 and inhibition of ERK signaling on maintenance of normal mitochondrial structure and function holds promise as a potential novel therapeutic strategy for AD

Inhibition of ERK-DLP1 signaling and mitochondrial division alleviates mitochondrial dysfunction in Alzheimer's disease cybrid cell

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease (AD). The underlying mechanisms and strategies to repair it remain unclear. Here, we demonstrate for the first time the direct consequences and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in AD. Using cytoplasmic hybrid (cybrid) neurons with incorporated platelet mitochondria from AD and age-matched non-AD human subjects into mitochondrial DNA (mtDNA)-depleted neuronal cells, we observed that AD cybrid cells had significant changes in morphology and function; such changes associate with altered expression and distribution of dynamin-like protein (DLP1) and mitofusin 2 (Mfn2). Treatment with antioxidant protects against AD mitochondria-induced extracellular signal-regulated kinase (ERK) activation and mitochondrial fission-fusion imbalances. Notably, inhibition of ERK activation not only attenuates aberrant mitochondrial morphology and function but also restores the mitochondrial fission and fusion balance. These effects suggest a role of oxidative stress-mediated ERK signal transduction in modulation of mitochondrial fission and fusion events. Further, blockade of the mitochondrial fission protein DLP1 by a genetic manipulation with a dominant negative DLP1 (DLP1K38A), its expression with siRNA-DLP1, or inhibition of mitochondrial division with mdivi-1 attenuates mitochondrial functional defects observed in AD cybrid cells. Our results provide new insights into mitochondrial dysfunction resulting from changes in the ERK-fission/fusion (DLP1) machinery and signaling pathway. The protective effect of mdivi-1 and inhibition of ERK signaling on maintenance of normal mitochondrial structure and function holds promise as a potential novel therapeutic strategy for AD

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Characterization of the Volatile Compounds of Onion with Different Fresh-Cut Styles and Storage Temperatures

The flavor of fresh onion and its processed products is an important index with which to evaluate its quality. In this study, the highly volatile compounds of onion with different fresh-cut styles (bulb, ring, and square) and different storage temperatures (4 °C, 20 °C, and 25 °C) were characterized at the molecular level, focusing in particular on the volatile sulfur compounds. Headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction-gas chromatography−mass spectrometry (HS-SPME-GC-MS) were employed. A total of 14 highly volatile compounds were identified in onion samples by HS-GC-IMS, and the square sample contained more volatile components. (E,E)-2,4-heptadianal, ethyl acetate, 2-methyl-1-pentanol, 2-pentylfuran, propyl acetate, and 2,6-dimethylpyrazine were produced in the ring and square samples when stored at higher temperatures, while pentanal, 2-heptenal, hexanal were decreased after cutting. Simultaneously, 16 sulfur compounds were identified in onions by HS-SPME-GC-MS. The sulfur compounds profile of the bulbs was significantly different from that of the rings and squares at any temperature. When stored at a low temperature (4 °C), cutting onions into a ring or square shape produced more sulfur. However, at higher temperatures (20 °C and 25 °C), fresh-cutting decreased the sulfur concentration. The total content of sulfur compounds was higher in the same cut style stored at higher temperatures (20 °C or 25 °C). 2-Mercapto-3,4-dimethyl-2,3-dihydrothiophene and 2,4-dimethylthiophene were formed during storage; however, (E)-1-(prop-1-en-1-yl)-3-propyltrisulfane, 1-(1-(methylthio)propyl)-2-propyldisulfane, (Z)-1-(1-propenyldithio)propyl disulfide, dipropyl trisulfide, and methyl 1-(1-propenylthio)propyl disulfide were lost from all samples after storage

Establishment of Self-incompatibility Gene cDNA Microarray to Identify S-genotypes of Pyrus pyrifolia

Based on the cDNA sequences from hyper variable (HV) regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase cDNA probes were designed, and a cDNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 cDNA probes, including all specific cDNA sequences from the HV regions of the S-alleles. Using the cDNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the cDNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli’, ‘Xiuyu’, ‘Midu Yuli’, ‘Baimianli’, and ‘Deshengxiang’ were similar to the known genotypes of all tested cultivars. The S-RNase cDNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of cDNA microarrays has further improved the pear S-RNase detection platform

Analysis on Composition and Content of Glucosinolates in Broccoli Flowers and Leaves

Glucosinolate composition and content were evaluated in flowers and leaves of 12 different broccoli varieties. The results indicated that there were 9 glucosinolates in broccoli, namely Glucoiberin (IBE); Progoitrin (PRO); Sinigrin (SIN); Glucoraphanin (RAA); Gluconapin (NAP); 4-Hydroxyglucobrassicin (4OH); Glucobrassicin (GBC); 4-Methoxyglucobrassicin (4ME); Neoglucobrassicin (NEO). Total glucosinolate content in flowers was 1-5 times higher than in leaves. The predominant glucosinolate in broccoli was glucoraphanin

Studies on the Simultaneous Determination of Cr(III) and Cr(VI) by Ion Chromatography (IC)

The research aimed to develop and validate an ion chromatography method for the simultaneous analysis of Cr(III) and Cr(VI) from pumpkin. A new analytical method based on ion chromatography techniques was developed by the Cr(III) pre-column derivatization and Cr(VI) post-column derivatization. The ion chromatography condition was optimized and the detection sensitivity was improved. Cr(III) and Cr(VI) were determined by 365 and 530 nm, respectively. The temperature of water bath, the heating time for pre-column derivatization, and the flow rate of post-column derivative liquid were screened on the basis of single factor experiment, the effects of various factors were determined by the method of L16(43) orthogonal experiment design. Considering the results of orthogonal experiments and the variation tendency of peak area under different factors, the optimum derivatization conditions were chose as follows: the flow rate of post-column derivative liquid is 0.5 mL/min, the temperature of water bath for pre-column derivatization is 100 ℃ and the heating time is 5 min. The conditions were optimized by means of orthogonal experiments under the pH of leacheate ranged from 6.5 to 6.8 and the I– concentration of 5 mmol/L. Under optimized derivatization conditions, the detection limits for Cr(III) and Cr(VI) were 0.17 and 0.019 mg, respectively. And the average recoveries of Cr(III) in pumpkin were in the range of 82%-85%. Results indicated that pumpkin does not contain Cr(VI) according to this ion chromatography method of simultaneously determination for Cr(III) and Cr(VI)

Determination of 18 Intact Glucosinolates in Brassicaceae Vegetables by UHPLC-MS/MS: Comparing Tissue Disruption Methods for Sample Preparation

Glucosinolates (GSLs) are important precursor compounds with anticancer activities in Brassicaceae vegetables and are readily hydrolyzed by myrosinase. Given the diversity of these species, establishing an accurate and universal method to quantify intact GSLs in different plant tissues is necessary. Here, we compared and optimized three tissue disruption methods for sample preparation. After microwave treatment for 90 s, 13 GSLs in homogenized Chinese cabbage samples were recovered at 73–124%. However, a limitation of this method was that different tissues could not be processed under the same microwave conditions. Regarding universality, GSLs in Brassicaceae vegetables could be extracted from freeze-dried sample powder with 70% methanol (v/v) or frozen-fresh sample powder with 80% methanol (v/v). Moreover, heating extraction is necessary for GSLs extracted from frozen-fresh sample powder. Average recoveries of the two optimized methods were 74–119% with relative standard deviations ≤ 15%, with the limits of quantification 5.72–17.40 nmol/g dry weight and 0.80–1.43 nmol/g fresh weight, respectively. Notably, the method for analyzing intact GSLs was more efficient than that for desulfo-GSLs regarding operational complexity, detection speed and quantification accuracy. The developed method was applied to identify the characteristic GSLs in 15 Brassicaceae vegetables, providing a foundation for further research on GSLs

Effects of fresh-cut and storage on glucosinolates profile using broccoli as a case study

Glucosinolates (GLS) contribute to the unique flavour, nutrition, and plant defence of the Cruciferous vegetables. Understanding the GLS changes through postharvest processing is essential for defined preservation. In this study, four different fresh-cut types, whole flower (W), floret (F), quarterly cut floret (QF) and shredded floret (FS) of broccoli, were stored for 0, 1, 2 and 3 day (s) to explore GLS responses to postharvest treatments. As a result, seven GLS were identified, mainly including glucoraphanin (RAA), neoglucobrassicin (NEO), and glucobrassicin (GBC) and accounting for 52.69%, 20.12% and 14.99% of the total GLS (21.92 ± 0.48) μmol · g-1DW, respectively. FS had the sharpest decrease in GLS after three days of storage (6.55 ± 0.37) μmol · g-1DW, while QF had the least (10.16 ± 0.33) μmol · g-1DW. All GLS components decreased over storage, except for 4-methoxyglucobrassicin (4 ME) in FS and QF, suggesting its key role in serious wound defence. The results suggested certain postharvest approaches influenced the flavour and nutrition of broccoli