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1.  Quantification of Arachidonic Acid and Its Metabolites in Rat Tissues by UHPLC-MS/MS: Application for the Identification of Potential Biomarkers of Benign Prostatic Hyperplasia 
PLoS ONE  2016;11(11):e0166777.
To evaluate the potential relationship between benign prostatic hyperplasia (BPH) and the arachidonic acid (AA) metabolome, a UHPLC—MS/MS method has been developed and validated for simultaneous determination of AA and its cyclooxygenase(COX) and lipoxygenase(LOX) pathway metabolites (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) in rat tissues. The analytes were extracted from tissue samples with a protein precipitation procedure and then separated on a Shim-pack XR-ODSC18 column with 0.05% formic acid in water (pH adjusted with dilute ammonia) and methanol:acetonitrile (20:80, v/v). Detection was performed on a UHPLC—MS/MS system with electrospray negative ionization (ESI) and a multiple reaction-monitoring mode. The lower limits of quantification (LLOQ) were 0.25–50 ng/mL for all of the analytes in the prostate, seminal, bladder, liver and kidney tissues. The absolute recoveries of the analytes from all of the tissues were more than 50%. By means of the method developed, the AA metabolites in tissue samples from Sham and BPH group rats were determined. The eleven biomarkers in the BPH group prostate, seminal, bladder, liver and kidney tissues were significantly higher than those of the sham group, indicating that BPH fortified the inducible expression of COX and LOX, as well as increased the production of AA and eicosanoids. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in vivo.
PMCID: PMC5125601  PMID: 27893755
2.  Identification and Characterization of an Aeromonas hydrophila Oligopeptidase Gene pepF Negatively Related to Biofilm Formation 
Bacterial biofilms are involved in adaptation to complex environments and are responsible for persistent bacterial infections. Biofilm formation is a highly complex process during which multifarious genes work together regularly. In this study, we screened the EZ-Tn5 transposon mutant library to identify genes involved in biofilm formation of Aeromonas hydrophila. A total of 24 biofilm-associated genes were identified, the majority of which encoded proteins related to cell structure, transcription and translation, gene regulation, growth and metabolism. The mutant strain TM90, in which a gene encoding oligopeptidase F (pepF) was disturbed, showed significant upregulation of biofilm formation compared to the parental strain. The TM90 colony phenotype was smaller, more transparent, and splendent. The adhesive ability of TM90 to HEp-2 cells was significantly increased compared with the parental strain. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the enhanced-biofilm mutant TM90 was highly attenuated relative to the wild-type strain. In conclusion, the pepF gene is demonstrated for the first time to be a negative factor for biofilm formation and is involved in A. hydrophila pathogenicity.
PMCID: PMC5032638  PMID: 27713736
Aeromonas hydrophila; biofilm; transposon mutant library; oligopeptidase F (pepF); virulence
3.  Targeting procaspase-3 with WF-208, a novel PAC-1 derivative, causes selective cancer cell apoptosis 
Caspase-3 is a critical effector caspase in apoptosis cascade, and is often over-expressed in many cancer tissues. The first synthesized procaspase-3 activator, PAC-1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase-3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF-208 stood out by its high cytotoxicity against procaspase-3 overexpressed HL-60 cells. Compared with PAC-1, WF-208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF-208 activated procaspase-3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase-3-dependent cell death in tumour cells, which possibly because of the zinc-chelating properties. WF-208 also showed greater antitumour activity than PAC-1 in murine xenograft model. In conclusion, we have discovered WF-208 as a promising procaspase-3 activating compound, with higher activity and higher cell selectivity than PAC-1.
PMCID: PMC4549042  PMID: 25754465
procaspase-3 activator; anticancer; new derivative; PAC-1
4.  Metabonomics study on Polygonum multiflorum induced liver toxicity in rats by GC-MS 
Polygonum multiflorum, a traditional Chinese medicinal herb, is widely used in liver and liver nourishing. Recent years, drug regulatory departments reported that Polygonum multiflorum caused serious adverse reaction in clinic, especially liver injury. In this study, we detected the changes in rat serum and liver tissue metabolites through gas chromatography-mass spectrometry (GC-MS). Mass spectrometry, partial least squares-discriminate analysis (PLS-DA) and other diversified techniques were used to analyze the differences among their metabolites. Compared to the control group, the serum concentrations of L-threonine and serine in water extraction groups increased. The serum concentrations of 9,12-octadecadienoic acid, hexadecanoic acid, oleic acid, D-glucose and octadecanoic acid in alcohol extraction groups increased, while lactic acid decreased to a great extent. For liver tissue, compared to the control group, the concentrations of myo-inositol, oleic acid and cholesterol in water extraction groups increased, while those of hexadecanoic acid, octadecanoic acid, ribitol and butanedioic acid decreased to a great extent. The concentrations of myo-inositol, phosphoric acid, uridine, oleic acid, cholesterol and butanoic acid in alcohol extraction groups increased to a great extent, while those of hexadecanoic acid, octadecanoic acid, ribitol and butanedioic acid decreased. The results indicate that Polygonum multiflorum induces the metabolic disorders of energy metabolism, amino acid and lipid metabolism. What’s more, liver injury of alcohol extraction group was more serious than group of water extraction.
PMCID: PMC4565277  PMID: 26379894
GC-MS; polygonum multiflorum; metabonomics
5.  Profiling and initial validation of urinary microRNAs as biomarkers in IgA nephropathy 
PeerJ  2015;3:e990.
Background. MicroRNAs (miRNAs) have been found in virtually all body fluids and used successfully as biomarkers for various diseases. Evidence indicates that miRNAs have important roles in IgA nephropathy (IgAN), a major cause of renal failure. In this study, we looked for differentially expressed miRNAs in IgAN and further evaluated the correlations between candidate miRNAs and the severity of IgAN.
Methods. Microarray and RT-qRCR (real-time quantitative polymerase chain reaction) were sequentially used to screen and further verify miRNA expression profiles in urinary sediments of IgAN patients in two independent cohorts. The screening cohort consisted of 32 urine samples from 18 patients with IgAN, 4 patients with MN (membranous nephropathy), 4 patients with MCD (minimal changes disease) and 6 healthy subjects; the validation cohort consisted of 102 IgAN patients, 41 MN patients, 27 MCD patients and 34 healthy subjects. The renal pathological lesions of patients with IgAN were evaluated according to Lee’s grading system and Oxford classification.
Results. At the screening phase, significance analysis of microarrays analysis showed that no miRNA was differentially expressed in the IgAN group compared to all control groups. But IgAN grade I–II and III subgroups (according to Lee’s grading system) shared dysregulation of two miRNAs (miR-3613-3p and miR-4668-5p). At the validation phase, RT-qPCR results showed that urinary level of miR-3613-3p was significantly lower in IgAN than that in MN, MCD and healthy controls (0.47, 0.44 and 0.24 folds, respectively, all P < 0.01 by Mann–Whitney U test); urinary level of miR-4668-5p was also significantly lower in IgAN than that in healthy controls (0.49 fold, P < 0.01). Significant correlations were found between urinary levels of miR-3613-3p with 24-hour urinary protein excretion (Spearman r = 0.50, P = 0.034), eGFR (estimated glomerular filtration rate) (r = − 0.48, P = 0.043) and Lee’s grades (r = 0.57, P = 0.014). Similarly, miR-4668-5p was significantly correlated with eGFR (r = − 0.50, P = 0.034) and Lee’s grades (r = 0.57, P = 0.013). For segmental glomerulosclerosis according to Oxford classification, patients scored as S0 had significantly lower levels of urinary miR-3613-3p and miR-4668-5p than those scored as S1 (0.41 and 0.43 folds, respectively, all P < 0.05).
Conclusions. The expression profile of miRNAs was significantly altered in urinary sediments from patients with IgAN. Urinary expression of miR-3613-3p was down-regulated in patients with IgAN. Moreover, urinary levels of both miR-3613-3p and miR-4668-5p were correlated with disease severity. Further studies are needed to explore the roles of miR-3613-3p and miR-4668-5p in the pathogenesis and progression of IgA nephropathy.
PMCID: PMC4458130  PMID: 26056621
MicroRNA; Microarray; Biomarkers; IgA nephropathy
6.  Boron deficiency in woody plants: various responses and tolerance mechanisms 
Boron (B) is an essential microelement for higher plants, and its deficiency is widespread around the world and constrains the productivity of both agriculture and forestry. In the last two decades, numerous studies on model or herbaceous plants have contributed greatly to our understanding of the complex network of B-deficiency responses and mechanisms for tolerance. In woody plants, however, fewer studies have been conducted and they have not well been recently synthesized or related to the findings on model species on B transporters. Trees have a larger body size, longer lifespan and more B reserves than do herbaceous plants, indicating that woody species might undergo long-term or mild B deficiency more commonly and that regulation of B reserves helps trees cope with B deficiency. In addition, the highly heterozygous genetic background of tree species suggests that they may have more complex mechanisms of response and tolerance to B deficiency than do model plants. Boron-deficient trees usually exhibit two key visible symptoms: depression of growing points (root tip, bud, flower, and young leaf) and deformity of organs (root, shoot, leaf, and fruit). These symptoms may be ascribed to B functioning in the cell wall and membrane, and particularly to damage to vascular tissues and the suppression of both B and water transport. Boron deficiency also affects metabolic processes such as decreased leaf photosynthesis, and increased lignin and phenol content in trees. These negative effects will influence the quality and quantity of wood, fruit and other agricultural products. Boron efficiency probably originates from a combined effect of three processes: B uptake, B translocation and retranslocation, and B utilization. Root morphology and mycorrhiza can affect the B uptake efficiency of trees. During B translocation from the root to shoot, differences in B concentration between root cell sap and xylem exudate, as well as water use efficiency, may play key roles in tolerance to B deficiency. In addition, B retranslocation efficiency primarily depends on the extent of xylem-to-phloem transfer and the variety and amount of cis-diol moieties in the phloem. The B requirement for cell wall construction also contribute to the B use efficiency in trees. The present review will provide an update on the physiological and molecular responses and tolerance mechanisms to B deficiency in woody plants. Emphasis is placed on the roles of B reserves that are more important for tolerance to B deficiency in trees than in herbaceous plants and the possible physiological and molecular mechanisms of differential B efficiency in trees. We propose that B may be used to study the relationship between the cell wall and the membrane via the B-bridge. Transgenic B-efficient tree cultivars have considerable potential for forestry or fruit rootstock production on low B soils in the future.
PMCID: PMC4621400  PMID: 26579163
boron efficiency; boron reserves; cell wall; lignin; transporter; trees
7.  Direct Characterization of Bulk Samples by Internal Extractive Electrospray Ionization Mass Spectrometry 
Scientific Reports  2013;3:2495.
A straight-forward analytical strategy called internal extractive electrospray ionization mass spectrometry (iEESI-MS), which combines solvent extraction of chemicals inside a bulk sample with in situ electrospray ionization mass spectrometry, has been established to directly characterize the interior of a bulk sample with molecular specificity. The method allows both qualitative and quantitative analysis of analytes distributed in a 3-dimensional volume (e.g., 1 ~ 100 mm3) of various synthetic and biological matrices (e.g., chewing gum, leaves, fruits, roots, pork, lung tissues) without either mashing the sample or matrix separation. Using different extraction solvents, online chromatographic separation of chemicals inside the sample volume was observed during iEESI-MS analysis. The presented method is featured by the high speed of analysis, high sensitivity, low sample consumption and minimal sample preparation and/or degradation, offering unique possibilities for advanced applications in plant science, clinical diagnosis, catalyst studies, and materials science.
PMCID: PMC3750536  PMID: 23970067
8.  Reference-free SNP calling: improved accuracy by preventing incorrect calls from repetitive genomic regions 
Biology Direct  2012;7:17.
Single nucleotide polymorphisms (SNPs) are the most abundant type of genetic variation in eukaryotic genomes and have recently become the marker of choice in a wide variety of ecological and evolutionary studies. The advent of next-generation sequencing (NGS) technologies has made it possible to efficiently genotype a large number of SNPs in the non-model organisms with no or limited genomic resources. Most NGS-based genotyping methods require a reference genome to perform accurate SNP calling. Little effort, however, has yet been devoted to developing or improving algorithms for accurate SNP calling in the absence of a reference genome.
Here we describe an improved maximum likelihood (ML) algorithm called iML, which can achieve high genotyping accuracy for SNP calling in the non-model organisms without a reference genome. The iML algorithm incorporates the mixed Poisson/normal model to detect composite read clusters and can efficiently prevent incorrect SNP calls resulting from repetitive genomic regions. Through analysis of simulation and real sequencing datasets, we demonstrate that in comparison with ML or a threshold approach, iML can remarkably improve the accuracy of de novo SNP genotyping and is especially powerful for the reference-free genotyping in diploid genomes with high repeat contents.
The iML algorithm can efficiently prevent incorrect SNP calls resulting from repetitive genomic regions, and thus outperforms the original ML algorithm by achieving much higher genotyping accuracy. Our algorithm is therefore very useful for accurate de novo SNP genotyping in the non-model organisms without a reference genome.
This article was reviewed by Dr. Richard Durbin, Dr. Liliana Florea (nominated by Dr. Steven Salzberg) and Dr. Arcady Mushegian.
PMCID: PMC3472322  PMID: 22682067
Next-generation sequencing; single nucleotide polymorphism; genotyping; maximum likelihood; mixed Poisson/normal model
9.  PtdIns (3,4,5) P3 Recruitment of Myo10 Is Essential for Axon Development 
PLoS ONE  2012;7(5):e36988.
Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker. Knocking down Myo10 gene transcription impaired outgrowth of axon with loss of Tau-1-positive phenotype. Interestingly, inhibition of actin polymerization by cytochalasin D rescued the defect of axon outgrowth. Furthermore, ectopic expression of Myo10 with enhanced green fluorescence protein (EGFP) labeled Myo10 mutants induced multiple axon-like neurites in a motor-independent way. Mechanism studies demonstrated that the recruitment of Myo10 through its PH domain to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) was essential for axon formation. In addition, in vivo studies confirmed that Myo10 was required for neuronal morphological transition during radial neuronal migration in the developmental neocortex.
PMCID: PMC3349655  PMID: 22590642

Results 1-9 (9)