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1.  Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria 
PLoS ONE  2014;9(2):e87250.
Background
As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH.
Methodology
We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation.
Results
Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them.
Conclusion
Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.
doi:10.1371/journal.pone.0087250
PMCID: PMC3911924  PMID: 24498303
2.  Oxidative stress induces gastric submucosal arteriolar dysfunction in the elderly 
AIM: To evaluate human gastric submucosal vascular dysfunction and its mechanism during the aging process.
METHODS: Twenty male patients undergoing subtotal gastrectomy were enrolled in this study. Young and elderly patient groups aged 25-40 years and 60-85 years, respectively, were included. Inclusion criteria were: no clinical evidence of cardiovascular, renal or diabetic diseases. Conventional clinical examinations were carried out. After surgery, gastric submucosal arteries were immediately dissected free of fat and connective tissue. Vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were measured by isolated vascular perfusion. Morphological changes in the gastric mucosal vessels were observed by hematoxylin and eosin (HE) staining and Verhoeff van Gieson (EVG) staining. The expression of xanthine oxidase (XO) and manganese-superoxide dismutase (Mn-SOD) was assessed by Western blotting analysis. The malondialdehyde (MDA) and hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined according to commercial kits.
RESULTS: The overall structure of vessel walls was shown by HE and EVG staining, respectively. Disruption of the internal elastic lamina or neointimal layers was not observed in vessels from young or elderly patients; however, cell layer number in the vessel wall increased significantly in the elderly group. Compared with submucosal arteries in young patients, the amount of vascular collagen fibers, lumen diameter and media cross-sectional area were significantly increased in elderly patients. Ach- and SNP-induced vasodilatation in elderly arterioles was significantly decreased compared with that of gastric submucosal arterioles from young patients. Compared with the young group, the expression of XO and the contents of MDA and H2O2 in gastric submucosal arterioles were increased in the elderly group. In addition, the expression of Mn-SOD and the activities of SOD and GSH-Px in the elderly group decreased significantly compared with those in the young group.
CONCLUSION: Gastric vascular dysfunction and senescence may be associated with increased oxidative stress and decreased antioxidative defense in the aging process.
doi:10.3748/wjg.v19.i48.9439
PMCID: PMC3882420  PMID: 24409074
Aging; Vascular dysfunction; Gastric blood flow; Oxidative stress; Human
3.  Mosquitoes of Western Yunnan Province, China: Seasonal Abundance, Diversity, and Arbovirus Associations 
PLoS ONE  2013;8(10):e77017.
Objective
The western borderland between Yunnan Province, China, and Myanmar is characterized by a climate that facilitates year-round production of mosquitoes. Numerous mosquito-transmitted viruses, including Japanese encephalitis virus circulate in this area. This project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations.
Methods
Mosquitoes were collected in Mangshi and Ruili cities of Dehong Prefecture near the border of China and Burma in Yunnan Province, the Peoples Republic of China in 2010. We monitored mosquito species abundance for a 12-month period using ultraviolet light, carbon dioxide baited CDC light and gravid traps; and tested the captured mosquitoes for the presence of virus to evaluate mosquito-virus associations in rural/agricultural settings in the area.
Results
A total of 43 species of mosquitoes from seven genera were collected, including 15 Culex species, 15 Anopheles spp., four Aedes spp., three Armigeres spp., one Mimomyia spp., two Uranotaenia spp. and three Mansonia spp.. Species richness and diversity varied between Mangshi and Ruili. Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Anopheles peditaeniatus were the most abundant species in both sampling sites. Ultraviolet light traps collected more specimens than CDC light traps baited with dry ice, though both collected the same variety of mosquito species. The CDC gravid trap was the most effective trap for capture of Culex quinquefasciatus, a species underrepresented in light trap collections. A total of 26 virus strains were isolated, which included 13 strains of Japanese encephalitis virus, four strains of Getah virus, one strain of Oya virus, one strain from the orbivirus genus, and seven strains of Culex pipien pallens densovirus.
Conclusions
The present study illustrates the value of monitoring mosquito populations and mosquito-transmitted viruses year-round in areas where the climate supports year-round adult mosquito activity.
doi:10.1371/journal.pone.0077017
PMCID: PMC3795637  PMID: 24146951
4.  An association study of TOLL and CARD with leprosy susceptibility in Chinese population 
Human Molecular Genetics  2013;22(21):4430-4437.
Previous genome-wide association studies (GWASs) identified multiple susceptibility loci that have highlighted the important role of TLR (Toll-like receptor) and CARD (caspase recruitment domain) genes in leprosy. A large three-stage candidate gene-based association study of 30 TLR and 47 CARD genes was performed in the leprosy samples of Chinese Han. Of 4363 SNPs investigated, eight SNPs showed suggestive association (P < 0.01) in our previously published GWAS datasets (Stage 1). Of the eight SNPs, rs2735591 and rs4889841 showed significant association (P < 0.001) in an independent series of 1504 cases and 1502 controls (Stage 2), but only rs2735591 (next to BCL10) showed significant association in the second independent series of 938 cases and 5827 controls (Stage 3). Rs2735591 showed consistent association across the three stages (P > 0.05 for heterogeneity test), significant association in the combined validation samples (Pcorrected = 5.54 × 10−4 after correction for 4363 SNPs tested) and genome-wide significance in the whole GWAS and validation samples (P = 1.03 × 10−9, OR = 1.24). In addition, we demonstrated the lower expression of BCL10 in leprosy lesions than normal skins and a significant gene connection between BCL10 and the eight previously identified leprosy loci that are associated with NFκB, a major regulator of downstream inflammatory responses, which provides further biological evidence for the association. We have discovered a novel susceptibility locus on 1p22, which implicates BCL10 as a new susceptibility gene for leprosy. Our finding highlights the important role of both innate and adaptive immune responses in leprosy.
doi:10.1093/hmg/ddt286
PMCID: PMC3792694  PMID: 23784377
5.  Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies 
Genome Biology  2010;11(1):R8.
Twelve cDNA libraries from two species of catfish have been sequenced, resulting in the generation of nearly 500,000 ESTs.
Background
Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.
Results
A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.
Conclusions
This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.
doi:10.1186/gb-2010-11-1-r8
PMCID: PMC2847720  PMID: 20096101
6.  Interocular Shift of Visual Attention Enhances Stereopsis and Visual Acuities of Anisometropic Amblyopes beyond the Critical Period of Visual Development: A Novel Approach 
Journal of Ophthalmology  2014;2014:615213.
Aims. Increasing evidence shows that imbalanced suppressive drive prior to binocular combination may be the key factor in amblyopia. We described a novel binocular approach, interocular shift of visual attention (ISVA), for treatment of amblyopia in adult patients. Methods. Visual stimuli were presented anaglyphically on a computer screen. A square target resembling Landolt C had 2 openings, one in red and one in cyan color. Through blue-red goggles, each eye could only see one of the two openings. The patient was required to report the location of the opening presented to the amblyopic eye. It started at an opening size of 800 sec of arc, went up and down in 160 sec of arc step, and stopped when reaching the 5th reversals. Ten patients with anisometropic amblyopia older than age 14 (average age: 26.7) were recruited and received ISVA treatment for 6 weeks, with 2 training sessions per day. Results. Both Titmus stereopsis (z = −2.809, P = 0.005) and Random-dot stereopsis (z = −2.317, P = 0.018) were significantly improved. Average improvement in best corrected visual acuity (BCVA) was 0.74 line (t = 5.842, P < 0.001). Conclusions. The ISVA treatment may be effective in treating amblyopia and restoring stereoscopic function.
doi:10.1155/2014/615213
PMCID: PMC4279785  PMID: 25580280
7.  Enalapril inhibits tubulointerstitial inflammation and NLRP3 inflammasome expression in BSA-overload nephropathy of rats 
Acta Pharmacologica Sinica  2014;35(10):1293-1301.
Aim:
Proteinuria is not only a common marker of renal disease, but also involved in renal tubulointerstitial inflammation and fibrosis. The aim of this study was to investigate the mechanisms underlying the protective effects of enalapril, an ACEI, against nephropathy in rats.
Methods:
Wistar rats underwent unilateral right nephrectomy, and then were treated with BSA (5 g·kg−1·d−1, ip), or BSA plus enalapril (0.5 g·kg−1·d−1, po) for 9 weeks. The renal lesions were evaluated using histology and immunohistochemistry. The expression of NLRP3, caspase-1, IL-1β and IL-18 was analyzed using immunohistochemistry, RT-PCR and Western blot.
Results:
BSA-overload resulted in severe proteinuria, which peaked at week 7, and interstitial inflammation with prominent infiltration of CD68+ cells (macrophages) and CD3+ cells (T lymphocytes), particularly of CD20+ cells (B lymphocytes). BSA-overload markedly increased the expression of NLRP3, caspase-1, IL-1β and IL-18 in the proximal tubular epithelial cells, and in inflammatory cells as well. Furthermore, the expression of IL-1β or IL-18 was significantly correlated with proteinuria (IL-1β: r=0.757; IL-18: r=0.834). These abnormalities in BSA-overload rats were significantly attenuated by concurrent administration of enalapril.
Conclusion:
Enalapril exerts protective effects against BSA-overload nephropathy in rats via suppressing NLRP3 inflammasome expression and tubulointerstitial inflammation.
doi:10.1038/aps.2014.66
PMCID: PMC4186985  PMID: 25152022
proteinuria; tubulointerstitial inflammation; NLRP3 inflammasome; enalapril
8.  FTY720 Induces Apoptosis of M2 Subtype Acute Myeloid Leukemia Cells by Targeting Sphingolipid Metabolism and Increasing Endogenous Ceramide Levels 
PLoS ONE  2014;9(7):e103033.
The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia.
doi:10.1371/journal.pone.0103033
PMCID: PMC4106898  PMID: 25050888
9.  Surgery for Complete Vertical Rectus Paralysis Combined with Horizontal Strabismus 
Journal of Ophthalmology  2014;2014:828919.
Aims. To report outcomes of the simultaneous surgical correction of vertical rectus paralysis combined with moderate-to-large angle horizontal strabismus. Methods. If a preoperative forced duction test was positive, antagonist muscle weakening surgery was performed, and then augmented partial rectus muscle transposition (APRMT) + partial horizontal rectus recession-resection was performed 2 months later. If a preoperative forced duction test was negative, APRMT + partial horizontal rectus recession-resection was performed. Antagonistic muscle weakening surgery and/or conventional recession-resection of the horizontal and/or vertical muscles of the contralateral eye was performed 2 months later, as needed. Results. Ten patients with a mean age of 22.3 ± 13.0 years were included and mean follow-up was 7.1 months. The mean vertical deviation that APRMT corrected was 21.4 ± 3.7 PD (prism diopter). The absolute deviation in horizontal significantly decreased from a preoperative value of 48.5 ± 27.4 PD to a value of 3.0 ± 2.3 PD 6 months postoperatively. The movement score decreased from a value of −5 ± 0 preoperatively to a value of −2.7 ± 0.8 at 6 months postoperatively. Conclusion. For patients with complete vertical rectus paralysis combined with a moderate- to-large angle of horizontal strabismus, combined APRMT and partial horizontal rectus recession-resection is safe and effective for correcting vertical and horizontal strabismus.
doi:10.1155/2014/828919
PMCID: PMC4026980  PMID: 24883204
10.  Whole genome comparative analysis of channel catfish (Ictalurus punctatus) with four model fish species 
BMC Genomics  2013;14:780.
Background
Comparative mapping is a powerful tool to study evolution of genomes. It allows transfer of genome information from the well-studied model species to non-model species. Catfish is an economically important aquaculture species in United States. A large amount of genome resources have been developed from catfish including genetic linkage maps, physical maps, BAC end sequences (BES), integrated linkage and physical maps using BES-derived markers, physical map contig-specific sequences, and draft genome sequences. Application of such genome resources should allow comparative analysis at the genome scale with several other model fish species.
Results
In this study, we conducted whole genome comparative analysis between channel catfish and four model fish species with fully sequenced genomes, zebrafish, medaka, stickleback and Tetraodon. A total of 517 Mb draft genome sequences of catfish were anchored to its genetic linkage map, which accounted for 62% of the total draft genome sequences. Based on the location of homologous genes, homologous chromosomes were determined among catfish and the four model fish species. A large number of conserved syntenic blocks were identified. Analysis of the syntenic relationships between catfish and the four model fishes supported that the catfish genome is most similar to the genome of zebrafish.
Conclusion
The organization of the catfish genome is similar to that of the four teleost species, zebrafish, medaka, stickleback, and Tetraodon such that homologous chromosomes can be identified. Within each chromosome, extended syntenic blocks were evident, but the conserved syntenies at the chromosome level involve extensive inter-chromosomal and intra-chromosomal rearrangements. This whole genome comparative map should facilitate the whole genome assembly and annotation in catfish, and will be useful for genomic studies of various other fish species.
doi:10.1186/1471-2164-14-780
PMCID: PMC3840565  PMID: 24215161
Catfish; Genome; Comparative mapping; Linkage mapping; Conserved synteny
11.  Correction: Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition 
PLoS ONE  2013;8(10):10.1371/annotation/84e623b5-d1a7-47b7-96d7-4f5464b6f4b3.
doi:10.1371/annotation/84e623b5-d1a7-47b7-96d7-4f5464b6f4b3
PMCID: PMC3794066
12.  Black rice anthocyanidins prevent retinal photochemical damage via involvement of the AP-1/NF-κB/Caspase-1 pathway in Sprague-Dawley Rats 
Journal of Veterinary Science  2013;14(3):345-353.
The effects of black rice anthocyanidins (BRACs) on retinal damage induced by photochemical stress are not well known. In the present study, Sprague-Dawley rats were fed AIN-93M for 1 week, after which 80 rats were randomly divided into two groups and treated with (n = 40) or without BRACs (n = 40) for 15 days, respectively. After treatment, both groups were exposed to fluorescent light (3,000 ± 200 lux; 25℃), and the protective effect of dietary BRACs were evaluated afterwards. Our results showed that dietary BRACs effectively prevented retinal photochemical damage and inhibited the retinal cells apoptosis induced by fluorescent light (p < 0.05). Moreover, dietary BRACs inhibited expression of AP-1 (c-fos/c-jun subunits), up-regulated NF-κB (p65) expression and phosphorylation of IκB-α, and decreased Caspase-1 expression (p < 0.05). These results suggest that BRACs improve retinal damage produced by photochemical stress in rats via AP-1/NF-κB/Caspase-1 apoptotic mechanisms.
doi:10.4142/jvs.2013.14.3.345
PMCID: PMC3788161  PMID: 23820171
apoptosis; black rice anthocyanidins; Caspase-1; photochemical damage; retina
13.  Correction: Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients 
PLoS ONE  2013;8(8):10.1371/annotation/6d657d25-6835-40ee-9751-3f8fbc40e2b8.
doi:10.1371/annotation/6d657d25-6835-40ee-9751-3f8fbc40e2b8
PMCID: PMC3748241  PMID: 23976934
14.  Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition 
PLoS ONE  2013;8(6):e66356.
Epithelial–mesenchymal transition (EMT) is thought to contribute to the progression of renal tubulointerstitial fibrosis. Norcantharidin (NCTD) is a promising agent for inhibiting renal interstitial fibrosis. However, the molecular mechanisms of NCTD are unclear. In this study, a unilateral ureteral obstruction (UUO) rat model was established and treated with intraperitoneal NCTD (0.1 mg/kg/day). The UUO rats treated with NCTD showed a reduction in obstruction-induced upregulation of α-SMA and downregulation of E-cadherin in the rat kidney (P<0.05). Human renal proximal tubule cell lines (HK-2) stimulated with TGF-β1 were treated with different concentrations of NCTD. HK-2 cells stimulated by TGF-β1 in vitro led to downregulation of E-cadherin and increased de novo expression of α-SMA; co-treatment with NCTD attenuated all of these changes (P<0.05). NCTD reduced TGF-β1-induced expression and phosphorylation of Smad2/3 and downregulated the expression of Snail1 (P<0.05). These results suggest that NCTD antagonizes tubular EMT by inhibiting the Smad pathway. NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.
doi:10.1371/journal.pone.0066356
PMCID: PMC3692527  PMID: 23825538
15.  Effect of Nutrient Restriction and Re-Feeding on Calpain Family Genes in Skeletal Muscle of Channel Catfish (Ictalurus punctatus) 
PLoS ONE  2013;8(3):e59404.
Background
Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions.
Methodology/Principal Findings
In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15–20 g) for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05), clpn2 (1.3-fold increase, P<0.05), and clpn3 (13.0-fold decrease, P<0.05), whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01) after 17 and 35 days of starvation, respectively.
Conclusion/Significance
We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.
doi:10.1371/journal.pone.0059404
PMCID: PMC3602173  PMID: 23527186
16.  Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery 
Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.
doi:10.7150/ijbs.6100
PMCID: PMC3831115  PMID: 24250247
chronic kidney disease; exosome; miRNA; urine; biomarker.
17.  Effects of cumulus cells removal after 6 h co-incubation of gametes on the outcomes of human IVF 
Purpose
To investigate the effects of cumulus cells removal after 6 h co-incubation of gametes on the fertilization, polyspermy, multinucleation and clinical pregnancy rates in human IVF.
Methods
A total of 1,200 IVF-ET cycles undergoing 6 h co-incubation of gametes in 2009 were included in this study. Inclusion criteria were: female age <38 years, first IVF treatment, with bi-ovary and normal ovarian response, e.g., 4 ~ 20 oocytes could be obtained. A 6 h period of co-incubation was applied in all IVF cycles. According to the history of infertility, cumulus cells were mechanically removed either 6 h post-insemination or 20 h post-insemination. For couples with primary infertility, or unexplained infertility, or mild oligospermia or asthenospermia, the cumulus cells were removed at 6 h of insemination for the polar body observation (6 h group, n = 565). Of these, 80 cycles received early rescue ICSI due to fertilization failure or low fertilization rate at 6 h of insemination. For couples with secondary infertility and normal semen analysis, the cumulus cells were removed at 20 h of insemination as routine (20 h group, n = 635). Of these, three cycles received late rescue ICSI due to fertilization failure at 20 h of insemination. Normal fertilization, polyspermy (≥3PN), multinucleation and clinical pregnancy rates were compared between the two groups (rescue ICSI cycles were not included in the comparison in both groups).
Results
Significant difference (P < 0.05) was observed between the groups regarding polyspermy rates (7.48% in 6 h group and 9.22% in 20 h group). No difference was observed between the groups regarding normal fertilization rates (2PN rate) (64.89% in 6 h group and 65.74% in 20 h group). No difference was observed between the groups regarding multinucleation and clinical pregnancy rates (11.01% and 65.15% in 6 h group, 10.75% and 66.93% in 20 h group, respectively). The clinical pregnancy rate was 51.43% in cycles receiving early rescue ICSI, while no clinical pregnancy was obtained in cycles receiving late rescue ICSI.
Conclusion
The present results indicate that cumulus cells removal at 6 h of insemination is a relatively safe operation, which yielded comparable normal fertilization rate, multinucleation and clinical pregnancy rates compared with 20 h group. This protocol may be beneficial for early obsevation of fertilization failure and make early rescue ICSI possible.
doi:10.1007/s10815-011-9630-9
PMCID: PMC3241845  PMID: 21898104
In-vitro fertilization; Short co-incubation; Cumulus cells removal; Polyspermy; Clinical pregnancy; Rescue ICSI
18.  Inflammation Disrupts the LDL Receptor Pathway and Accelerates the Progression of Vascular Calcification in ESRD Patients 
PLoS ONE  2012;7(10):e47217.
Background
Chronic inflammation plays a crucial role in the progression of vascular calcification (VC). This study was designed to investigate whether the low-density lipoprotein receptor (LDLr) pathway is involved in the progression of VC in patients with end-stage renal disease (ESRD) during inflammation.
Methods and Results
Twenty-eight ESRD patients were divided into control and inflamed groups according to plasma C-reactive protein (CRP) level. Surgically removed tissues from the radial arteries of patients receiving arteriovenostomy were used in the experiments. The expression of tumour necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) of the radial artery were increased in the inflamed group. Hematoxylin-eosin and alizarin red S staining revealed parallel increases in foam cell formation and calcium deposit formation in continuous cross-sections of radial arteries in the inflamed group compared to the control, which were closely correlated with increased LDLr, sterol regulatory element binding protein-2 (SREBP-2), bone morphogenetic proteins-2 (BMP-2), and collagen I protein expression, as shown by immunohistochemical and immunofluorescent staining. Confocal microscopy confirmed that inflammation enhanced the translocation of the SREBP cleavage-activating protein (SCAP)/SREBP-2 complex from the endoplasmic reticulum to the Golgi, thereby activating LDLr gene transcription. Inflammation increased alkaline phosphatase protein expression and reduced α-smooth muscle actin protein expression, contributing to the conversion of the vascular smooth muscle cells in calcified vessels from the fibroblastic to the osteogenic phenotype; osteogenic cells are the main cellular components involved in VC. Further analysis showed that the inflammation-induced disruption of the LDLr pathway was significantly associated with enhanced BMP-2 and collagen I expression.
Conclusions
Inflammation accelerated the progression of VC in ESRD patients by disrupting the LDLr pathway, which may represent a novel mechanism involved in the progression of both VC and atherosclerosis.
doi:10.1371/journal.pone.0047217
PMCID: PMC3480367  PMID: 23115640
19.  miRNA589 Regulates Epithelial-Mesenchymal Transition in Human Peritoneal Mesothelial Cells 
Background. microRNA (miRNA, miR) are thought to interact with multiple mRNAs which are involved in the EMT process. But the role of miRNAs in peritoneal fibrosis has remained unknown. Objective. To determine if miRNA589 regulates the EMT induced by TGFβ1 in human peritoneal mesothelial cell line (HMrSV5 cells). Methods. 1. Level of miR589 was detected in both human peritoneal mesothelial cells (HPMCs) isolated from continuous ambulatory peritoneal dialysis (CAPD) patients' effluent and HMrSV5 cells treated with or without TGFβ1. 2. HMrSV5 cells were divided into three groups: control group, TGFβ1 group, and pre-miR-589+TGFβ1 group. The level of miRNA589 was determined by realtime PCR. The expressions of ZO-1, vimentin, and E-cadherin in HPMCs were detected, respectively. Results. Decreased level of miRNA589 was obtained in either HPMCs of long-term CAPD patients or HMrSV5 cells treated with TGFβ1. In vitro, TGFβ1 led to upregulation of vimentin and downregulation of ZO-1 as well as E-cadherin in HMrSV5 cells, which suggested EMT, was induced. The changes were accompanied with notably decreased level of miRNA589 in HMrSV5 cells treated with TGFβ1. Overexpression of miRNA589 by transfection with pre-miRNA589 partially reversed these EMT changes. Conclusion. miRNA589 mediates TGFβ1 induced EMT in human peritoneal mesothelial cells.
doi:10.1155/2012/673096
PMCID: PMC3479401  PMID: 23118514
20.  Second-Generation Genetic Linkage Map of Catfish and Its Integration with the BAC-Based Physical Map 
G3: Genes|Genomes|Genetics  2012;2(10):1233-1241.
Construction of high-density genetic linkage maps is crucially important for quantitative trait loci (QTL) studies, and they are more useful when integrated with physical maps. Such integrated maps are valuable genome resources for fine mapping of QTL, comparative genomics, and accurate and efficient whole-genome assembly. Previously, we established both linkage maps and a physical map for channel catfish, Ictalurus punctatus, the dominant aquaculture species in the United States. Here we added 2030 BAC end sequence (BES)-derived microsatellites from 1481 physical map contigs, as well as markers from singleton BES, ESTs, anonymous microsatellites, and SNPs, to construct a second-generation linkage map. Average marker density across the 29 linkage groups reached 1.4 cM/marker. The increased marker density highlighted variations in recombination rates within and among catfish chromosomes. This work effectively anchored 44.8% of the catfish BAC physical map contigs, covering ∼52.8% of the genome. The genome size was estimated to be 2546 cM on the linkage map, and the calculated physical distance per centimorgan was 393 Kb. This integrated map should enable comparative studies with teleost model species as well as provide a framework for ordering and assembling whole-genome scaffolds.
doi:10.1534/g3.112.003962
PMCID: PMC3464116  PMID: 23050234
catfish; linkage map; physical map; genome; map integration
21.  A Pilot Trial Assessing Urinary Gene Expression Profiling with an mRNA Array for Diabetic Nephropathy 
PLoS ONE  2012;7(5):e34824.
Background
The initiation and progression of diabetic nephropathy (DN) is complex. Quantification of mRNA expression in urinary sediment has emerged as a novel strategy for studying renal diseases. Considering the numerous molecules involved in DN development, a high-throughput platform with parallel detection of multiple mRNAs is needed. In this study, we constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers.
Methods
mRNA array containing 88 genes were fabricated and its performance was evaluated. A pilot study with 9 subjects including 6 DN patients and 3 normal controls were studied with the array. DN patients were assigned into two groups according to their estimate glomerular rate (eGFR): DNI group (eGFR>60 ml/min/1.73 m2, n = 3) and DNII group (eGFR<60 ml/min/1.73 m2, n = 3). Urinary cell pellet was collected from each study participant. Relative abundance of these target mRNAs from urinary pellet was quantified with the array.
Results
The array we fabricated displayed high sensitivity and specificity. Moreover, the Cts of Positive PCR Controls in our experiments were 24±0.5 which indicated high repeatability of the array. A total of 29 mRNAs were significantly increased in DN patients compared with controls (p<0.05). Among these genes, α-actinin4, CDH2, ACE, FAT1, synaptopodin, COL4α, twist, NOTCH3 mRNA expression were 15-fold higher than those in normal controls. In contrast, urinary TIMP-1 mRNA was significantly decreased in DN patients (p<0.05). It was shown that CTGF, MCP-1, PAI-1, ACE, CDH1, CDH2 mRNA varied significantly among the 3 study groups, and their mRNA levels increased with DN progression (p<0.05).
Conclusion
Our pilot study demonstrated that mRNA array might serve as a high-throughput and sensitive tool for detecting mRNA expression in urinary sediment. Thus, this primary study indicated that mRNA array probably could be a useful tool for searching new biomarkers for DN.
doi:10.1371/journal.pone.0034824
PMCID: PMC3356359  PMID: 22629296
22.  Parthenolide attenuates LPS-induced activation of NF-κB in a time-dependent manner in rat myocardium☆ 
Journal of Biomedical Research  2012;26(1):37-43.
Parthenolide (PTN), a selective nuclear factor kappa B (NF-κB) inhibitor, has been used extensively to inhibit NF-κB activation. The duration of the inhibitory effect of PTN on NF-κB in vivo remains unclear. This study was to determine whether a lipopolysaccharide (LPS) challenge 6, 12 and 24 h after the administration of PTN could activate NF-κB. Rats were devided into five groups. The rats in the PTN, PTN+LPS and DMSO groups were injected intraperitoneally with PTN or DMSO. After 6, 12 or 24 h, LPS was administered in LPS and PTN+LPS groups. The expressions of NF-κB p50, IκBα and p-IκBα were inhibited in both PTN and PTN+LPS group at end of 6 and 12 h and no effects at 24 h. In summary, myocardial NF-κB expression occurs 1 h after the administration of LPS. PTN blocks this effect given at 6 h and no inhibitory effect 24 h after administration in vivo.
doi:10.1016/S1674-8301(12)60005-0
PMCID: PMC3596078  PMID: 23554728
parthenolide; nuclear-factor-κB; lipopolysaccharide; myocardium
23.  Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition 
PLoS Pathogens  2011;7(10):e1002249.
The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.
Author Summary
The influenza neuraminidase (NA) enzyme is the most successful drug target against the seasonal and pandemic flu. The 2009 H1N1 flu pandemic led to record sales of the NA inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza). Recently, a new drug, laninamivir (Inavir), has been approved for use in Japan can also be administered effectively using a single dose via its octanoate prodrug (CS-8958), however its effectiveness against oseltamivir-resistant influenza infection has not been demonstrated in clinical studies. In this study we comprehensively evaluate the effectiveness of laninamivir and its prodrug using NA from different groups with different active site features. We expressed and purified a group 2 NA from the 1957 pandemic H2N2, an atypical group 1 NA from the 2009 H1N1 pandemic and a group 1 NA from avian H12N5. NA inhibition was assayed and NAs were further crystallized with each inhibitor to determine the structural basis of their action. We found that laninamivir inhibition is highly potent for each NA, however binding and inhibition of laninamivir and its prodrug showed group specific preferences. Our results provide the structural and functional basis of NA inhibition using classical and novel inhibitors, with NAs from multiple serotypes with different properties.
doi:10.1371/journal.ppat.1002249
PMCID: PMC3197600  PMID: 22028647
24.  Pharmacokinetics of ligustrazine ethosome patch in rats and anti-myocardial ischemia and anti-ischemic reperfusion injury effect 
The objective of this study was to investigate the pharmacokinetics of the ligustrazine ethosome patch and antimyocardial ischemia and anti-ischemic reperfusion injury effect. Male Sprague Dawley rats were divided randomly into 3 groups: Group A (intragastric ligustrazine), Group B (transdermal ligustrazine ethosome patch), and Group C (conventional transdermal ligustrazine patch). After treatment, samples of blood and of various tissues such as heart, liver, spleen, lung, kidney, brain, and muscle samples were taken at different time points. Drug concentration was measured with HPLC, and the drug concentration–time curve was plotted. Pharmacokinetic software 3p97 was applied to calculate pharmacokinetic parameters and the area under the drug concentration–time curve (AUC) in various tissues. The rat model of acute myocardial ischemia was constructed with intravenous injection of pituitrin and the model of myocardial ischemia-perfusion injury was constructed by tying off the left anterior descending coronary artery of rats to observe the effect of ligustrazine ethosome patches on ischemic myocardium and ischemia-reperfusion injury. Results showed that AUC was highest in the transdermal drug delivery group of ligustrazine ethosome patch. There were significant differences in whole blood viscosity, plasma viscosity, hematocrit, red blood cell aggregation index, and deformation index between ligustrazine the ethosome patch group and ischemic control group (P < 0.01). Moreover, ligustrazine ethosome patches could reduce the scope of myocardial infarction induced by long-term ischemia. Ligustrazine ethosome patches have a sustained-release property. They can maintain stable and sustained blood drug concentration, increase bioavailability, and reduce administration times. The drug patch can decrease hemorheological indices of myocardial ischemia in rats, as well as protect acute ischemic myocardium and ischemia-reperfusion injured myocardium.
doi:10.2147/IJN.S20263
PMCID: PMC3133529  PMID: 21760733
ligustrazine; ethosome; patch; pharmacokinetics; myocardial ischemia; ischemia- reperfusion injury
25.  Preparation of a ligustrazine ethosome patch and its evaluation in vitro and in vivo 
Background
The purpose of this study was to develop a transdermal ligustrazine patch containing a stable formulation and with good entrapment efficiency, release rate, and transdermal absorption.
Methods
Ligustrazine ethosomes were prepared by ethanol injection-sonication, with entrapment efficiency as an indicator. Using acrylic resin as the primary constituent, the ligustrazine ethosome patch was prepared by adding succinic acid as a crosslinking agent and triethyl citrate as a plasticizer. In vitro release and transdermal permeation studies were carried out. Finally, a pharmacokinetic study was carried out in rats to explore relative bioavailability. The formulations of ligustrazine ethosome were 1% (w/v) phospholipid, 0.4% (w/v) cholesterol, and 45% (v/v) ethanol.
Results
Ligustrazine ethosomes were obtained with an average particle size of 78.71 ± 1.23 nm and an average entrapment efficiency of 86.42% ± 1.50%. In vitro transdermal testing of the ligustrazine ethosome patches showed that the cumulative 24-hour amount of ligustrazine was up to 183 ± 18 μg/cm2. The pharmacokinetic results revealed that the relative bioavailability was 209.45%.
Conclusion
Compared with conventional ligustrazine administration, ligustrazine ethosome patches could promote better drug absorption and increase bioavailability. This study demonstrates that the transdermal action of the ligustrazine ethosome patch was comparatively good.
doi:10.2147/IJN.S16044
PMCID: PMC3075898  PMID: 21499422
ligustrazine; ethosomes; patch

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