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1.  Two unrelated patients with rare Crigler-Najjar syndrome type I: two novel mutations and a patient with loss of heterozygosity of UGT1A1 gene 
Crigler-Najjar syndrome type I (CN-I) is the most severe type of hereditary unconjugated hyperbilirubinemia. It is caused by homozygous or compound heterozygous mutations of the UDP-glycuronosyltransferase gene (UGT1A1) on chromosome 2q37. Two patients clinically diagnosed with CN-I were examined in this paper. We sequenced five exons and their flanking sequences, specifically the promoter region of UGT1A1, of the two patients and their parents. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the UGT1A1 gene copy number of one patient. In patient A, two mutations, c.239_245delCTGTGCC (p.Pro80HisfsX6; had not been reported previously) and c.1156G>T (p.Val386Phe), were identified. In patient B, we found that this patient had lost heterozygosity of the UGT1A1 gene by inheriting a deletion of one allele, and had a novel mutation c.1253delT (p.Met418ArgfsX5) in the other allele. In summary, we detected three UGT1A1 mutations in two CN-I patients: c.239_245delCTGTGCC (p.Pro80HisfsX6), c.1253delT (p.Met418ArgfsX5), and c.1156G>T (p.Val386Phe). The former two mutations are pathogenic; however, the pathogenic mechanism of c.1156G>T (p.Val386Phe) is unknown.
doi:10.1631/jzus.B1300233
PMCID: PMC4076604  PMID: 24793765
Crigler-Najjar syndrome type I (CN-I); Hyperbilirubinemia; UDP-glycuronosyltransferase gene (UGT1A1); Mutation; Loss of heterozygosity
2.  Common Variants in LRP2 and COMT Genes Affect the Susceptibility of Gout in a Chinese Population 
PLoS ONE  2015;10(7):e0131302.
Gout is a common inflammation disease resulting from an increase in serum uric acid. Nearly 70% of uric acid is excreted via the kidneys. To date, evidence for an association between genetic loci and gout is absent, equivocal or not replicated. Our study aims to test variants in two genes abundantly expressed in the kidney, LRP2 and COMT, for their association with uric acid and gout. In total, 1318 Chinese individuals were genotyped for rs2544390 in LRP2 and rs4680 in COMT. These LRP2 and COMT gene polymorphisms showed no significant effect on uric acid (P = 0.204 and 0.188, separately); however, rs2544390 in LRP2 did influence uric acid levels in individuals with BMI ≥ 25 (P = 0.009). In addition, the allele frequency distributions of the two loci showed a significant difference between gout patients and healthy controls. A missense variation in rs4680 (G > A) decreased the risk of gout (OR = 0.77, P = 0.015), whereas the T allele of rs2544390 was associated with gout pathogenesis risk (OR = 1.26, P = 0.020). The present study provides the first evidence for an association between COMT and gout. Rs2544390 in LRP2 only influenced uric acid levels in individuals with BMI ≥ 25, which might explain the discrepant results among previous studies. In addition, we are the first to identify the association between LRP2 and gout in a Chinese population and to confirm this association in Asians.
doi:10.1371/journal.pone.0131302
PMCID: PMC4493088  PMID: 26147675
3.  Purification, crystallization and preliminary X-ray crystallographic study of the tRNA pseudouridine synthase TruB from Streptococcus pneumoniae  
The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine at residue 55 of elongator tRNAs. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported.
The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine (Ψ) at residue 55 of elongator tRNAs. In order to better elucidate the functions of TruB in the formation of pseudouridine, the three-dimensional structure of full-length TruB was determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported. The crystal belonged to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 Å, β = 102.05°, and diffracted to a resolution of 1.7 Å. The crystal is most likely to contain one molecule in the asymmetric unit, with a V M value of 2.40 Å3 Da−1.
doi:10.1107/S1744309113012487
PMCID: PMC3702319  PMID: 23832202
TruB; Streptococcus pneumoniae
4.  Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis 
Protein & Cell  2015;6(8):589-598.
Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.
doi:10.1007/s13238-015-0171-3
PMCID: PMC4506285  PMID: 26091621
systemic sclerosis; fibrosis; collagen; heat shock protein 47; anti-centromere antibody; therapeutic target
5.  Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis 
Protein & Cell  2015;6(8):589-598.
Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.
doi:10.1007/s13238-015-0171-3
PMCID: PMC4506285  PMID: 26091621
systemic sclerosis; fibrosis; collagen; heat shock protein 47; anti-centromere antibody; therapeutic target
6.  Association between subjective well-being and exceptional longevity in a longevity town in China: a population-based study 
Age  2014;36(3):9632.
To examine the associations of cognitive and emotional facets (measured by life satisfaction [LS], positive affect [PA], negative affect [NA], and affect balance [AB]) of subjective well-being (SWB) with exceptional longevity (EL), we conducted a population-based study with 463 EL individuals (95+, EL group) recruited from a longevity town of Rugao, China (N = 755, with a response rate of 71.6 %), and 926 elderly individuals (60–69, elderly/control group). The population-based controls were sampled from the resident registry according to the gender ratio of the EL group. We found that the EL group had significantly higher levels of LS (30.74 vs. 28.93), PA (3.91 vs. 3.67), and AB (7.89 vs. 7.40) and a lower level of NA (1.02 vs. 1.27) than the elderly group. Multivariate logistic regression analysis revealed that higher levels of LS, PA, AB, and NA were significantly associated with EL, with odds ratios (ORs) of 1.98 (95 % CI, 1.36–2.89), 2.35 (95 % CI, 1.59–3.48), 2.56 (95 % CI, 1.75–3.75), and 0.50 (95 % CI, 0.33–0.74), respectively. Stratification analysis showed that the associations were significant in the healthy subsample, with the following ORs: LS = 2.31, PA = 2.53, AB = 3.05, and NA = 0.39. In conclusion, SWB, with high cognitive and emotional facets, was associated with EL in the healthy Rugao population. The findings imply that interventions that aim to improve elderly individuals’ SWB may promote their quality of life and, ultimately, EL.
doi:10.1007/s11357-014-9632-5
PMCID: PMC4082570  PMID: 24590626
Exceptional longevity; Subjective well-being; Life satisfaction; Positive affect; Negative affect; Affect balance
7.  Selective Radical Amination of Aldehydic C(sp2)–H Bonds with Fluoroaryl Azides via Co(II)-Based Metalloradical Catalysis: Synthesis of N-Fluoroaryl Amides from Aldehydes under Neutral and Nonoxidative Conditions 
The Co(II) complex of the D2h-symmetric amidoporphyrin 3,5-DitBu-IbuPhyrin, [Co(P1)], has proven to be an effective metalloradical catalyst for intermolecular amination of C(sp2)–H bonds of aldehydes with fluoroaryl azides. The [Co(P1)]-catalyzed process can employ aldehydes as the limiting reagents and operate under neutral and non-oxidative conditions, generating nitrogen gas as the only byproduct. The metalloradical aldehydic C–H amination is suitable for different combinations of aldehydes and fluoroaryl azides, producing the corresponding N-fluoroaryl amides in good to excellent yields. A series of mechanistic studies support a stepwise radical mechanism for the Co(II)-catalyzed intermolecular C–H amination.
doi:10.1039/C4SC00697F
PMCID: PMC4111259  PMID: 25071929
8.  Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells 
AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR).
METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry.
RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy.
CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.
doi:10.3748/wjg.v21.i17.5250
PMCID: PMC4419065  PMID: 25954098
Autophagy; Augmenter of liver regeneration; Apoptosis; HepG2 cells
9.  Ancient DNA Reveals That the Genetic Structure of the Northern Han Chinese Was Shaped Prior to 3,000 Years Ago 
PLoS ONE  2015;10(5):e0125676.
The Han Chinese are the largest ethnic group in the world, and their origins, development, and expansion are complex. Many genetic studies have shown that Han Chinese can be divided into two distinct groups: northern Han Chinese and southern Han Chinese. The genetic history of the southern Han Chinese has been well studied. However, the genetic history of the northern Han Chinese is still obscure. In order to gain insight into the genetic history of the northern Han Chinese, 89 human remains were sampled from the Hengbei site which is located in the Central Plain and dates back to a key transitional period during the rise of the Han Chinese (approximately 3,000 years ago). We used 64 authentic mtDNA data obtained in this study, 27 Y chromosome SNP data profiles from previously studied Hengbei samples, and genetic datasets of the current Chinese populations and two ancient northern Chinese populations to analyze the relationship between the ancient people of Hengbei and present-day northern Han Chinese. We used a wide range of population genetic analyses, including principal component analyses, shared mtDNA haplotype analyses, and geographic mapping of maternal genetic distances. The results show that the ancient people of Hengbei bore a strong genetic resemblance to present-day northern Han Chinese and were genetically distinct from other present-day Chinese populations and two ancient populations. These findings suggest that the genetic structure of northern Han Chinese was already shaped 3,000 years ago in the Central Plain area.
doi:10.1371/journal.pone.0125676
PMCID: PMC4418768  PMID: 25938511
10.  Postoperative continuous wound infusion of ropivacaine has comparable analgesic effects and fewer complications as compared to traditional patient-controlled analgesia with sufentanil in patients undergoing non-cardiac thoracotomy 
Objective: To compare the postoperative analgesic effects of continuous wound infusion of ropivacaine with traditional patient-controlled analgesia (PCA) with sufentanil after non-cardiac thoracotomy. Methods: One hundred and twenty adult patients undergoing open thoracotomy were recruited into this assessor-blinded, randomized study. Patients were randomly assigned to receive analgesia through a wound catheter placed below the fascia and connected to a 2 ml/h ropivacaine 0.5% (RWI group) or sufentanil PCA (SPCA group). Analgesia continued for 48 h. Visual analogue scores (VAS) at rest and movement, Ramsay scores and adverse effects were recorded at 2, 8, 12, 24, 36 and 48 h after surgery. Three months after discharge, patient’s satisfaction, residual pain and surgical wound complications were assessed. Results: General characteristics of patients were comparable between two groups. There were no statistical differences in the VAS scores and postoperative pethidine consumption between two groups (P > 0.05). However, when compared with SPCA group, the incidences of drowsiness, dizziness and respiratory depression, ICU stay and hospital expenditure reduced significantly in RWI group (P < 0.05). Patients’ satisfaction with pain management was also improved markedly in RWI group (P < 0.05). Conclusion: Continuous wound infusion with ropivacaine is effective for postoperative analgesia and has comparable effects to traditional PCA with sufentanil. Furthermore, this therapy may also reduce the incidences of drowsiness, dizziness, respiratory depression and decrease the ICU stay and hospital expenditure.
PMCID: PMC4483959  PMID: 26131121
Ropivacaine; sufentanil; patient-controlled analgesia; continuous wound infusion; non-cardiac thoracotomy
11.  Quantitating and Dating Recent Gene Flow between European and East Asian Populations 
Scientific Reports  2015;5:9500.
Historical records indicate that extensive cultural, commercial and technological interaction occurred between European and Asian populations. What have been the biological consequences of these contacts in terms of gene flow? We systematically estimated gene flow between Eurasian groups using genome-wide polymorphisms from 34 populations representing Europeans, East Asians, and Central/South Asians. We identified recent gene flow between Europeans and Asians in most populations we studied, including East Asians and Northwestern Europeans, which are normally considered to be non-admixed populations. In addition we quantitatively estimated the extent of this gene flow using two statistical approaches, and dated admixture events based on admixture linkage disequilibrium. Our results indicate that most genetic admixtures occurred between 2,400 and 310 years ago and show the admixture proportions to be highly correlated with geographic locations, with the highest admixture proportions observed in Central Asia and the lowest in East Asia and Northwestern Europe. Interestingly, we observed a North-to-South decline of European gene flow in East Asians, suggesting a northern path of European gene flow diffusing into East Asian populations. Our findings contribute to an improved understanding of the history of human migration and the evolutionary mechanisms that have shaped the genetic structure of populations in Eurasia.
doi:10.1038/srep09500
PMCID: PMC4382708  PMID: 25833680
12.  Fourier transform infrared spectroscopy for the distinction of MCF-7 cells treated with different concentrations of 5-fluorouracil 
Background
In order to provide personalized treatment to patients with breast cancer, an accurate, reliable and cost-efficient analytical technique is needed for drug screening and evaluation of tumor response to chemotherapy.
Methods
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used as a tool to assess cancer cell response to chemotherapy. MCF-7 cells (human breast adenocarcinoma cell line) were treated with different concentrations of 5-fluorouracil (5-FU). The inhibition of cell proliferation was monitored by MTT, and apoptosis rates were determined by flow cytometry. Finally, spectra of the cell populations were acquired by ATR-FTIR.
Results
The cell response to 5-FU was detectable at different concentrations by ATR-FTIR. First, a band observed at 1741 cm−1, representing membrane phospholipids, was enhanced with increasing 5-FU concentrations. In addition, the MCF-7 cell spectrum shifted progressively from 1153 to 1170 cm−1 with increasing drug doses. Finally, the normalized band intensity of 1741 cm−1/Amide I was highly correlated with the percentage of apoptotic cells as assessed by partial correlation analysis.
Conclusions
These findings suggest that the effects of different concentrations of drugs can be monitored by ATR-FTIR, which may help evaluate the response to chemotherapy and improve treatment strategies.
doi:10.1186/s12967-015-0468-2
PMCID: PMC4391530  PMID: 25884618
FTIR spectroscopy; 5-fluorouracil; MCF-7 cell; Chemotherapy; Phosphatidylserine
13.  NKD1 down-regulation is associated with poor prognosis in breast invasive ductal carcinoma 
As a negative modulator of the canonical Wnt signaling pathway, Naked1 (NKD1) is widely expressed in many normal tissues. However, the expression and clinicopathological significance of NKD1 in patients with breast cancer is still unclear. The aim of this study was to evaluate NKD1 expression in breast cancer and to investigate the question of whether reduced expression of NKD1 may have any pathological significance in breast cancer development or progression. In this study, we performed western blotting and immunohistochemistry to evaluate the expression of NKD1 and relevance with clinicopathological factors in the breast invasive ductal carcinoma. Reduction of NKD1 was significantly correlated with lymph node metastasis, histological grade and ER expression in breast cancer. Patients with negative NKD1 expression had significantly lower cumulative postoperative 5 year survival rate than those with positive NKD1 expression. This interpretation is in keeping with the results obtained from our in vitro experiments on MDA-MB-231 cells, we demonstrated that upregulation of NKD1 expression by infect with an adenovirus containing a NKD1 vector significantly reduced the migration of breast cancer cells. These data suggest that NKD1 plays an important role in invasion in human breast cancer and it appears to be a potential prognostic marker for patients with breast cancer.
PMCID: PMC4466976  PMID: 26097589
Breast carcinoma; NKD1; prognostic marker; metastasis
14.  Combination of CD4+CD25+CD127- regulatory T cells with MLC-BE and BE-Ab2: an efficient evaluation of the therapy of paternal lymphocyte induced immunization in unexplained recurrent spontaneous abortion patients 
The aim of this retrospective study was to compare the immune tolerance status of patients suffered from unexplained spontaneous abortion (URSA) before and after treatment with paternal lymphocyte induced immunization (PLII) four times, and its relationship to the pregnancy outcome. 168 URSA patients were included in the present study. Among 168 couples, 138 couples were conceived again, of whom 86 were successfully pregnant till 20 gestational weeks, 31 cases again failed in the first trimester, 21 cases were still under follow-up, another 30 cases still had not conceived. Both the level of one way mixed lymphocyte culture blocking efficiency (MLC-BE) and anti-idio blocking antibody (BE-Ab2) were markedly elevated in succeeded group after PLII. In contrast, although a significant increase could be observed in the failed group after treatment, the elevation of BE-Ab2 was much lower than that in successful group. PLII therapy significantly up-regulated the percentage of peripheral CD4+CD25+CD127- regulatory T cells (Tregs) in successfully pregnant women; however, there was no significant change of Tregs in pregnancy loss cases although receiving PLII therapy. These results suggested a positive correlation between higher frequency of Tregs and rate of successful pregnancies. The sensitivity and specificity of combination of Tregs with MLC-BE and BE-Ab2 were 81.8% and 81.3%, respectively. Therefore, the percentage of Tregs in peripheral blood may hopefully serve as a potential biomarker for monitoring the efficacy of therapy in URSA patients. Combination of Tregs with MLC-BE and BE-Ab2 may expect to better evaluate the efficacy of PLII in URSA patients.
PMCID: PMC4466977  PMID: 26097590
URSA; PLII; MLC-BE; BE-Ab2; Tregs; blocking antibody
15.  Cloning and Functional Characterization of SAD Genes in Potato 
PLoS ONE  2015;10(3):e0122036.
Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.
doi:10.1371/journal.pone.0122036
PMCID: PMC4380360  PMID: 25825911
16.  ILDR1 deficiency causes degeneration of cochlear outer hair cells and disrupts the structure of the organ of Corti: a mouse model for human DFNB42 
Biology Open  2015;4(4):411-418.
Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Mutations in ILDR1 are responsible for DFNB42, but the pathogenesis of hearing loss caused by ILDR1 mutations remains to be elucidated. To explore the role of ILDR1 in hearing, we created Ildr1 knockout mice. In heterozygous mice, ILDR1 expression was found in outer hair cells (OHCs) and inner hair cells (IHCs) of the organ of Corti. ILDR1-deficient mice are profoundly deaf by postnatal day 21 (P21). No significant difference was observed in the supporting cells and IHCs of ILDR1-deficient mice, but progressive degeneration of OHCs occurred at P15 and disruption of the tunnel running through the organ of Corti was noticeable at P21. By P28, there were no OHCs visible in any of the turns of the organ of Corti, and the tunnel of the organ of Corti was entirely destroyed. ILDR1 deficiency affects expression of tricellulin in vivo, and this provides a possible explanation to hearing loss. To further elucidate the mechanism of deafness related to ILDR1 deficiency, we pursued a differential proteomic approach to comprehensively assess differential protein expression in the cochleae of Ildr1+/− and Ildr1−/− mice at P21. Altogether, 708 proteins were up-regulated (fold change >1.5) and 114 proteins were down-regulated (fold change <0.5) in the Ildr1−/− mice compared with Ildr1+/− mice. Gene ontology classification indicated that a number of differentially expressed proteins are involved in cell adhesion, protein and vesicle-mediated transport, cell death, membrane organization, and cellular homeostasis. A few of these proteins are closely related to hearing development. Taken together, our data suggest that ILDR1 is important for the survival of OHCs and provide novel insights into the pathogenesis of human deafness DFNB42 deafness.
doi:10.1242/bio.201410876
PMCID: PMC4400585  PMID: 25819842
ILDR1; Deafness; Genetics
17.  AMACO is a novel component of the basement membrane associated Fraser complex 
Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly and other malformations resulting from mutations in FRAS1, FREM2 and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1 and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane (BM). However, several cases of FS could not be attributed to mutations in FRAS1, FREM2 or GRIP1, while Fraser syndrome displays high clinical variability, suggesting there is an additional genetic, possibly modifying contribution to this disorder. AMACO, encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1 deficient zebrafish and mice and that Fras1 and AMACO interact directly via their CSPG and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a novel member of the Fraser complex.
doi:10.1038/jid.2013.492
PMCID: PMC4361737  PMID: 24232570
18.  MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro 
Scientific Reports  2015;5:8689.
Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.
doi:10.1038/srep08689
PMCID: PMC4346788  PMID: 25732513
19.  Crystallization and preliminary X-ray crystallographic studies of DnaJ from Streptococcus pneumoniae  
DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported.
DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I212121 and the diffraction resolution is 3.0 Å with unit-cell parameters a = 47.68, b = 104.45, c = 234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a V M value of 3.24 Å3 Da−1 and a solvent content of 62.1%.
doi:10.1107/S1744309113001668
PMCID: PMC3606571  PMID: 23519801
DnaJ; Streptococcus pneumoniae
20.  Associations of PI3KR1 and mTOR Polymorphisms with Esophageal Squamous Cell Carcinoma Risk and Gene-Environment Interactions in Eastern Chinese Populations 
Scientific Reports  2015;5:8250.
Single nucleotide polymorphisms (SNPs) in the PI3K/PTEN/AKT/mTOR signaling pathway may contribute to carcinogenesis. We genotyped five potentially functional PIK3R1 and mTOR SNPs in 1116 esophageal squamous cell cancer (ESCC) patients and 1117 cancer-free controls to assess their associations with ESCC risk. We observed no association with ESCC risk for any of the selected SNPs. However, the combined analysis of these SNPs revealed that subjects with one-to-three risk genotypes had an increased ESCC risk. Stratified analysis by body mass index (BMI) found that ESCC risk was significantly associated with each of three mTOR SNPs among subjects with BMI < 25.0. Specifically, we found that subjects carrying ≥ 1 risk genotypes had significantly increased ESCC risk, particularly for males, ever-smokers, ever-drinkers, and those with age > 60, or BMI < 25.0. Moreover, three mTOR haplotypes were associated with an increase in ESCC risk. Our meta-analysis of mTOR rs2295080 and cancer risk provided further evidence that mTOR SNPs might modulate cancer susceptibility. In this population, such risk effects might be modified by other risk factors, highlighting the importance of gene-environment interaction in esophageal carcinogenesis. Additional, larger studies are warranted to validate our findings.
doi:10.1038/srep08250
PMCID: PMC4318264  PMID: 25654238
21.  Identification and validation of the methylation biomarkers of non-small cell lung cancer (NSCLC) 
Clinical Epigenetics  2015;7(1):3.
Background
DNA methylation was suggested as the promising biomarker for lung cancer diagnosis. However, it is a great challenge to search for the optimal combination of methylation biomarkers to obtain maximum diagnostic performance.
Results
In this study, we developed a panel of DNA methylation biomarkers and validated their diagnostic efficiency for non-small cell lung cancer (NSCLC) in a large Chinese Han NSCLC retrospective cohort. Three high-throughput DNA methylation microarray datasets (458 samples) were collected in the discovery stage. After normalization, batch effect elimination and integration, significantly differentially methylated genes and the best combination of the biomarkers were determined by the leave-one-out SVM (support vector machine) feature selection procedure. Then, candidate promoters were examined by the methylation status determined single nucleotide primer extension technique (MSD-SNuPET) in an independent set of 150 pairwise NSCLC/normal tissues. Four statistical models with fivefold cross-validation were used to evaluate the performance of the discriminatory algorithms. The sensitivity, specificity and accuracy were 86.3%, 95.7% and 91%, respectively, in Bayes tree model. The logistic regression model incorporated five gene methylation signatures at AGTR1, GALR1, SLC5A8, ZMYND10 and NTSR1, adjusted for age, sex and smoking, showed robust performances in which the sensitivity, specificity, accuracy, and area under the curve (AUC) were 78%, 97%, 87%, and 0.91, respectively.
Conclusions
In summary, a high-throughput DNA methylation microarray dataset followed by batch effect elimination can be a good strategy to discover optimal DNA methylation diagnostic panels. Methylation profiles of AGTR1, GALR1, SLC5A8, ZMYND10 and NTSR1, could be an effective methylation-based assay for NSCLC diagnosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-014-0035-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13148-014-0035-3
PMCID: PMC4318209  PMID: 25657825
Non-small cell lung cancer; DNA methylation; Biomarker; Batch effect elimination; Diagnosis
22.  Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans 
Scientific Reports  2014;4:7465.
EPAS1 involves in the hypoxic response and is suggested to be responsible for the genetic adaptation of high-altitude hypoxia in Tibetans. However, the detailed molecular mechanism remains unknown. In this study, a single nucleotide polymorphism rs56721780:G>C and an insertion/deletion (indel) polymorphism −742 indel in the promoter region showed divergence between Tibetans and non-Tibetan lowlanders. rs56721780:G>C regulated the transcription of EPAS1 by IKAROS family zinc finger 1 (IKZF1), which was identified as a new transcriptional repressor for EPAS1 gene. It demonstrated that the C allele of rs56721780:G>C decreased the binding of IKZF1, leading to the attenuated transcriptional repression of EPAS1 gene. The insertion at −742 indel provided a new binding site for Sp1 and was related to the activation of EPAS1 promoter. Further functional analysis revealed that lysyl oxidase (LOX) gene, which was reported to be responsible for extracellular matrix protein cross-linking of amnion previously, was a direct target of EPAS1. The CC genotype at rs56721780:G>C and the insertion genotype at −742 indel were found associated with higher EPAS1 and LOX expression levels in amnion, as well as higher birth weight of Tibetan newborns, suggesting that EPAS1 gene might play important roles in the development of amnion, fetus growth and high-altitude adaptation of Tibetans.
doi:10.1038/srep07465
PMCID: PMC4264014  PMID: 25501874
23.  Ultrasound contrast-enhanced imaging and in vitro antitumor effect of paclitaxel-poly(lactic-co-glycolic acid)-monomethoxypoly (ethylene glycol) nanocapsules with ultrasound-targeted microbubble destruction 
Molecular Medicine Reports  2014;11(4):2413-2420.
A combination of diagnostic and therapeutic ultrasound (US) techniques may be able to provide the basis of specific therapeutic protocols, particularly for the treatment of tumors. Nanotechnology may aid the progression towards the use of US for tumor diagnosis and targeted therapy. The current study investigated in vivo and in vitro US contrast imaging using nanocapsules (NCs), and also US and US-targeted microbubble destruction (UTMD) therapy using drug-loaded NCs for pancreatic cancer in vitro. In the current study, the NCs were made from the polymer nanomaterial poly(lactic-co-glycolic acid)-monomethoxypoly(ethylene glycol) (PLGA-mPEG), encapsulated with paclitaxel (PTX), to create PTX-PLGA-mPEG NCs. The PTX-PLGA-mPEG NCs were used as a US contrast agent (UCA), which produced satisfactory US contrast-enhanced images in vitro and in vivo of the rabbit kidneys, with good contrast compared with lesions in the peripheral regions. However, clear contrast-enhanced images were not obtained using PTX-PLGA-mPEG NCs as a UCA, when imaging the superficial pancreatic tumors of nude mice in vivo. Subsequently, fluorescence and flow cytometry were used to measure the NC uptake rate of pancreatic tumor cells under various US or UTMD conditions. An MTT assay was used to evaluate the efficiency of PTX and PTX-PLGA-mPEG NCs in killing tumor cells following 24 or 48 h of US or UTMD therapy, compared with controls. The specific US or UTMD conditions had been previously demonstrated to be optimal through repeated testing, to determine the conditions by which cells were not impaired and the efficiency of uptake of nanoparticles was highest. The current study demonstrated high cellular uptake rates of PLGA-mPEG NCs and high tumor cell mortality with PTX-PLGA-mPEG NCs under US or UTMD optimal conditions. It was concluded that the use of NCs in US-mediated imaging and antitumor therapy may provide a novel application for US.
doi:10.3892/mmr.2014.3072
PMCID: PMC4337512  PMID: 25500683
nanocapsules; paclitaxel-poly(lactic-co-glycolic acid)-monomethoxy poly(ethylene glycol); ultrasound contrast-enhanced imaging; ultrasound-targeted microbubble destruction; antitumor
24.  A new method of building permanent A-V block model: ablating his-bundle potential through femoral artery with pre-implanted biventricular pacemaker 
Background
To explore the feasibility of a new method of achieving a permanent A-V block animal model.
Methods
16 beagles were randomly divided into two groups based on the method of their pre-implanted biventricular pacemakers. (1) In the first group (8 beagles), the A-V block model was achieved by ablating his-bundle potential at the site of the left ventricular superior-septum, under the aortic sinus, through femoral artery. (2) In the second group (8 beagles), the A-V block model was achieved by ablating his-bundle potential at the triangle of Koch, through femoral vein. A complete A-V block model was achieved as a standard in this study. The success rates, intraoperative arrhythmias, operative and X-ray exposure time, intraoperative bleeding amount were assessed in this two groups, both animal models were followed up for four weeks and then fasted to monitor myocardial pathological changes.
Results
The success rate of the first group, which with fewer intraoperative arrhythmias, and less operative and X-ray exposure time, was significantly higher than the second group.
Conclusions
Compared with traditional animal method, our new method of ablating his-bundle potential at the left ventricle from the femoral artery has a higher success rate, fewer occurrence of malignant arrhythmias, and less operation and X-ray time. Thus, our new method should be preferred in the building of Permanent A-V Block Model.
doi:10.1186/1471-2261-14-164
PMCID: PMC4246545  PMID: 25410685
A-V block; His-bundle potential; Biventricular pacemaker; Radiofrequency ablation; Beagle; Disease models

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