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Objective We investigated the application of high-resolution microarray-based comparative genomic hybridisation (array CGH) on a fetus showing increased nuchal translucency (NT). Design Case study. Setting Tertiary referral obstetrics unit. Sample Pregnant woman attended the antenatal clinic. Methods Conventional karyotyping and genetic test was carried out for the alpha-globin gene. High-resolution array CGH using the high-density 244K Agilent microarray was performed on fetal blood sample by cordocentesis to investigate the possibility of any genomic imbalance. Main outcome measures Detection of chromosomal abnormality. Results Karyotyping analysis showed 46,XY. Molecular genetic diagnosis confirms the fetus has Hb-H constant spring disease but cannot explain the increased NT to 3.2 mm. Array CGH analysis discovered a 1.32-Mb microdeletion on chromosome 16p13.11. Deletion at 16p13.11 has been implicated to predispose to autism and/or mental retardation. Baby was delivered at 40 weeks of gestation, and follow up was carried out at 3 months of age without sign of mental retardation/developmental delay. Conclusions This case study demonstrated that array CGH can accurately calibrate the size and identify de novo interstitial chromosome imbalances. However, the presence of chromosome copy variants with unknown clinical significance currently limits its wider scale application in prenatal diagnosis and needs further investigations.

Glycogen storage disease type la (GSD1a) is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase (G6Pase). We analyzed the G6Pase genes of two unrelated Chinese families with GSD1a. DNA sequencing of all five exons and the exon-intron boundaries revealed a G T transversion at nucleotide 727 (727G-->T) in exon 5, which has previously been reported to cause abnormal splicing. In one family, the subject and her affected sister were confirmed to be homozygous for this mutation and their parents to be heterozygotes. In the other family, the proband was identified to be heterozygous for this mutation, and a novel mutation, the 341delG in exon 2, was identified. This mutation alters the reading frame and creates a stop codon TAA 15 codons downstream from the mutation, resulting in a truncated protein. Family studies revealed that the father was heterozygous for the 727G-->T mutation and that the mother was heterozygous for the 341delG mutation. This is the first time that the 727G T mutation has been found in Chinese patients or outside Japan. Since we only tested two GSD1a families and found 727G-->T in both, we believe that this mutation may also be prevalent in our local Chinese population. To investigate allele frequencies, we screened 385 Chinese healthy volunteers and found two asymptomatic carriers. Our findings suggest that the 727G-->T mutation is indeed prevalent in Hong Kong.

Microscopic forms of karyotyping and cytogenetic analysis by means of G-banded chromosome analysis and rapid FISH (fluorescencein situhybridization) on amniotic fluids or chorionic villus samples are at present regarded as the gold standard for prenatal diagnosis of chromosomal anomalies. Nevertheless, up to now the resolution of conventional chromosomal analysis was limited to approximately 4–5 Mb and not smaller than 2 Mb for FISH. Thus numerous common microdeletion syndromes are not detectable by conventional karyotyping. In addition, prenatal cells yield lower band resolution by conventional karyotyping than peripheral white blood cells making detection of subtle abnormalities even more difficult. With the advances in molecular-based techniques, a collaborative effort has led to the standardized method for detection of a restricted set of common chromosomal aneuploidies and microdeletion syndromes such as Down's syndrome, DiGeorge or Angelman syndrome either by rapid FISH and/or quantitative fluorescent PCR (QF-PCR). Even if the presence of particular phenotypic features of microdeletion or duplication syndromes may direct the use of syndrome-specific FISH tests in the postnatal period, syndrome-specific FISH analysis still has a very limited potential and application in the prenatal period due to the limitation in prenatal morphological or imaging diagnosis of many of the syndromes.

Intraocular pressure (IOP) elevation is an important cause of glaucoma. Animal models of ocular hypertension have been widely used to mimic glaucoma to investigate the mechanisms underlying retinal ganglion cell (RGC) death and search for possible cure. The aim of the present study was to examine the role of JAK/STAT pathway in RGC viability in normal condition or after acute IOP elevation. Retinal explants obtained from intact or IOP-elevated eyes were firstly used to examine the effect of the JAK/STAT pathway inhibitors, AG490 and Jak Inhibitor I, on RGC viability in vitro. The role of this signal pathway was further investigated and confirmed in vivo. AG490 and Jak Inhibitor I were applied into the left eye on days 3, 9, and 15 post 2-h IOP elevation at 110mmHg. Fluorescence dye Fluorogold was used to retrogradely label surviving RGCs. Because macrophage recruitment was seen in the IOP-elevated eyes after inhibition of this pathway, clodronate liposomes were used to remove phagocytic cells in the eye and examine the role of JAK/STAT pathway in RGC survival independent of macrophages. Activities and location of JAK/STAT pathway in the retina were examined using Western blotting and immunohistochemistry. We found that inhibition of JAK/STAT pathway did not affect RGC survival in the retinal explants derived from intact eye but caused RGC death in the retinal explants that were derived from IOP-elevated eye. Importantly, the detrimental effect of JAK/STAT pathway inhibition on RGC survival was also observed in vivo following acute IOP elevation, but not in intact eye. In addition, both in vitro and in vivo experiments confirmed a detrimental action of phagocytic cells following acute IOP elevation and the pathway inhibition. Compatible with what were observed in vivo, Western blotting and immunohistochemistry showed that JAK/STAT activities were not present in intact retina, but acute IOP elevation activated JAK/STAT pathway in the retina, in the regions of inner nuclear layer and ganglion cell layer, including RGCs. The IOP elevation-induced JAK/STAT activities were effectively abolished by intravitreal application of AG490. This study thus shows that (1) acute IOP elevation activates JAK/STAT pathway in RGCs, and (2) JAK/STAT pathway mediates RGC survival following IOP elevation but not under normal condition.

Purpose To investigate the penetration of ofloxacin and moxifloxacin into the aqueous and vitreous after topical administration one hour before vitrectomy surgery. Design Prospective, randomized, double-blind case series study. Methods Twenty-seven patients undergoing vitrectomy were randomized to receive either topical ofloxacin 0.3% or moxifloxacin 0.5% every 10 minutes for one hour before surgery. Aqueous and vitreous samples were obtained and analyzed using high-performance liquidation chromatography. Results The moxifloxacin aqueous (1.576 ± 0.745 μg/ml) and vitreous (0.225 ± 0.013 μg/ml) levels were significantly higher than the ofloxacin aqueous (0.816 ± 0.504 μg/ml) (P = .0009) and vitreous (0.225 ± 0.013 μg/ml) [P = .0054] levels, respectively. The mean moxifloxacin aqueous and vitreous levels exceeded the minimum inhibitory concentration for 90% of isolates (MIC90) for a wide variety of bacteria implicated in endophthalmitis. In contrast, the aqueous level of ofloxacin exceeded the MIC90 of only a few organisms. Conclusions Moxifloxacin applied every 10 minutes during the hour before vitrectomy penetrated the eye significantly better than ofloxacin.

We sought to investigate the utility of chromosomal microarray analysis (CMA) for prenatal diagnosis of oral clefts, as compared with traditional chromosome analysis, for improved prenatal genetic counseling and discovery of a potential correlation between genotype and oral cleft. This retrospective analysis encompassed 270 prenatal oral cleft cases with documented detailed ultrasound findings and CMA results from four referral centers. Detection rates for pathogenic copy-number variants (CNVs) were calculated and compared with cases for which chromosome analysis was also performed. The overall detection rate was 14.8% (40/270) for pathogenic CNVs by CMA, 7.2% (9/125) for the nonsyndromic cases, and 21.4% (31/145) for the syndromic cases. Of the nonsyndromic cases with ultrasound soft markers, 20% (5/25) were identified with pathogenic CNVs. CMA showed an improved detection rate of 15.3% (29/190) compared with 10.5% (20/190) for chromosome analysis. This study not only highlights the improved detection of chromosomal defects by CMA in prenatal oral clefts but also deepens our understanding of oral clefts. The results suggest that CMA is highly recommended in prenatal invasive genetic testing not only for syndromic oral cleft cases but also for nonsyndromic cases with soft markers. Candidate genes including CRKL, AKAP8, SYDE1, BRD4 are worthy of further investigation regarding their role in human palatogenesis. Genet Med 18 10, 1052–1055.

Proteases have been shown to modulate pain signaling in the spinal cord and may contribute to the development of chronic postsurgical pain. By using peripheral inflammation in rats as a chronic pain model, the authors identified the deregulation of proteases and their inhibitors as a hallmark of chronic pain development using a genome-wide screening approach.A microarray analysis was performed and identified spinal cathepsin G (CTSG) as the most up-regulated gene in rats with persistent hyperalgesia after intraplantar injection of complete Freund's adjuvant (n = 4). Further experiments were performed to elucidate the mechanisms of CTSG-induced hyperalgesia by intrathecally applying specific CTSG inhibitor (n = 10). The authors also evaluated the association between CTSG gene polymorphisms and the risk of chronic postsurgical pain in 1,152 surgical patients.CTSG blockade reduced heat hyperalgesia, accompanied by a reduction in neutrophil infiltration and interleukin 1β levels in the dorsal horns. In the gene association study, 246 patients (21.4%) reported chronic postsurgical pain at 12-month follow-up. Patients with AA genotypes at polymorphisms rs2070697 (AA-15.3%, GA-24.1%, and GG-22.3%) or rs2236742 (AA-6.4%, GA-20.4%, and GG-22.6%) in the CTSG gene had lower risk for chronic postsurgical pain compared with wild-types. The adjusted odds ratios were 0.67 (95% CI, 0.26 to 0.99) and 0.34 (95% CI, 0.21 to 0.98), respectively.This study demonstrated that CTSG is a pronociceptive mediator in both animal model and human study. CTSG represents a new target for pain control and a potential marker to predict patients who are prone to develop chronic pain after surgery.

The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs-on-Beads(TM) (PNBoBs(TM) ) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBs(TM) under different prenatal indications.A total of 9648 prenatal samples were prospectively analyzed by karyotyping plus PNBoBs(TM) and classified by prenatal indication. The frequencies of the genomic defects and their 95%CIs were calculated for each indication.The overall incidence of cryptic imbalances was 0.7%. The majority involved the DiGeorge syndrome critical region (DGS). The additional diagnostic yield of PNBoBs(TM) in the population with a low a priori risk was 1/298. The prevalences of DGS microdeletion and microduplication in the low-risk population were 1/992 and 1/850, respectively.The constant a priori risk for common pathogenic cryptic imbalances detected by this technology is estimated to be ~0.3%. A prevalence higher than that previously estimated was found for the 22q11.2 microdeletion. Their frequencies were independent of maternal age. These data have implications for cell-free DNA screening tests design and justify prenatal screening for 22q11 deletion, as early recognition of DGS improves its prognosis.

Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis.We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multicenter series of 42 persons with 16p11.2 deletions.We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P3.8×10(-6)). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P1.1×10(-6)). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis.Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed. (Funded by the National Basic Research Program of China and others.).

We carried out a pharmacokinetic study to determine the levels and profiles of catechins in pregnant rats and their fetuses after ingestion of green tea extract (GTE). We measured total catechin levels after enzyme digestions. Dams, at 15.5 days of gestation, were fed with GTE and catechins were measured in the maternal plasma, placenta, and fetus 0, 0.5, 1, 2, 3, 5, 8, 10, 12, 16, and 20 h after maternal GTE intake. The pharmacokinetic changes were analyzed by non compartmental models. We found that maternal plasma concentrations of catechins were about 10 times higher than in placenta and 50-100 times higher than in the fetus. AUC and Cmax levels of (-)-epicatechin (EC) were the highest in plasma while the levels of (-)-epigallocatechin gallate (EGCG) were the highest in the placenta and the fetus. The exposure level of catechin derivatives was higher than the gallate derivatives in maternal plasma after normalization but reversed in the placenta and fetus. The absorption of epi-isomers in plasma was found to be more favorable than their non epi-isomer counterparts. EGCG had the highest level of exposure (AUC) and the highest Cmax in the fetus, implying it may have potential for in utero antioxidant protection.

Objectives: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. Design and methods: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. Results: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including αA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). Conclusions: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

To determine the aqueous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in patients with active polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and pathologic myopia.Prospective, comparative control study.Aqueous humors were collected from 32 eyes of 32 patients for either active PCV or CNV. Among them, 11 eyes had active and symptomatic PCV, 12 eyes had active CNV secondary to AMD, and nine eyes had active CNV of pathologic myopia. Levels of VEGF and PEDF were determined by commercially available enzyme-linked immunosorbent assay kits. A group of 10 aqueous samples from 10 patients who underwent cataract surgery without other ocular or systemic diseases comprised the controls.VEGF concentrations in aqueous humor were markedly increased in patients with PCV, CNV of AMD, and CNV of myopia when compared with the controls (analysis of variance [ANOVA], P.001). VEGF levels in eyes with PCV were, however, significantly lower than those of exudative AMD (P = .045). The PEDF levels were also significantly different among the groups (ANOVA, P = .001), and we observed increased levels in PCV, CNV of AMD, and CNV of myopia.VEGF and PEDF factors were coexpressed and increased with positive correlation in aqueous humor of eyes with active PCV. The different levels of both factors in eyes of PCV and AMD might suggest distinct clinical entities or different angiogenesis courses between PCV and AMD.

BACKGROUND: Estrogen replacement therapy (ERT) has been used in the treatment of pelvic organ prolapse (POP) but clinical results are inconclusive. The purpose of this study was to investigate the effect of 17β-estradiol (E 2 ) on the proliferation of fibroblasts derived from cardinal ligaments in women with or without POP. METHODS: Fibroblasts were derived from seven patients with POP and seven age-matched controls. The growth rate of POP fibroblasts was compared with that of control by 3-(4,5,-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Four cell strains from each patient and control group were treated with different concentrations of E 2 (10 -4 , 10 -8 , 10 -9 and 10 -10 mol/l). The effect of E 2 on cell proliferation was then measured by MTT assay. RESULTS: The overall growth rate of POP fibroblasts was significantly slower than that of controls under normal culture conditions. Addition of E 2 suppressed cell proliferation of all the fibroblasts, especially in POP fibroblasts. POP fibroblasts showed a significantly lower proliferative rate than that of controls at all E 2 concentrations, with the most prominent inhibitory effect at physiological concentration (10.83 ± 34.41% versus 81.56 ± 48.10% at 10 -8 mol/l). CONCLUSIONS: Our results suggest that decreased fibroblast turnover may contribute to the development of POP; and ERT may not be an effective POP treatment.

We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2'-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of the retinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.

The optical coherence tomography (OCT) findings, clinical features, and mutations in the RS1 gene of two unrelated patients with X-linked retinoschisis (XLRS) are reported herein. Two Chinese patients with early onset XLRS were given a comprehensive ophthalmologic examination and OCT investigation. The RS1 gene was screened for sequence alterations in all exons and splice regions. The two patients presented with different phenotypic features and OCT findings. One patient with more severe clinical presentation had a RS1 exon 1 deletion and a P193S mutation was found in the other patient with mild macular involvement. OCT demonstrates the markedly different features of XLRS patients with different RS1 mutations. This study strengthens the role of OCT in the diagnosis and monitoring of XLRS.

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β- d -glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10–2000 and 10–5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.

A quick, simple, and reliable HPLC method for analysis of lignocaine and its metabolite xylidine in aqueous humor was developed. Aqueous humor was precipitated by 30% 5-sulfosalicylic acid and acetonitrile, then buffered by 4 M phosphate buffer at pH 4.0, with bupivacaine as internal standard. The chromatographic method employed a reverse C18 silica AccQ-Tag column and gradient mobile phase AccQ-Tag Eluent A and acetonitrile for separation at 37°C. The sample molecules were quantified at 220 nm. The analytical recoveries ranged from 96–109% and inter-assay imprecision less than 3.5%. In the analysis of 200 aqueous humor samples, no xylidine was detected after lignocaine gel application.

The human retina is the peripheral portion of the visual system. It contains a great diversity of neuronal cell types, including six major neuronal cell types and one glial cell type organized in a laminar structure. Current models for retinal neuron differentiation suggest that the formation of a specific retinal neuron is determined by the intrinsic properties of the retinal progenitor and extrinsic cues from the retinal environment, and yet few intrinsic gene products have been identified that direct this process. Through recent developments in mouse and human genetics, and also by the use of molecular biological techniques, we are beginning to understand the genetic control of the differentiation and function of these molecules in the retina. Several transcription factors, such as the Pax6 gene, and neural transcription factors (bHLH) together with the Notch and Delta signaling pathway have been implicated in retinal cell subtype specification. The aim of this review is to summarize the current status of molecular genetic findings in the developing retina.

Supplemental digital content is available in the text., Background Ischemia-reperfusion injury (IRI) is commonly encountered after liver surgery. This study evaluated the hepatoprotective effects of topically applied adipose-derived mesenchymal stromal cells (ADMSCs) on hepatic IRI in a rat model. Methods ADMSCs from transgenic green fluorescent protein Sprague-Dawley rats were topically applied to the liver surface of Sprague-Dawley rats after hepatic IRI and fixed in position by fibrin glue (group A, n = 24). An equivalent amount of ADMSCs were administered through the portal (group B, n = 24) or tail vein (group C, n = 24). In the control group (group D, n = 20), no treatment was given to the IRI liver. Results All the rats in group A and group D survived. Within 2 days after hepatic IRI, only 50% of rats survived in group B, and ADMSCs were detected in thromboemboli within large vessels. 62.5% of the rats died in group C because most of the ADMSCs were trapped in the lungs. ADMSCs migrated across the liver capsule and homed to the injured liver parenchyma 3 days after topical application in group A. The homed ADMSCs expressed hepatocyte nuclear factor-4α and hepatocyte nuclear factor-1. Compared with group D, the rate of hepatic regeneration in group A was enhanced with less inflammation, smaller necrotic areas, and improved liver function. Proinflammatory cytokines IL-6, IL-21, and CD70 were significantly downregulated in group A by 6.3-, 2.7-, and 12.7-fold, respectively (P < 0.05). The neurogenic locus NOTCH homolog protein pathway was activated in the topical ADMSCs. Conclusions Topically applied adipose-derived mesenchymal stromal cells demonstrated hepatoprotective effects on hepatic IRI in an animal model.

Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive. High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole-genome linkage analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known ADNSHL genes and shared by the sequenced samples: c.328CA in COCH (DFNA9) resulting in a p.Q110K substitution and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs*2. The pathogenicity of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co-segregation with phenotype in the pedigree and in light of established genotype-phenotype correlations. The frameshift deletion in MYO6 was confirmed as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the linkage scan. Our clinical data also supported the emerging genotype-phenotype correlation for DFNA22.

Delivery of drugs and biomolecules into skin has significant advantages. To achieve this, herein, a nanomaterial-strengthened dissolving microneedle patch for transdermal delivery is reported. The patch comprises thousands of microneedles, which are composed of dissolving polymers, nanomaterials, and drug/biomolecules in their interior. With the addition of nanomaterials, the mechanical property of generally weak dissolving polymers can be dramatically improved without sacrificing dissolution rate within skin. In this experiments, layered double hydroxides (LDH) nanoparticles are incorporated into sodium carboxymethylcellulose (CMC) to form a nanocomposite. The results show that, by adding 5 wt% of LDH nanoparticles into CMC, the mechanical strength significantly increased. Small and densely packed CMC-LDH microneedles penetrate human and pig skin more reliably than pure CMC ones and attractively the nanocomposite-strengthened microneedles dissolve in skin and release payload within only 1 min. Finally, the application of using the nanocomposite-strengthened microneedle arrays is tested for in vivo vaccine delivery and the results show that significantly stronger antibody response could be induced when compared with subcutaneous injection. These data suggest that nanomaterials could be useful for fabricating densely packed and small polymer microneedles that have robust mechanical properties and rapid dissolution rates and therefore potential use in clinical applications. Refereed/Peer-reviewed

Objective To evaluate the diagnostic performance of the BACs-on-Beads™ (BoBs™) assay for prenatal detection of chromosomal abnormalities. Design Retrospective study. Setting Tertiary prenatal diagnosis centre. Population Women referred for prenatal diagnosis. Methods We retrieved 2153 archived DNA samples collected between January 2010 and August 2011 for the BoBs™ assay. These samples had previously been tested by quantitative fluorescence polymerase chain reaction (QF-PCR) and karyotyping. In the BoBs™ assay a sample was defined as normal disomic when the ratio of the fluorescence intensities in a chromosome locus lay within the threshold (mean ratio ± 2SD), and as deleted or duplicated when the ratio was below the lower threshold (0.6–0.8) or above the upper threshold (1.3–1.4), respectively. The BoBs™ results were further validated by microarray and compared in a blinded manner with the original QF-PCR and karyotyping results. Main outcome measures Concordance of any numerical, structural, and submicroscopic chromosomal abnormalities between the methods. Results BACs-on-Beads™ was similar to karyotyping and QF-PCR in detecting trisomy 13, trisomy 18, trisomy 21, and sex chromosomal aneuploidies, and superior to QF-PCR in detecting major structural abnormalities (53.3 versus 13.3%) and mosaicism (28.6 versus 0%) involving chromosomal abnormalities other than the common aneuploidies. BoBs™ detected six microdeletion syndromes missed by karyotyping and QF-PCR; however, BoBs™ missed two cases of triploidy identified by QF-PCR. Therefore, the sensitivity of BoBs™ is 96.7% (95% CI 92.6–98.7%), and its specificity is 100% (95% CI 99.8–100%). Conclusions BACs-on-Beads™ can replace QF-PCR for triaging in prenatal diagnosis, and gives a better diagnostic yield than current rapid aneuploidy tests.

Background Evidence implicated the diagnostic significance of microRNAs in whole urine/urine sediments in urothelial carcinoma of the bladder (UCB). However, the contaminated blood cells in patients with haematouria significantly altered the expression profiles of urinary microRNA, influencing the test accuracy. Methods MicroRNA profiles of the urine supernatants of UCB patients and controls without any malignancy and profiles of malignant and corresponding normal mucosa tissues from the patients were determined by microRNA microarray and compared to identify differentially expressed microRNAs. The differential expression was verified in the tissues of an independent patient cohort by RT-qPCR. The diagnostic significance of selected microRNAs as biomarkers in the urine supernatant was investigated in the expanded cohorts. Results MicroRNA-99a and microRNA-125b were down-regulated in the urine supernatants of UCB patients. The degree of down-regulation was associated with the tumor grade. A diagnostic model was developed using a combined index of the levels of microRNA-99a and microRNA-125b in the urine supernatant with a sensitivity of 86.7%, a specificity of 81.1% and a positive predicted value (PPV) of 91.8%. Discriminating between high- and low-grade UCB, the model using the level of microRNA-125b alone exhibited a sensitivity of 81.4%, a specificity of 87.0% and a PPV of 93.4%. Conclusions The results revealed a unique microRNA expression signature in the urine supernatants of UCB patients for the development of molecular diagnostic tests. An effective cell-free urinary microRNA-based model was developed using a combined index of the levels of microRNA-99a and microRNA-125b to detect UCB with good discriminating power, high sensitivity and high specificity.

The aim of this study was to investigate urine cytokine and chemokine levels in symptomatic ketamine abusers compared with age-matched controls. Midstream urine specimens were collected in a prospective study of 23 ketamine abusers and 27 controls who had never used ketamine. Their basic demographic and urinary symptoms were compared. The urine was analyzed by a multiplex panel screen for 19 cytokines/chemokines: EGF, GM-CSF, GRO, IL-1Ra, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IP-10, MCP-1, MIP-1b, sCD40L, sIL-2Ra, VEGF, MCP-4, and TARC using Luminex™ xMAP® technology. Protein concentration values were normalized to urine creatinine concentrations. Mean age of the control group was 21.1 ± 4.3 years (n = 27) and of the ketamine group was 20.6 ± 3.7 years (n = 23). All participants were women. The urine cytokine analysis showed a significant elevation in EGF levels in the ketamine group with lower urinary tract symptoms (LUTS) compared with the control group (p

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. HCC cell lines established from different patients would be useful in elucidating the molecular pathogenesis. However, success of HCC primary culture establishment remains at low rate. We aim to establish and characterize HCC primary culture and the derived cell line. METHODS: Fresh tumor tissues were collected from 30 HCC patients. Culture conditions were optimized for the attachment and growth of the isolated hepatocytes. Granulin-epithelin precursor (GEP), a growth factor reported to associate with cancer stem cell properties, was examined by flow cytometry to elucidate its role on primary culture establishment. The primary cell line was characterized in detail. RESULTS: Cells isolated from 16 out of 30 HCC cases (53%) had viability more than 70% and were subject to subsequent in vitro culture. 7 out of 16 cases (44%) could give rise to cells that were able to attach and grow in culture. GEP expression levels significantly correlated with the viability of isolated hepatocytes and success rate of subsequent primary culture establishment. Cells from HCC patient 21 grew and expanded rapidly in vitro and was selected to be further characterized. The line, designated HCC21, derived from a Hong Kong Chinese female patient with HCC at Stage II. The cells exhibited typical epithelial morphology and expressed albumin, AFP and HBV antigens. The cell line was authenticated by short tandem repeat analysis. Comparative genome hybridization analysis revealed chromosomal loss at 1p35-p36, 1q44, 2q11.2-q24.3, 2q37, 4q12-q13.3, 4q21.21-q35.2, 8p12-p23, 15q11.2-q14, 15q24-q26, 16p12.1-p13.3, 16q, 17p, 22q and gain at 1q21-q43 in both HCC21 cells and the original clinical tumor specimen. Sequence analysis revealed p53 gene mutation. Subcutaneous injection of HCC21 cells into immunodeficient mice showed that the cells were able to form tumors at the primary injection sites and metastatic tumors in the peritoneal cavity. CONCLUSIONS: The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability., published_or_final_version

High-throughput screening for single nucleotide polymorphisms (SNPs) or mutations can be achieved by inexpensive technologies. We modified the original protocols of conformation-sensitive gel electrophoresis (CSGE) to increase throughput several fold to 1.3 samples/min, which is about five times faster than denaturing high-performance liquid chromatography (DHPLC). The modifications include decreasing the gel thickness, increasing the number of lanes to 96, and increasing the number of samples per lane to seven. This high-throughput CSGE method is fast, robust, and as simple as the original protocols. Together with a two-stage strategy for screening homozygotes and the replacement of ethidium bromide with SYBR® Gold DNA dye staining, this protocol is a reliable and cost-effective alternative for laboratories that require high-throughput screening.

Inherited mutations in the tumor susceptibility genes BRCA1 and BRCA2 account for about 70%–90% of familial breast cancer in most Caucasian populations while BRCA1 mutations alone contribute to about half of the cases (1– 3). Epidemiologic data from Caucasians show that BRCA1 mutations occur in the general population at a frequency between 0.05% and 0.2%. BRCA1 mutations account for about 5% of all breast cancers and up to 12% of earlyonset breast cancers (3,4). The relative contribution of BRCA2 may be slightly lower (2,3). The BRCA1 tumor suppressor gene is composed of 22 coding and two noncoding exons (5,6). The BRCA1 protein may play a role in cell cycle regulation (7) and control of DNA repair (8,9). More than 300 heritable mutations in the BRCA1 gene have been found spread throughout the whole coding sequence (10). All known BRCA1 mutations are germline and more than 85% are nonsense or frameshift mutations leading to premature termination of protein translation (2). Ancient and founder BRCA1 mutations have been identified, such as 185delAG mutation in exon 2, which is found in almost 1% of the Ashkenazi Jewish population (11,12). Studies of the BRCA1 gene in Oriental populations are few. There is no information on the frequency of BRCA1 mutation in the Chinese population. However, a lower prevalence of BRCA1 mutations has been suggested in the Japanese population (13–15). Although the incidence of breast cancer among Chinese women is about half of that seen in Caucasians, the proportion of familial cases and incidence of early-onset cancer are similar (16,17). Here, we report our investigation of mutations in BRCA1 among unselected consecutive samples of archived tumor tissues to determine an unbiased estimation of the prevalence of BRCA1 mutations among Chinese patients with breast cancer. Frozen tumor tissues were obtained for BRCA1 mutation analysis from 130 female Chinese patients, who underwent mastectomy for carcinoma of breast at the Prince of Wales Hospital in Hong Kong during the period of 1992 through 1993. As informed consent was not available for these stored samples, mutation analysis was performed in an anonymous manner. The patient identifier codes of these specimens had been removed before mutation analysis was performed. This procedure conforms to the consensus statement on use of stored samples in genetic research (18). Estrogen receptor and progesterone receptor statuses were determined by immunohistochemistry employing commercially available antibodies (DAKO, Glostrup, Denmark). In addition, truncating and missense mutations identified from this series of 130 tissue samples were also screened for in another group of 56 patients with early-onset breast cancers who were diagnosed with cancer before 45 years of age. This prospective group of patients had consented to participate in an ongoing BRCA1/BRCA2 genetics study. The study protocol was approved by a local Institutional Research Ethics Committee. The entire BRCA1 coding region was covered by 35 amplicons amplified by polymerase chain reaction (PCR) from genomic DNA (19). Mutations were screened by single-strand conformational polymorphism (SSCP) analysis under three different electrophoresis conditions. PCR products showing mobility shift on SSCP were sequenced by cycle sequencing in both sense and antisense directions (Life Technologies, Inc., Gaithersburg, MD). The chisquared test was used to compare frequency of alleles. Fisher’s exact test was used to compare clinical parameters between patients with and without BRCA1 mutation. Exact 95% confidence intervals (CIs) of prevalence data were determined by StatXact software (CYTEL, Cambridge, MA). Three nonsense (589delCT, Q356X, and Q1458X) and one presumptive disease-related (P1150S) BRCA1 mutations were found in five of 130 patients, which represented a prevalence of 3.8% (95% confidence interval [CI] 4 1.3%– 8.8%). Three nonsense mutations were identified in four patients (Table 1). Mutation 589delCT was found in two patients aged 24 years and 34 years, respectively, with infiltrating ductal carcinoma (IDC). The other two mutations were novel. The Q1458X mutation was found in a 44-year-old patient having a medullary carcinoma. Q356X was found in an 80-year-old patient with IDC. A missense mutation P1150S was detected in a 31-year-old woman with IDC. Fifty of 130 patients had breast cancer before the age of 45 years, and the prevalence of BRCA1 mutation among this subgroup of early-onset patients was 8.0% (95% CI 4 2.2%–19.2%). Additional screening specifically for these four mutations among another group of 56 patients with early-onset breast cancer further identified one more patient who was a heterozygote for 589delCT. The hometowns of these three unrelated patients sharing 589delCT mutation were different cities within the Guangdong Province of Southern China, but, as far as we know, the patients were not related. Although P1150S was reported as a disease-associated mutation in the Breast Cancer Information Core, the original report that described P1150S in a Japanese patient with a positive family history (15) did not include details on familial segregation of this mutation. As restricted by the retrospective nature of our analysis, we could not further our investigation into the inheritance of P1150S in our patient. Therefore, this

Low density lipoprotein receptor related protein (LRP) is a receptor for protease complexes, and may function in cell growth and repair, and in tumor invasiveness. LRP expression increases in glioblastomas compared to lower grade astrocytomas. Two potential mechanisms for this increased expression were investigated. The LRP C766T polymorphism is protective against Alzheimer's disease, perhaps through alteration of LRP expression. The frequency of the polymorphism was measured in astrocytoma patients and controls, but no significant difference was found. Differential PCR revealed LRP gene amplification in four of 25 high-grade gliomas and 0 of 23 other brain tumors. Co-amplification with epidermal growth factor receptor (EGFR) occurred in all four of the LRP-amplified tumors. Thus, LRP amplification may be partly responsible for increased LRP expression in astrocytomas, and may often occur in conjunction with EGFR amplification.

Background Complex chromosomal rearrangements and chromosomal deletion and duplication syndromes are commonly associated with abnormal clinical phenotypes. The 2q31.1 microdeletion syndrome is a rare cytogenetic event that leads to limb and multi-internal organ anomalies. In this study we investigated the genetic basis of the physical and mental symptoms exhibited by a 4-year-old boy with a suspected 2q31.1 deletion. Methods Cytogenetic and molecular techniques including karyotyping, array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH) and real-time PCR were used to identify the nature and extent of chromosome abnormalities in the patient. Results A 3.6 Mb interstitial microdeletion of 2q31.1 was identified in association with complex balanced genomic structural rearrangements involving chromosomes 2, 3, 6, 15 and 18. The 2q31.1 deletion resulted in the loss of one copy of several known disease genes, including GAD1 , DCAF17 , SLC25A12 and ITGA6 associated with mental retardation and facial abnormalities and DLX1 / DLX2 partially associated with limb abnormalities. Two additional genes, HOXD13 and CHN1 , required for normal limb and eye development that map immediately distal to the 2q31.1 deletion had normal copy numbers, although CHN1 was found to express at a lower level in patient's lymphocytes. Conclusions We speculated that the 2q31.1 deletion and/or translocation may have a positional effect which reduces expression of HOXD13 and CHN1 causing haplo-insufficiency, and in combination with the hemizygous expression of the disease genes at 2q31.1, provides a plausible explanation for the diverse clinical symptoms exhibited by the patient.

To examine the additive effect of the z-4 microsatellite polymorphism of aldose reductase gene (ALR2) and glycaemic control on risk of cataract in a prospective cohort of Chinese type 2 diabetic patients.The (CA)n microsatellite polymorphism of ALR2 was determined using PCR followed by capillary gel electrophoresis. Cataract was defined by presence of lens opacity on direct ophthalmoscopy or history of cataract surgery. A non-linear curve approach was used to identify the threshold of glycated hemoglobin (HbA1c) at which the odds ratio (OR) for cataract started to increase. The association of z-4 allele with cataract, above and below this threshold, was assessed using multiple logistic regression analysis.Of the 5823 patients analyzed, 28.1% had cataracts. After adjusting for conventional risk factors and using non-z-4 carriers with HbA1c8.0% as referent group (n = 3173), the OR (95% confidence intervals) for cataract was highest in z-4 carriers with HbA1c ≥ 8.0% [1.43 (1.05-1.96), n = 244], compared to non-z-4 carriers with HbA1c ≥ 8.0 [1.27 (1.10-1.47), n = 1836] and z-4 carriers with HbA1c8.0%[1.01 (0.77-1.29), n = 420, P(trend)0.001]. This additive association remained significant after additional adjustments for drug use (P(trend) = 0.002) and renal function (P(trend) = 0.01).In type 2 diabetic patients with suboptimal glycaemic control, the z-4 allele of ALR2 (CA)n polymorphism was independently associated with increased susceptibility to cataracts.

Disease associated gene deficient embryonic stem cells can serve as valuable in vitro models to study disease mechanisms and screen drugs. Smad3 mediated TGF-β/Activin/Nodal signaling plays important roles in many biological processes. Despite numerous studies regarding Smad3 function, the role of Smad3 in mouse ES cells is not well studied. To understand the function of Smad3 in mouse ES cells, we derived Smad3−/− ES cells and wild type ES cells. Smad3−/− ES cells display no defect on self-renewal. They express similar level of pluripotent genes and lineage genes compared to wild type ES cells. However, Smad3 ablation results in transient difference in germ layer marker expression during embryoid body formation. Mesoderm lineage marker expression is significantly reduced in the embryoid body formed by Smad3−/− ES cells compared to wild type ES cells. Intriguingly, subcutaneous injection of Smad3−/− ES cells into nude mice leads to formation of malignant immature teratomas, whilst wild type ES cells tend to form mature teratomas. Smad3−/− ES cell formed teratomas can therefore provide a new model for the study of the mechanism of malignant teratomas.

During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges.

As a distinct type of head and neck cancer, non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF-κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV-associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF-κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV-positive NPC tumours. siRNA or chemical inhibition of NF-κB signalling significantly inhibited the growth of EBV-positive NPC cells C666-1. Gene expression profiling identified a number of NF-κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF-κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF-κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF-κB activation in EBV-associated NPC. Except for LMP1-expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF-κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF-κB signals are constitutively activated in EBV-positive NPC cells by either multiple genetic changes or EBV latent genes.

Because of difficulty in measuring each antioxidant component separately and interactions among antioxidants, methods have been developed to assess the total antioxidant capacity of serum or plasma. The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity (TEAC) assay (1), the oxygen radical absorbance capacity (ORAC) assay (2), and the ferric reducing ability of plasma (FRAP) assay (3) are commonly used and have been extensively evaluated. Although comparable mean results have been obtained with the TEAC and ORAC assays (4)(5), no correlation has been found between ORAC and TEAC values or between FRAP and TEAC values (6). This lack of correlation between TEAC and other assays is likely attributable to underestimation of overall antioxidant capacity. Underestimation may be related to the effects of dilution (7) and to premature measurement of inhibition percentage at a fixed time of 3 min (6). In fact, both the ORAC and TEAC assays are inhibition methods: a sample is added to a free- radical-generating system, and the inhibition of the free radical action is measured. This inhibition is related to the antioxidant capacity of the sample. In addition, both assay methods measure antioxidants in serum or plasma proteins, including albumin (6). In this study we investigated the performance of the TEAC assay, modified the procedure, and then reevaluated the TEAC for comparison with the ORAC assay. The TEAC assay, commercialized by Randox Laboratories Ltd., is based on the suppression of the absorbance of radical cations of 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) by antioxidants in the test sample when ABTS incubates with a peroxidase (metmyoglobin) and H2O2 (1). If the inhibition time is fixed at 3 min, as stated in the manufacturer’s instructions, the added antioxidants quench ABTS radicals in a nonlinear dose–response fashion (6). To optimize the incubation period for the complete inhibition of ABTS radical …

To report secondary or additional findings arising from introduction of non-invasive prenatal testing (NIPT) for aneuploidy by whole genome sequencing as a clinical service.Five cases with secondary findings were reviewed.In Case 1, NIPT revealed a large duplication in chromosome 18p, which was supported by arrayCGH of amniocyte DNA, with final karyotype showing mosaic tetrasomy 18p. In Case 2, a deletion in the proximal long arm of chromosome 18 of maternal origin was suspected and confirmed by arrayCGH of maternal white cell DNA. In Case 3, NIPT was negative for trisomies 21 and 18. In-depth analysis for deletions/duplications was requested when fetal structural anomalies were detected at routine scan. A deletion in the proximal long arm of chromosome 3 was found and confirmed by karyotyping. In Case 4, NIPT correctly predicted confined placental mosaicism with triple trisomy involving chromosomes X, 7 and 21. In Case 5, NIPT correctly detected a previously unknown maternal mosaicism for 45X.Non-invasive prenatal testing is able to detect a wide range of fetal, placental and maternal chromosomal abnormalities. This has important implications on patient counseling when an abnormality is detected by NIPT.

To assess the value of spectral karyotyping (SKY) combined with microarray-based comparative genomic hybridization (array-CGH) for the diagnosis of complex ring chromosome aberration.For an 8-year-old boy featuring growth retardation, G-banding analysis has indicated a 46,XY,r(15)? karyotype, which was delineated by SKY in combination with array-CGH.The ring chromosome has originated from chromosome 15 according to SKY analysis. Position of the breakpoint (15q26.3) and a 594 kb deletion were revealed by array-CGH.Molecular cytogenetic technologies are efficient tools for clarifying complex chromosomal abnormality, which has provided a powerful tool for conventional cytogenetic analysis.

Adult stem cells are critical for the healing process in regenerative medicine. However, cigarette smoking inhibits stem cell recruitment to tissues and delays the wound-healing process. This study investigated the effect of nicotine, a major constituent in the cigarette smoke, on the regenerative potentials of human mesenchymal stem cells (MSC) and periodontal ligament-derived stem cells (PDLSC). The cell proliferation of 1.0 μM nicotine-treated MSC and PDLSC was significantly reduced when compared to the untreated control. Moreover, nicotine also retarded the locomotion of these adult stem cells. Furthermore, their osteogenic differentiation capabilities were reduced in the presence of nicotine as evidenced by gene expression (RUNX2, ALPL, BGLAP, COL1A1, and COL1A2), calcium deposition, and alkaline phosphatase activity analyses. In addition, the microRNA (miRNA) profile of nicotine-treated PDLSC was altered; suggesting miRNAs might play an important role in the nicotine effects on stem cells. This study provided the possible mechanistic explanations on stem cell-associated healing delay in cigarette smoking.

Purpose We investigated the in vitro amyloid aggregation ability of TGFBI (transforming growth factor beta-induced) mutants causing corneal dystrophies (CDs). Methods Peripheral blood samples were collected from 42 unrelated Chinese CD patients and 185 healthy subjects for mutation screening in all TGFBI coding exons and flanking introns. The expression vector pCMV6_TGFBI containing wild-type, Arg-124, or Arg-555 mutations was transfected to HEK293 cells. Cell-free media was incubated with amyloid-beta (Aβ) (1-40) peptides with or without a chemical osmolyte, trimethylamine N-oxide (TMAO), for different time intervals. After ultracentrifugation, protein aggregates were analyzed by denatured gel electrophoresis. The effect of TMAO on chemical and morphological properties of Aβ aggregation was examined. Results TGFBI sequencing analysis showed c.Arg124Cys in all 6 lattice CD patients, c.Arg555Glu in all 11 granular CD type 1 patients, and c.Arg124His in 22 of 25 granular CD type 2 patients. Double heterozygosity (c.307-308delCT and c.Arg124His) was detected in one GCD2 patient. After transfection, cell-free media containing Arg-124 TGFBIp led to Aβ aggregation within 12 hours, whereas wild-type and Arg-555 mutant displayed aggregation after 24 hours. Western blot and Congo red binding assays showed that TMAO dose-dependently suppressed Arg-124-induced Aβ aggregation. Transmission electron microscopy showed that TMAO reduced the fibrillar aggregates caused by Aβ and c.124R > H mutated TGFBIp. Conclusions TGFBI sequence heterogeneity was observed in Chinese CD patients. TMAO reduced amyloid aggregation caused by Arg-124 mutants, which suggests a potential chemical-based treatment for CDs.

Purpose This study investigated the relationship between B lymphoma Mo-MLV insertion region 1 (BMI-1)—a polycomb protein for stem cell self-renewal and proliferation—and the clinicopathological parameters of human retinoblastomas, including differentiation status and retinal tissue invasion, as well as the effects of BMI-1 on retinoblastoma Y79 cells. Methods Thirty-four archived human retinoblastoma samples were recruited for BMI-1 immunohistochemistry. The percentage of BMI-1-expressing cells was scored by independent pathologists and the data were correlated with the clinical features. Y79 cells were transfected to overexpress or specifically inhibit BMI-1 for cell proliferation, propidium iodide cell cycle and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis analyses, multicellular sphere formation assay, and gene expression study. Results BMI-1 was widely expressed in human retinoblastomas. Higher percentages of BMI-1-expressing cells were selectively limited to undifferentiated tumors and those tumors undergoing invasion to the optic nerve and choroid. However, there was no difference in BMI-1 expression in retinoblastoma retinas with or without tumor invasion. In Y79 cells, BMI-1 stimulated cell proliferation and suppressed apoptosis with reduced p14ARF and p16INK4 expression, along with upregulation of proliferating cell nuclear antigens cyclin D1 and D2. In contrast, silencing BMI-1 reversed these changes. It also upregulated CHX10 and Rx, but not other retinal development-related genes, including nestin and neurofilament M. Conclusions Our work indicates that BMI-1 might render important oncogenic property of retinoblastomas and it could be a therapeutic target for the cancer treatment.

Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples.

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.

Objectives To assess the prevalence of levator ani muscle injury in Chinese women after their first delivery and investigate associated factors. Methods A prospective observational study was conducted involving Chinese nulliparous women recruited in the first trimester of pregnancy. Translabial ultrasound was performed at 35–38 weeks' gestation and 8 weeks postpartum and three-dimensional volume datasets were obtained. Offline analysis to detect levator ani muscle injury was performed by investigators blinded to the delivery details. Results 339 women, with a mean age of 30.6 ± 3.9 years, completed the study. Overall, 201 (59.3%) had a spontaneous vaginal delivery, 62 (18.3%) an operative vaginal delivery (48 ventouse extraction and 14 forceps delivery), 14 (4.1%) an elective Cesarean section and 62 (18.3%) an emergency Cesarean section. No levator ani muscle injury was detected in any woman antenatally. After vaginal delivery, 57 (21.7% (95% CI, 16.7–26.7%)) women had levator ani muscle injury. The rates of injury for spontaneous vaginal delivery, ventouse extraction and forceps delivery were 15.4%, 33.3% and 71.4%, respectively. There was no levator ani muscle injury in the Cesarean section groups. Logistic regression analysis showed that only operative vaginal delivery (odds ratio, 3.09) was associated with an independent increase in the likelihood of levator ani muscle injury. Intrapartum epidural analgesics, duration of second stage of labor and infant birth weight were not independently associated with levator ani muscle injury. Conclusions The prevalence of levator ani muscle injury in Chinese women after their first vaginal delivery was 21.7% (95% CI, 16.7–26.7%). Operative vaginal delivery was found to increase the likelihood of women suffering such injury. A longer follow-up of these women and future studies on the effects of episiotomy are proposed. Copyright © 2012 ISUOG. Published by John Wiley & Sons, Ltd.

X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.

BackgroundEpigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Methodology/principal findingsHuman LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.Conclusion/significanceWe found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.