Application of real-time PCR-based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses.

BACKGROUND: To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program. METHODS: A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated. RESULTS: Except for initiation codon (T > G) (HBB:c.2T > G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay. CONCLUSION: MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.

Compound heterozygous mutation of the ASXL3 gene causes autosomal recessive congenital heart disease.

To explore mutations in the additional sex combs-like 3 (ASXL3) gene in two Chinese families with congenital heart disease (CHD). Whole-exome sequencing (WES) was used to reveal a novel compound heterozygous mutation in the ASXL3 gene that was associated with CHD. Sanger sequencing of a further 122 CHD patients was used to determine an additional compound heterozygous mutation in the ASXL3 gene. Cell apoptosis was examined by MTS assay and flow cytometry. The cardiac structure was identified via hematoxylin-eosin (HE), Masson's trichrome, and ultrasound scanning. RNA sequencing was performed to identify a series of differentially expressed mRNAs. The mRNA and protein expressions were identified by quantitative real-time PCR and western blotting, respectively. A compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) in the ASXL3 gene associated with CHD was identified. Overexpression of this compound heterozygous mutation in HL-1 cells resulted in increased apoptosis and reduced cell viability. Moreover, it affected cardiac structure and fibrosis in mice. There were 126 downregulated mRNAs and 117 upregulated mRNAs between the ASXL3 compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) mice and wild-type mice. Ezh2, Slc6a4, and Socs3, which could interact with ASXL3 through proteins, were all upregulated. Another compound heterozygous mutation c.3526C > T (p.Arg1176Trp) and c.4643A > G (p.Asp1548Gly) in the ASXL3 gene was identified by screening a further 122 patients with CHD. The ASXL3 gene is important in cardiac development and may exert this influence by affecting the expression of mRNAs associated with cell apoptosis and cell proliferation.

LncRNA-HGBC stabilized by HuR promotes gallbladder cancer progression by regulating miR-502-3p/SET/AKT axis.

BACKGROUNDS: Long non-coding RNAs (lncRNAs) are essential factors that regulate tumor development and metastasis via diverse molecular mechanisms in a broad type of cancers. However, the pathological roles of lncRNAs in gallbladder carcinoma (GBC) remain largely unknown. Here we discovered a novel lncRNA termed lncRNA Highly expressed in GBC (lncRNA-HGBC) which was upregulated in GBC tissue and aimed to investigate its role and regulatory mechanism in the development and progression of GBC. METHODS: The expression level of lncRNA-HGBC in GBC tissue and different cell lines was determined by quantitative real-time PCR. The full length of lncRNA-HGBC was obtained by 5' and 3' rapid amplification of the cDNA ends (RACE). Cellular localization of lncRNA-HGBC was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. In vitro and in vivo assays were preformed to explore the biological effects of lncRNA-HGBC in GBC cells. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay were used to identify lncRNA-HGBC-interacting proteins. Dual luciferase reporter assays, AGO2-RIP, and MS2-RIP assays were performed to verify the interaction between lncRNA-HGBC and miR-502-3p. RESULTS: We found that lncRNA-HGBC was upregulated in GBC and its upregulation could predict poor survival. Overexpression or knockdown of lncRNA-HGBC in GBC cell lines resulted in increased or decreased, respectively, cell proliferation and invasion in vitro and in xenografted tumors. LncRNA-HGBC specifically bound to RNA binding protein Hu Antigen R (HuR) that in turn stabilized lncRNA-HGBC. LncRNA-HGBC functioned as a competitive endogenous RNA to bind to miR-502-3p that inhibits target gene SET. Overexpression, knockdown or mutation of lncRNA-HGBC altered the inhibitory effects of miR-502-3p on SET expression and downstream activation of AKT. Clinically, lncRNA-HGBC expression was negatively correlated with miR-502-3p, but positively correlated with SET and HuR in GBC tissue. CONCLUSIONS: Our study demonstrates that lncRNA-HGBC promotes GBC metastasis via activation of the miR-502-3p-SET-AKT cascade, pointing to lncRNA-HGBC as a new prognostic predictor and a therapeutic target.

Development of an all-sky imaging system for cloud cover assessment.

This study develops a novel automatic all-sky imaging system, namely, an all-sky camera (ASC) system, for cloud cover assessment. The proposed system does not require conventional solar occulting devices and can capture complete hemispheric sky images. Cloud detection is performed innovatively using a convolutional neural network model (i.e., the optimized U-Net model). Experiments demonstrate that the optimized U-Net model can effectively detect clouds from sky images. In terms of cloud cover, the estimation results of the ASC system exhibit a high correlation with those obtained via manual observation, thereby indicating the applicability of the ASC system in ground-based cloud observation and analysis.

Chromosome microarray analysis in the investigation of children with congenital heart disease.

BACKGROUND: Our study was aimed to explore the clinical implication of chromosome microarray analysis (CMA) in genetically etiological diagnosis of children with congenital heart disease (CHD). METHODS: A total of 104 children with CHD with or without multiple congenital anomalies (MCA) or intellectual disabilities/developmental delay (ID/DD) but normal karyotype were investigated using Affymetrix CytoScan HD array. RESULT: Pathogenic copy number variations (PCNVs) were identified in 29 children (27.9%). The detection rates in children with simple CHD and complex CHD were 31.1% (19/61) and 23.2% (10/43), respectively. The detection rates of PCNVs were 17.9% (7/39), 20% (5/25), 63.2% (12/19) and 23.8% (5/21) in isolated CHD, CHD plus MCA, CHD plus ID/DD, CHD plus MCA and ID/DD, respectively. The PCNVs rate of CHD plus ID/DD was significantly higher than that of isolated CHD. Two genomic loci including 15q11.2 deletion and 1q43-q44 deletion were considered as CHD locus. The DVL1, SKI, STIM1, CTNNA3 and PLN were identified as candidate genes associated with CHD phenotypes. CONCLUSION: CMA can increase the diagnostic rate and improve the etiological diagnosis in children with CHD. We suggest CMA as a first-tier test in children with CHD, especially in children with CHD plus ID/DD.

[Early intellectual developmental outcome of late preterm infants].

OBJECTIVE: To investigate the early intellectual developmental outcome of late preterm infants. METHODS: A total of 106 late preterm infants with a gestational age of 34-36+6 weeks who were admitted to the neonatal ward between January 2012 and January 2015, cured, discharged, and regularly followed up at the outpatient service for high-risk children were enrolled as the preterm group. A total of 120 healthy full-term infants during the same period were randomly selected as the term group. Neonatal behavioral neurological assessment (NBNA) was performed for late preterm infants at a corrected gestational age of 40 weeks and full-term infants at a gestational age of 40 weeks. The Gesell Developmental Scale was used for late preterm infants at a corrected age of 3, 6, and 12 months and full-term infants at an age of 3, 6, and 12 months. RESULTS: The preterm group had an NBNA score of <37 and a significantly lower NBNA score than the term group (P<0.05). At the corrected age of 3 months, the preterm group had significantly lower scores of gross motor, fine motor, and social competence than the term group (P<0.05). At the corrected age of 6 months, the preterm group had significantly lower scores of adaptability, gross motor, and fine motor than the term group (P<0.05). At the corrected age of 12 months, the preterm group had significantly lower scores of adaptability, gross motor, and social competence than the term group (P<0.05). CONCLUSIONS: Late preterm infants have early intellectual developmental delay. It is necessary to perform neurodevelopmental monitoring for late preterm infants.

The prevalence of non-detectable chromosomal abnormalities by QF-PCR in amniocentesis for certain referral indications: experience at a mainland Chinese hospital.

PURPOSE: To study the prevalence of non-detectable chromosomal abnormalities by quantitative fluorescent polymerase chain reaction (QF-PCR) in a Chinese population referred for amniocentesis. METHODS: The karyotype results were reviewed in 8,466 amniotic fluid cultures performed for positive fetal Down syndrome screening or advanced maternal age between January 2002 and June 2012. The karyotype results were classified as detectable or not detectable by QF-PCR, using the assumption that all tests were conducted by this rapid molecular method. RESULTS: Of the 8,466 karyotypes obtained, 211 abnormal karyotypes were found (2.5%). Out of these, 168 cases of common aneuploidies were identified by QF-PCR, and 43 cases of chromosomal abnormalities were missed. The 43 cases missed by QF-PCR included 31 cases predicted to confer no increased risk and 12 with a potential clinical significance. When QF-PCR shows a normal result, the overall residual risk is 0.1% for any clinically significant chromosomal abnormality. CONCLUSIONS: A normal QF-PCR result predicts a very low residual risk for patients who are referred solely for an increased risk of a common trisomy.

Discordant results between fetal karyotyping and non-invasive prenatal testing by maternal plasma sequencing in a case of uniparental disomy 21 due to trisomic rescue.

Uniparental disomy (UPD) is an uncommon chromosome condition, but UPD involving chromosome 21 is rarely reported. We reported here a case who had first trimester screening test for Down syndrome, chorionic villus sampling for fetal karyotyping, quantitative fluorescence polymerase chain reaction (QF-PCR), as well as non-invasive prenatal testing (NIPT) by maternal plasma sequencing. There were discordant results between fetal karyotyping and NIPT due to UPD 21combined with confined placental mosaicism of trisomy 21. This demonstrated that it is possible to detect placental mosaicism by NIPT, but further studies are required to confirm its sensitivity. Therefore, all positive NIPT results must be confirmed by conventional invasive test and karyotyping. QF-PCR has the additional benefit in diagnosing UPD.

Peeling mechanism of tomato induced by HHAIB: Microscopic, ultrastructure, chemical, physical and mechanical properties perspectives.

In order to better manage the peeling degree and avoid unnecessary losses, the current work aimed to explore the peeling mechanism of a novel peeling technology, high-humidity hot air impingement blanching (HHAIB). The relationships between HHAIB peeling performance and the changes in skin temperature, skin structure, water state, pectin fractions content, and skin mechanical properties of tomatoes were analyzed. Results showed, after HHAIB treatment, the epicuticular wax was disrupted, the skin exhibited more and longer random cracks, the degradation of inner skin tissue was observed by transmission electron microscopy, the free water percentage increased resulting in water loss in the whole tomato, the water-soluble pectin contents decreased in tomato fleshes, while the contents of chelate-soluble pectin and sodium-carbonate-soluble pectin increased. HHAIB heating reduced the elongation at break, and increased Young's Modulus of tomato peel. This study revealed the HHAIB peeling mechanism and provided new insights for developing HHAIB peeling technology.