Noninvasive prenatal testing (NIPT) using sequencing of fetal cellfree DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/ duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.noninvasive prenatal testing | NIPT | maternal plasma DNA | cell-free DNA | semiconductor sequencing G enomic disorders are defined by loss, gain, or translocation of chromosomal material. Deletion/duplication syndromes are known to be associated with a wide range of structural and functional abnormalities (1), such as Cri du Chat Syndrome (5p deletion) (2) and DiGeorge Syndrome (22q11.2 deletion) (3). Such deletion/duplication syndromes can be reliably diagnosed prenatally from the DNA of fetal cells; fetal DNA may be assessed for chromosomal abnormalities by karyotyping, FISH, comparative genomic hybridization (CGH), and array-based technologies (4). G-banded karyotyping is the predominant technique for diagnosis of chromosomal abnormalities, but it is limited to resolution of 5-10 Mb (5, 6). Genomic disorders of a smaller size are more reliably detected by chromosomal microarray analysis (CMA), of which array CGH (aCGH) is an example.According to The American Society of Human Genetics, CMA has replaced the standard metaphase karyotype in postnatal assessment of individuals with developmental delay, intellectual disability, congenital anomalies, and autism (7). In December of 2013, The American Congress of Obstetricians and Gynecologists and the Society of Maternal Fetal-Medicine recommended pr...
-Aflatoxin B 1 (AFB 1 ) and zearalenone (ZEA) are the secondary toxic metabolites of fungi which contaminate a wide range of food and feedstuffs. Limiting exposure of humans and livestock to them is very essential. Among numerous methods of mycotoxin-degradation, biodegradation by microorganisms and enzymes is an effective and promising approach to eliminate their hazards. The present study aims to optimize the proportion of different species of beneficial microbes by means of response surface methodology (RSM) and its combination with mycotoxin-degradation enzymes. The results indicated that AFB 1 and ZEA degradation rates were 38.38% and 42.18% by individual Bacillus subtilis (P < 0.05); however, AFB 1 and ZEA degradation rates reached 45.49% and 44.90% (P < 0.05) when three probiotic species such as Bacillus subtilis, Lactobacillus casein and Candida utilis were at a ratio of 1:1:1, corresponding with the predictive value of the RSM model. The further experiment showed that AFB 1 and ZEA degradation rates were 63.95% and 73.51% (P < 0.05) when the compound of three probiotic species was combined with mycotoxin-degradation enzymes from Aspergillus oryzae at a ratio of 3:2. This result indicated that the combination of probiotics with mycotoxin-degradation enzymes is a promising new approach for synchronous detoxification of AFB 1 and ZEA.
Zearalenone (ZEA) and aflatoxin B1 (AFB1) are two main kinds of mycotoxins widely existing in grain and animal feed that cause a lot of economic loss and health problems for animals and humans. In order to alleviate the cytotoxic effects of AFB1 and ZEA on swine jejunal epithelial cells (IPEC-J2), the combination of a cell-free supernatant of compound probiotics (CFSCP) with mycotoxin degradation enzymes (MDEs) from Aspergillus oryzae was tested. The results demonstrated that coexistence of AFB1 and ZEA had synergetic toxic effects on cell viability. The cell viability was decreased with mycotoxin concentrations increasing, but increased with incubation time extension. The necrotic cell rates were increased when 40 µg/L AFB1 and/or 500 µg/L ZEA were added, but the addition of CFSCP + MDE suppressed the necrotic effects of AFB1 + ZEA. The viable cell rates were decreased when AFB1 and/or ZEA were added: However, the addition of CFSCP + MDE recovered them. The relative mRNA abundances of Bcl-2, occludin, and ZO-1 genes were significantly upregulated, while Bax, caspase-3, GLUT2, ASCT2, PepT1, and IL6 genes were significantly downregulated by CFSCP + MDE addition, compared to the groups containing 40 µg/L AFB1 and 500 µg/L ZEA. This research provided an effective strategy in alleviating mycotoxin cytotoxicity and keeping normal intestinal cell structure and animal health.
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