Identification and quantification of Meiguihua oral solution using liquid chromatography combined with hybrid quadrupole-orbitrap and triple quadrupole mass spectrometers.

Meiguihua oral solution (MOS), a classical Chinese medicinal formula, was approved by the China Food and Drug Administration for production. However, the quality evaluation of MOS has not been reported. In this present study, qualitative and quantitative analysis of MOS were conducted by ultra high performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS) and high performance liquid chromatography-tri-quadrupole linear ion trap mass spectrometry (HPLC-tri-Q-LIT-MS). Totally 46 phenolic compounds (21 flavonoids and 25 tannins) were identified in the MOS, among them 14 polyphenols were not reported in raw plant materials of MOS. The simultaneous quantification of ten compounds including gallic acid, quercetin-3-O-sophoroside, ellagic acid, sophoraflavonoloside, hyperoside, isoquercitrin, avicularin, astragalin, quercitrin and juglanin, which were completed in 16 min in the negative electrospray ionization (ESI) mode under multiple reaction monitoring (MRM) method. Linearity was reached fine determination coefficient (r2 > 0.9995). Precisions, repeatability, stability (inter-day and intra-day), and recovery were validated and the relative standard deviations (RSD) were less than 2.9%, 4.7%, 3.6% and 1.79%, respectively. This result proved the high sensitivity and efficiency of the method. The quantitative and qualitative analysis of MOS would provide the substantial basis for further quality control and medicinal values.

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform.

BACKGROUND: Recent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S. METHODS: The artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH). RESULTS: Our SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable. CONCLUSIONS: Our studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Detection of fetal duplication 16p11.2q12.1 by next-generation sequencing of maternal plasma and invasive diagnosis.

OBJECTIVE: The objective of study is to report the feasibility of non-invasive prenatal screening (NIPS) combined with invasive detection by chromosomal analysis in identifying fetal duplication, providing clinical performance of NIPS on copy number variations (CNVs) detection. MATERIAL AND METHODS: NIPS was offered to a 35-year-old pregnant woman. Amniocentesis was performed to confirm the positive screening result. Fetal sample was detected by karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA). Parental karyotyping was also conducted. RESULTS: NIPS result was positive for chromosome 16, indicating an extra copy of chromosome 16. FISH and chromosomal karyotyping revealed that the fetus had a marker chromosome derived from chromosome 16. CMA further demonstrated an approximately 19-Mb duplication in chromosome 16. The final fetal karyotype was 47,XY,+mar. ish der (16)(D16Z3+).arr 16p11.2q12.1 (30 624 186-49 696 337 × 3). Ultrasound scan and MRI showed some structure malformations. CONCLUSIONS: A protocol for CNVs detection by combining a series of genetic methods was presented in this study and a novel marker duplication 16p11.2q12.1 was reported. With the ability to identify subchromosomal deletions and duplications in fetus, NIPS could reduce the possibility of invasive diagnosis. The followed confirmation test for positive sample is necessary and ensures the accuracy of the diagnosis.

Detection of fetal subchromosomal aberration with cell-free DNA screening led to diagnosis of parental translocation: Review of 11344 consecutive cases in a university hospital.

BACKGROUND: Fetal chromosome aberrations and sub-chromosomal copy number variations (CNVs) are not rare. There are several ways to detect duplications and deletions; cell-free DNA screening (cfDNA screening) is nowadays an accurate and safe detection method. The objective of this study is to report the feasibility of cfDNA screening as an indicator of parental balanced chromosome translocation. RESULTS: From February 2015 to March 2016, cfDNA screening was offered to 11344 pregnant women. 137 out of 11344 individuals tested positive for aneuploidies using cfDNA screening were confirmed by karyotyping. 6 additional cases also tested positive for other deletion/duplication were confirmed by chromosomal microarray analysis (CMA). 11201 patients tested negative and 10342 of them were confirmed through interviews after delivery. Among the 137 cases that were screened positive in cfDNA screening, 91 were common trisomies (63 cases of trisomy 21, 25 cases of trisomy 18 and 3 cases of trisomy 13) and 46 cases were positive for sex-chromosomal abnormalities. In addition, 6 cases were positive for other deletion/duplication in which 2 were identified as terminal duplication and deletion on different chromosomes. The cfDNA screening findings were confirmed by CMA or karyotyping, and the origins of CNVs were validated afterward by karyotyping or fluorescence in situ hybridization (FISH) using parental blood samples. CONCLUSION: CfDNA screening may help identify deletions and duplications in fetus, which in some cases may indicate risk of a parent being a balanced rearrangement carrier, and that the diagnostic follow-up testing is necessary.

A feasible diagnostic approach for the translocation carrier from the indication of products of conception.

BACKGROUND: Chromosome translocations are rare but frequently associated with infertility. The objective of this study is to investigate the feasibility of using chromosomal microarray analysis (CMA) on products of conception (POC) samples as an indicator of parental balanced translocation. From January 2011 to December 2016, CMA using Affymetrix Cytoscan™750K array was performed on 1294 POC samples in our hospital. Karyotyping and fluorescence in situ hybridization (FISH) using parental blood samples were performed to validate the origin of subchromosomal copy number variations (CNVs). RESULTS: In the 1294 cases of POCs, we detected CNVs of terminal duplication and deletion that imply unbalanced translocation derivatives in 16 cases, and accurate diagnosis with the parental study was made in all the cases by karyotyping and/or FISH. In 10/16 (62.5%) of these cases, CNVs were inherited from one carrier parent of balanced translocation (Cases 1 to 10), while 6/16 (37.5%) cases occurred de novo (Cases 11 to 16). CONCLUSION: This study clearly illustrated the importance of the utilization of CMA on POC, followed by parental karyotyping and FISH to better characterize CNVs. This approach is especially useful for couples in whom one partner carries a cryptic/submicroscopic balanced translocation but has an apparently normal karyotype.

XCI-escaping gene KDM5C contributes to ovarian development via downregulating miR-320a.

Mechanisms underlying female gonadal dysgenesis remain unclarified and relatively unstudied. Whether X-chromosome inactivation (XCI)-escaping genes and microRNAs (miRNAs) contribute to this condition is currently unknown. We compared 45,X Turner Syndrome women with 46,XX normal women, and investigated differentially expressed miRNAs in Turner Syndrome through plasma miRNA sequencing. We found that miR-320a was consistently upregulated not only in 45,X plasma and peripheral blood mononuclear cells (PBMCs), but also in 45,X fetal gonadal tissues. The levels of miR-320a in PBMCs from 45,X, 46,XX, 46,XY, and 47,XXY human subjects were inversely related to the expression levels of XCI-escaping gene KDM5C in PBMCs. In vitro models indicated that KDM5C suppressed miR-320a transcription by directly binding to the promoter of miR-320a to prevent histone methylation. In addition, we demonstrated that KITLG, an essential gene for ovarian development and primordial germ cell survival, was a direct target of miR-320a and that it was downregulated in 45,X fetal gonadal tissues. In conclusion, we demonstrated that downregulation of miR-320a by the XCI-escaping gene KDM5C contributed to ovarian development by targeting KITLG.

Cost-effective imprinting combining macromolecular crowding and a dummy template for the fast purification of punicalagin from pomegranate husk extract.

The combination of molecular crowding and virtual imprinting was employed to develop a cost-effective method to prepare molecularly imprinted polymers. By using linear polymer polystyrene as a macromolecular crowding agent, an imprinted polymer recognizable to punicalagin had been successfully synthesized with punicalin as the dummy template. The resulting punicalin-imprinted polymer presented a remarkable selectivity to punicalagin with an imprinting factor of 3.17 even at extremely low consumption of the template (template/monomer ratio of 1:782). In contrast, the imprinted polymer synthesized without crowding agent, did not show any imprinting effect at so low template amount. The imprinted polymers made by combination of molecular crowding and virtual imprinting can be utilized for the fast separation of punicalagin from pomegranate husk extract after optimizing the protocol of solid-phase extraction with the recovery of 85.3 ± 1.2%.

KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma.

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

Hepatoprotective activities of a sesquiterpene-rich fraction from the aerial part of Cichorium glandulosum.

BACKGROUND: Cichorium glandulosum Boiss. et Huet is used for treatment of liver disorders, and its effects are attributed to sesquiterpenes. This study aims to investigate the hepatoprotective effects of a sesquiterpene-rich fraction (SRF) from the aerial part of C. glandulosum on carbon tetrachloride (CCl4)-induced acute hepatotoxicity in mice, and on priming with Bacillus Calmette-Guerin (BCG) followed by lipopolysaccharide (LPS)-induced immunological liver injury in mice. METHODS: SRF was suspended in water and administered to mice at 0.05, 0.10 and 0.20 g/kg body weight for 7 consecutive days. An active control drug (bifendate pills) was suspended in distilled water and administered to mice at 0.40 g/kg body weight for 7 consecutive days. Hepatotoxicity was induced by intraperitoneal injection of 0.1% CCl4 (0.2 mL/mouse) at 13 h before the last drug administration, or by tail intravenous injection of BCG (0.2 mL/mouse) before the first drug administration and LPS (0.2 mL/mouse; 8 µg) at 15 h before the last drug administration. Blood samples and the livers were collected for evaluation of the biochemical parameters of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (TBIL). RESULTS: SRF significantly reduced the impact of CCl4 toxicity. The highest dose of SRF (0.20 g/kg) was the most effective, reflected by significant reductions in the levels of AST (P = 0.001), ALT (P = 0.000) and TBIL (P = 0.009). The serum enzymatic levels induced by BCG and subsequent LPS injection were significantly and dose-dependently restored by SRF, reflected by significant reductions in the levels of AST (P = 0.003), ALT (P = 0.003) and TBIL (P = 0.007) for the highest dose of SRF (0.20 g/kg). CONCLUSION: SRF is hepatoprotective in animal models of chemical and immunological acute liver injury.

[Conventional and molecular cytogenetic analyses of a derivative X chromosome in amniocentesis].

OBJECTIVE: To analyze the aberrant der(X) chromosome using conventional and molecular cytogenetic approaches in a fetus of second trimester and to discuss its clinical effect. METHODS: Conventional cytogenetic procedures (GTG and CBG banding) were performed on cultured amniotic fluid cells. Three-color fluorescence in situ hybridization (FISH) consisting of X chromosome enumeration probes(CEPX), CEPY and Tel Xp/Yp was further performed to study the aberrant der(X) chromosome. RESULTS: Der(X) was a rare X/Y translocation. The final karyotypes of the fetus was designated as: 46,X,der(X)t(X;Y)(p22.3;q11.2). ish der(X)t(X;Y)(p22.3;q11.2)(X/Ypter-, DXZ1+, DYZ1+)mat. CONCLUSION: The combination of FISH and conventional cytogenetic techniques is a powerful tool to determine derivative chromosome and to offer an accurate genetic counseling. Identification of Xp; Yq rearrangement can help estimate the risk of fetus abnormalities and give a more precise prognosis.

[Sperm chromosome analysis and preimplantation genetic diagnos is in an infertile male with mosaic trisomy 18].

OBJECTIVE: To analyze the numerical aberration rate of X, Y and chromosome 18 in sperms from an oligozoospermic male with mosaic trisomy 18 and to perform preimplantation genetic diagnosis (PGD) for the couple. METHODS: G-banding and fluorescence in situ hybridization (FISH) were performed on metaphase chromosome. Sperm was analyzed in three-color FISH with a probe mixture containing CEP18, CEPY and Tel Xq/Yq. A healthy man with normal semen parameters was used as control. RESULTS: Significant difference in the rates of disomy for chromosome 18 (0.63% vs. 0.16%) and the gonosomes (0.945% vs. 0.35%) and diploidy (0.87% vs. 0.31%) was found in the spermatozoa between the patient and the control. After four embryos were biopsied in one PGD cycle, two embryos with XY1818 and XX1818 were selected for implanting and clinical pregnancy was ongoing. CONCLUSION: Sperm-FISH allows further understanding of aneuploidy rate and accurate genetic counseling. FISHPGD was effective for patient with mosaic trisomy 18.

[Genetic analysis of a complex chromosome rearrangement involving two chromosomes and four breakpoints in an azoospermic man].

OBJECTIVE: To perform genetic analysis of a complex chromosome rearrangement (CCR) 46,XY, t(3;11)(q27; q13), ins(11;3)(q13;p26p13) in an azoospermic man. METHODS: Peripheral blood lymphocytes we re obtained for karyotyping, and metaphases were studied by multicolor fluorescence in situ hybridization procedure, Y chromosomal microdeletions in the azoospermia factor (AZF) region were analyzed with multiplex polymerase chain reaction. RESULTS: The case was a complex chromosomal translocation between chromosomes 3 and 11 with four breakpoints, and accompanied with a band of chromosome 3 inserting into chromosome 11. No Y-chromosome microdeletions were identified at 6 STS sequences of the AZF loci. CONCLUSION: CCR can have a significant impact on male fertility. Molecular cytogenetic techniques may contribute to improving and personalizing reproductive counseling.

[Sperm-fluorescence in situ hybridization analysis in patients with pericentric inversions of Y chromosome].

OBJECTIVE: To analyze the sex chromosome meiotic segregation in inv(Y) patients by fluorescence in situ hybridization (FISH). METHODS: Conventional cytogenetic procedures (GTG and CBG banding) and FISH were performed on metaphase chromosome. Three-color FISH was performed on sperm samples using a probe mixture containing CEPX, Tel Xp/Yp and Tel Xq/Yq to investigate the sex chromosome segregation of five inv(Y) (p11.1q11.2) carriers. A healthy man with normal semen parameters was used as control. RESULTS: There was no statistical difference in the abnormal sex chromosome number and recombination frequencies in each spermatozoon from the patient in comparison with that in the control. CONCLUSION: There was no apparent sex chromosome abnormality in the sperm of the inv(Y) (p11.1q11.2) carriers. Sperm-FISH allows further understanding of the sex chromosome segregation pattern and an accurate genetic counseling.

[Purification technology of Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis in Fufang Xuelian dropping pills by macroporous resin].

OBJECTIVE: To study the purification technology of Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis in Fufang Xuelian dropping pills by macroporous resin. METHOD: Taking osthole, isomperatorin as index ingredients, the type of resin sampling amount and elution solvent were decided, and the influence of sample concentration pH of sample and ratio of diameter to height of column to adsorption were studied. RESULT: HPD400A was chosen to purify, the suitable sampling ratio of resin volume to raw material was 1:2; pH 3.5 (crude drug) and ratio of diameter to height was 1:7; 95% ethanol of the elution solvent was satisfactory eluant for desorption. CONCLUSION: HPD400A macroporous resin can be used to purify Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis.