The role of chromosomal microarray analysis among fetuses with normal karyotype and single system anomaly or nonspecific sonographic findings.

INTRODUCTION: Chromosomal microarray analysis is recommended as the first-tier test for the evaluation of fetuses with structural anomalies. This study aims to investigate the incremental diagnostic yield of chromosomal microarray over conventional karyotyping analysis in fetuses with anomalies restricted to one anatomic system and those with nonspecific anomalies detected by sonography. MATERIAL AND METHODS: This is a retrospective cohort analysis of 749 fetuses undergoing prenatal diagnosis for abnormal ultrasound findings isolated to one anatomic system and normal karyotype, utilizing chromosomal microarray. Overall, 495 (66%) fetuses had anomalies confined to one anatomic system and 254 (34%) had other nonspecific anomalies including increased nuchal translucency (≥3.5 mm), cystic hygroma, intrauterine growth restriction and hydrops fetalis. RESULTS: Fetuses with ultrasound anomalies restricted to one anatomic system had a 3.0% risk of carrying a pathogenic copy number variant; the risk varied dependent on the anatomic system affected. Fetuses with confined anomalies of the cardiac system had the highest diagnostic yield at 4.6%, but there were none in the urogenital system. Fetuses with nonspecific ultrasound anomalies had the highest diagnostic yield in fetuses with an intrauterine growth restriction at 5.9%. Overall, fetuses with a nonspecific ultrasound anomaly were affected with pathogenic copy number variants in 1.6% in the cases. CONCLUSIONS: The diagnostic yield of chromosomal microarray in fetuses with normal karyotype and ultrasound abnormality confined to a single anatomic system was highest if it involved cardiac defects or intrauterine growth restriction. This diagnostic yield ranges from 0% to 4.6% depending on the anatomic system involved. Chromosomal microarray has considerable diagnostic value in these pregnancies.

Characteristics and mode of inheritance of pathogenic copy number variants in prenatal diagnosis.

BACKGROUND: Microdeletions and microduplications can occur in any pregnancy independent of maternal age. The spectrum and features of pathogenic copy number variants including the size, genomic distribution, and mode of inheritance are not well studied. These characteristics have important clinical implications regarding expanding noninvasive prenatal screening for microdeletions and microduplications. OBJECTIVES: The aim was to investigate the spectrum and characteristics of pathogenic copy number variants in prenatal genetic diagnosis and to provide recommendations for expanding the scope of noninvasive prenatal screening for microdeletions and microduplications. STUDY DESIGN: This was a retrospective study of 1510 pregnant women who underwent invasive prenatal diagnostic testing by chromosomal microarray analysis. Prenatal samples were retrieved by amniocentesis or chorionic villus sampling and sent to our prenatal genetic diagnosis laboratory for chromosomal microarray analysis. The risk of carrying a fetus with pathogenic copy number variants is stratified by the patients' primary indication for invasive testing. We searched the literature for published prenatal chromosomal microarray data to generate a large cohort of 23,865 fetuses. The characteristics and spectrum of pathogenic copy number variants including the type of aberrations (gains or losses), genomic loci, sizes, and the mode of inheritance were studied. RESULTS: Overall, 375 of 23,865 fetuses (1.6%) carried pathogenic copy number variants for any indication for invasive testing, and 44 of them (11.7%) involve 2 or more pathogenic copy number variants. A total of 428 pathogenic copy number variants were detected in these fetuses, of which 280 were deletions and 148 were duplications. Three hundred sixty (84.1%) were less than 5 Mb in size and 68 (15.9%) were between 5 and 10 Mb. The incidence of carrying a pathogenic copy number variant in the high-risk group is 1 in 36 and the low-risk group is 1 in 125. Parental inheritance study results were available for 311 pathogenic copy number variants, 71 (22.8%) were maternally inherited, 36 (11.6%) were paternally inherited, and 204 (65.6%) occurred de novo. CONCLUSION: Collectively, pathogenic copy number variants are common in pregnancies. High-risk pregnancies should be offered invasive testing with chromosomal microarray analysis for the most comprehensive investigation. Detection limits on size, parental inheritance, and genomic distribution should be carefully considered before implementing copy number variant screening in expanded noninvasive prenatal screening.

Impact of preimplantation genetic testing for aneuploidy on obstetrical practice.

PURPOSE OF REVIEW: To provide updated information on preimplantation genetic testing for aneuploidy (PGT-A), focusing on its implications on prenatal diagnostic approaches after transferal of mosaic aneuploid embryos. RECENT FINDINGS: PGT-A is a technology to screen for chromosome aneuploidy or major chromosome structural rearrangement in embryos before implantation using different cytogenetic analyses. PGT-A has been shown to reduce the negative effect of increasing maternal age on in-vitro fertilization (IVF) outcomes. PGT-A also encourages clinicians and patients to accept single embryo transfer after IVF-PGT-A to reduce the chance of multiple pregnancies. However, mosaic aneuploid embryo may be encountered following PGT-A. Recent discussions have focused on the consideration of transferal of mosaic aneuploid embryos in couples with no euploid embryo following multiple trials of IVF-PGT-A. A risk score for each chromosome has been developed to prioritize which mosaic embryos should be considered for transfer. However, there is a lack of long-term outcome data following mosaic aneuploid embryo transfer. SUMMARY: Recent advances in PGT-A increase the detection of embryos with mosaicism, which is associated with an increased risk of miscarriage, fetal mosaic aneuploidy, and uniparental disomy. Strategy in prenatal diagnostic testing after mosaic aneuploid embryo transfer is discussed in this review.

Prenatal Diagnosis of a Retroesophageal Left Brachiocephalic Vein: Two Case Reports.

A retroesophageal left brachiocephalic vein is an extremely rare anomaly and has only been reported in 6 postnatal cases. Two prenatally diagnosed cases are reported. On the 3-vessel view, the vein appears as an aberrant vessel transversely coursing behind the aorta and trachea, which subsequently drains into the superior vena cava, giving rise to a U-shaped configuration. On color Doppler sonography, the U sign is bicolored. This anomaly should prompt the sonographer to carefully assess for other congenital heart defects, suggest consideration for genetic testing, and alert the cardiologist because it could affect central line procedures and cardiac interventions after delivery.

Increased bone turnover, osteoporosis, progressive tibial bowing, fractures, and scoliosis in a patient with a final-exon SATB2 frameshift mutation.

Haploinsufficiency of SATB2 causes cleft palate, intellectual disability with deficient speech, facial and dental abnormalities, and other variable features known collectively as SATB2-associated syndrome. This phenotype was accompanied by osteoporosis, fractures, and tibial bowing in two previously reported adult patients; each possessed SATB2 mutations either predicted or demonstrated to escape nonsense-mediated decay, suggesting that the additional bone defects result from a dominant negative effect and/or age-dependent penetrance. These hypotheses remain to be confirmed, as do the specific downstream defects causing bone abnormalities. We report a SATB2 mutation (c.2018dupA; p.(H673fs)) in a 15-year-old patient whose SATB2-associated syndrome phenotype is accompanied by osteoporosis, fractures, progressive tibial bowing, and scoliosis. As this homeodomain-disrupting and predicted truncating mutation resides within the final exon of SATB2, escape from nonsense-mediated decay is likely. Thus, we provide further evidence of bone phenotypes beyond those typically associated with SATB2-associated syndrome in individuals with potential dominant-negative SATB2 alleles, as well as evidence for age-dependence of bone features. Elevations in alkaline phosphatase, urinary N-telopeptide/creatinine ratio, and osteocalcin in the patient indicate increased bone turnover. We propose surveillance and treatment with osteoclast inhibitors to prevent fractures and to slow progressive bone deformities. © 2016 Wiley Periodicals, Inc.

Prenatal detection of 10q22q23 duplications: dilemmas in phenotype prediction.

OBJECTIVES: The phenotype for 10q22q23 duplication is diverse, ranging from intellectual disability and dysmorphism to normal development. Interpreting the clinical significance of the duplication identified in this region is difficult, especially in the prenatal setting. This study aimed to characterize the prenatal findings associated with this submicroscopic imbalance and discuss the dilemmas in predicting the phenotype of 10q22q23 duplications. METHODS: This is a retrospective study of three cases of 10q22q23 duplications diagnosed prenatally by chromosomal microarray analysis. Detailed pregnancy outcome and pediatric follow-up were documented. RESULTS: The genotypic and phenotypic features of the reported cases were discussed. 10q22q23 duplications are associated with an unpredictable and variable phenotypic outcome. Despite there was no phenotype found to be shared by 50% of the duplication cases, congenital heart defects, hypotelorism, and developmental delays including speech and motor delay seem to be more common. CONCLUSIONS: The phenotype of 10q22q23 duplication is highly variable prenatally and postnatally. Identification of additional affected individuals with similar duplications is needed to provide further insights into the pathogenesis of this microduplication. © 2016 John Wiley & Sons, Ltd.

Women's uptake of non-invasive DNA testing following a high-risk screening test for trisomy 21 within a publicly funded healthcare system: findings from a retrospective review.

OBJECTIVE: The objective of the study was to evaluate the uptake of non-invasive cell-free fetal DNA screening test (NIDT) after a high-risk screening result for trisomy 21 METHODS: Association between maternal and pregnancy characteristics on women's test choice was assessed after adjusting for confounding factors in Hong Kong Chinese women who had a high-risk (term risk ≥1:250) first-trimester or second-trimester screening test at three public hospitals. Main outcome measures were rate of declining further testing and obstetric and maternal factors impacting on patient's selection of testing options. RESULTS: Compared with the pre-NIDT period, the availability of NIDT resulted in a 45% (P < 0.001) reduction in the rate of refusal for further testing and a decrease from 92.2% to 66.7% in the use of invasive diagnostic test after a positive screening test. Nulliparous women with a spontaneous [adjusted odds ratio (aOR) = 2.18, 95% confidence interval (CI) 1.63-2.92] or assisted reproduction pregnancy (aOR = 3.95, 95% CI 1.6-9.32) were more likely to choose NIDT. Women with an adjusted risk of '>1:10' (aOR = 7.36, 95% CI 4.22-12.8) and '1:10 to 1:50' (aOR = 1.53, 95% CI 1.01-2.32) were more likely to opt for chorionic villi sampling or amniocentesis. CONCLUSIONS: NIDT reduced the refusal rate. Uptake of NIDT was highest in pregnancies of nulliparous women.

Patient's choice between a non-invasive prenatal test and invasive prenatal diagnosis based on test accuracy.

OBJECTIVE: To assess how pregnant women choose between a non-invasive DNA test (NIDT) and an invasive prenatal test (IPD) based on the accuracy of the test. MATERIALS AND METHODS: Pregnant women who attended for first-trimester combined screening assessment of risk of Down syndrome were invited to participate in an interviewer-administered survey. Women were asked to choose between NIDT (variable detection rate but no miscarriage risk) and IPD (∼100% detection rate but 0.5-1% miscarriage risk) if their screening test was positive for Down syndrome using the standard gamble technique. RESULTS: 358 women were approached of which 106 (29.6%) were unwilling to participate in the study as it had already been decided in advance which additional test they would have if they were screened positive. Of these 106 women, 70 (19.6%) would only choose IPD whereas 36 (10%) would only choose NIDT. Among those who agreed to undertake the gamble and participate in the study (n=252), 50% were willing to accept NIDT as an alternative to IPD provided that NIDT had a detection rate of 95%. CONCLUSION: The majority can accept NIDT as an alternative to IPD provided that the test is 95% accurate in the diagnosis of Down syndrome. Current evidence indicates that the detection rate of NIDT will be higher than this level. Health professionals should consider NIDT as an alternative to IPD when counseling women with a positive screening test.

Photogrammetric reconstruction of 3D carpological collection in high resolution for plants authentication and species discovery.

This study provides an accurate and efficient method to reconstruct detailed and high-resolution digital 3D models of carpological materials by photogrammetric method, in which only about 100 to 150 images are required for each model reconstruction. The 3D models reflect the realistic morphology and genuine color of the carpological materials. The 3D models are scaled to represent the true size of the materials even as small as 3 mm in diameter. The interfaces are interactive, in which the 3D models can be rotated in 360° to observe the structures and be zoomed to inspect the macroscopic details. This new platform is beneficial for developing a virtual herbarium of carpological collection which is thus the most important to botanical authentication and education.

Antiviral constituents against respiratory viruses from Mikania micrantha.

Phytochemical investigation of the dried aerial parts of Mikania micrantha led to the isolation of a new sesquiterpene, 3beta-acetoxy-1,10-epoxy-4-germacrene-12,8;15,6-diolide (1), along with six known constituents: 1,10-epoxy-4-germacrene-12,8;15,6-diolide (2), dihydromikanolide (3), potassium mikanin 3-sulfate (4), mikanin (5), alpinetin (6), and ergosta-7,22-dien-3beta-ol (7). Their structures were elucidated by spectroscopic methods, and the molecular structures and stereochemistry of sesquiterpene lactones 1-3 were revealed by single-crystal X-ray analysis. Compound 2 showed moderate activity against respiratory syncytial virus (IC(50) = 37.4 uM) and parainfluenza type 3 virus (IC(50) = 37.4 uM) with a therapeutic index (TI) of 16.0 for both compounds. Compound 4, the main component of M. micrantha, exhibited inhibitory activity against parainfluenza type 3 virus with IC(50) (19.7 uM) and TI (24.0) values comparable to those of ribavirin, serving as a positive control.

Miscarriage after invasive prenatal diagnostic procedures: how much risk our pregnant women are willing to take?

OBJECTIVES: To elicit the level of risk of prenatal diagnostic procedure-related miscarriage that Chinese pregnant women were willing to accept. METHODS: An interviewer-administered survey was conducted on 276 women who presented to the University Obstetric Unit. Using the standard gamble approach, subjects were asked to choose between a screening test with a 90% detection rate and a diagnostic test which is definitive but carries a finite risk of abortion. This probability of abortion was varied until the subject was indifferent between the two choices, and the value was called the utility score. RESULTS: When compared with a screening test with 90% detection rate, the median utility score was 0.989 (IQR: 0.970-0.999). The median risk of abortion below which the subjects would rather opt for an invasive test instead of a screening test was 1.1%. The percentage of patients who could accept a procedure-related miscarriage risk of 0.2, 0.5, 1 and 2% were 76, 67, 59.8 and 38.4%, respectively. CONCLUSIONS: Pregnant Chinese women agreed to trade a definitive chromosomal diagnostic test from a highly effective screening test with a small risk of undiagnosed aneuploidy provided that the procedure-related miscarriage risk was 1.1% or lower.

Chinese women's preferences for prenatal diagnostic procedure and their willingness to trade between procedures.

OBJECTIVE: To assess Chinese women's preference for the choice of a prenatal diagnosis test, karyotyping or rapid aneuploidy, and its relationship to maternal psychological state. STUDY DESIGN: Three hundred consenting women completed a self administered structured questionnaire which documented their psychological state and their preferred choice of diagnostic test for chromosomal abnormality using a discrete choice experiment design. Diagnostic tests were categorised according to three attributes: completeness of chromosomal information, procedure-to-result time interval and cost. RESULTS: Participants indicated a preference towards the karyotype test irrespective of cost and procedure-to-result time interval. The value of obtaining the extra information provided by karyotyping above that of rapid aneuploidy was pound267.82 (95% CI 226.71-323.31). Women would only accept the rapid aneuploidy test if results where available 18 days sooner than if they were undergoing a karyotyping test. Among women who traded between diagnostic tests, the value of extra chromosomal information was reduced to pound153.83 (95% CI 125.81-192.19). Women with high trait anxiety scores (>40) did not preferentially select one test. CONCLUSION: Chinese women showed a strong preference towards performing a full karyotype test and having a full chromosomal assessment performed for their pregnancy.

EDN1 Lys198Asn is associated with diabetic retinopathy in type 2 diabetes.

PURPOSE: We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR). METHODS: A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1-80C>A, IVS1-206G>C, IVS1-252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited. RESULTS: A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms. CONCLUSIONS: The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes.

Acceptance towards giving birth to a child with beta-thalassemia major - A prospective study.

OBJECTIVE: To explore the acceptance of pregnant Chinese women on giving birth to a child with beta-thalassemia major. MATERIALS AND METHODS: Women's acceptance on having a child with beta thalassemia major was assessed using standard gamble metrics during an interviewer-administered survey on 309 women recruited in the antenatal clinic. Utility scores were determined and the association with sociodemographic factors was assessed. RESULTS: The median utility score for having a child with beta-thalassemia major was 0.5 (0-0.65). Women having either higher educational level or family income had significantly higher utility scores (P < 0.05) corresponding to a higher acceptance. During the interview 59.9% participants indicated that they would elect to undergo a termination of pregnancy if their fetus was diagnosed with beta-thalassemia major but 26.5% participants were unable to decide what action they would take. CONCLUSION: Many Chinese pregnant women are ambivalent about giving birth to a baby with beta-thalassemia major. Women with higher educational level or higher family income had a higher acceptance towards the condition.

Isolation and chemotaxonomic significance of tuberostemospironine-type alkaloids from Stemona tuberosa.

An alkaloid named 6alpha-hydroxycroomine (1) as well as the known croomine (2), both belonging to the tuberostemospironine-alkaloid type, were isolated from Stemona tuberosa as the major components. The structure of 1 was elucidated through extensive spectroscopic analyses. Comparison of the HPLC profiles of the total alkaloids and the crude methanol extract showed that both compounds are naturally occurring. The first isolation of 1 and 2 from S. tuberosa has chemotaxonomic significance, confirming the close relationship between Stemona and Croomia. The trnL sequences of plants from the four genera of Stemonaceae cluster together as a clade, further lending support to retaining them in a single family.

Isolation of iridoid and secoiridoid glycosides and comparative study on Radix gentianae and related adulterants by HPLC analysis.

HPLC profile guided study led to the isolation of an acylated secoiridoid glycoside, named gentiotrifloroside (1), together with six known compounds, i.e., loganic acid (2), 6-O-beta-d-glucopyranosylgentiopicroside (3), swertiamarin (4), gentiopicroside (5), sweroside (6) and 2 -(o,m-dihydroxybenzyl)-sweroside (7) from Gentiana triflora and Gentiana rigescens. The structure of 1 was deduced from one- and two-dimensional NMR spectroscopic experiments. Compounds 1-7 were used successfully as chemical markers for the comparison of the four species of Gentiana used as Radix gentianae. Additionally, differentiation of Gentiana species mentioned and those used as adulterants was evaluated. The close similarity of chemical composition among the four genuine Gentiana species explain their popular usage as R. gentianae in Chinese medicine. We have also shown that the variation of chemical composition in R. gentianae and related adulterants agree well with their botanical phylogeny.

Prenatal diagnosis of maternally inherited X-linked Opitz G/BBB syndrome by chromosomal microarray in a fetus with complex congenital heart disease.

BACKGROUND: Prenatal sonographic diagnosis of Optiz G/BBB syndrome is difficult because the common clinical features, such as hypertelorism, hypospadias and abnormalities of midline structures, including laryngotracheoesophageal defects, are subtle. METHOD: Chromosomal microarray (CMA) analysis using a target enriched Fetal DNA Chip design was performed on the DNA of a fetus with congenital cardiac abnormalities. RESULTS: Fetal DNA chip revealed a 48Kb single copy number loss within chromosome region Xp22.2 (arr[hg18]Xp22.2(10,627,354-10,675,946)x0 mat). This deletion included the 3' UTR region of the MID1 gene predicted to cause the X-linked Opitz G/BBB syndrome. CONCLUSIONS: This case supports the use of CMA in prenatal diagnosis of fetuses with congenital heart disease. CMA allows prenatal diagnosis of genomic aberrations at a much higher resolution compared with conventional karyotyping, and such findings enable proper genetic counseling and decision making in the pregnancy.

Antitussive and central respiratory depressant effects of Stemona tuberosa.

AIMS OF THE STUDY: Stemona alkaloids with distinctly different chemical skeletons are recently reported as the active components in the antitussive herb Baibu derived from the root-tubers of Stemona tuberosa. This study aims to determine if alkaloids of this herb contribute equally to the antitussive functions, act on the same sites of cough reflex, and play any role in inducing central respiratory depressant effects. MATERIALS AND METHODS: Antitussive potency of four major alkaloids was evaluated on guinea pigs with citric acid aerosol to induce cough. The action sites of the alkaloids on cough reflex pathway were tested with electrical stimulation of the superior laryngeal nerve in guinea pigs. The central respiratory effects of croomine were also tested on guinea pigs. RESULTS: Croomine, neotuberostemonine and stemoninine showed similar antitussive potency, while tuberostemonine showed much weaker antitussive potency. Neotuberostemonine, tuberostemonine and stemoninine acted on the peripheral cough reflex pathway, while croomine acted on the central part. Croomine also showed obvious central respiratory depressant effects. CONCLUSIONS: The four major Stemona alkaloids in Stemona tuberosa do not contribute equally to antitussive potency in guinea pigs. Neotuberostemonine, tuberostemonine and stemoninine target on peripheral cough reflex pathway. Croomine acts on central sites in the cough reflex pathway and demonstrates central respiratory depressant effects, which can partly account for the adverse reactions reported for the herb.

The utility assessment of Chinese pregnant women towards the birth of a baby with Down syndrome compared to a procedure-related miscarriage.

OBJECTIVE: This study was performed to investigate the preferences of Chinese pregnant women for Down syndrome-affected birth compared to invasive test-related miscarriage, using the standard gamble approach, and to investigate whether there is a difference in Utility Score between general obstetric patients and those who request prenatal screening. METHODS: An interviewer-administered survey was conducted on 67 women who presented to the General Obstetric Clinic for booking visits and 69 women who presented to the first-trimester Combined Screening Clinic for fetal Down syndrome in a University Obstetric Unit. Preferences for Down syndrome-affected birth compared to invasive test-related miscarriage were assessed using the standard gamble approach. The differences in Utility Scores for the two outcomes and difference in scores between the two study groups were compared. RESULTS: There was no significant difference in any of the Utility Scores studied between the two study groups. Therefore the summary statistics were performed using the whole study population. The median Utility Score for a Down syndrome-birth was 0.20 (IQR: 0.10-0.40), which was significantly lower than that of 0.55 (IQR: 0.40-0.80) for a procedure-related miscarriage (p < 0.001). Also, the Utility Scores were neither found to be associated with any particular patient demographic characteristics nor their perception of the functional disability of individuals with Down syndrome. CONCLUSION: The Chinese pregnant women in Hong Kong consider a Down syndrome-affected birth as a much worse health state and life event than a miscarriage. Whether or not to have a screening test appeared to be a result of accessibility and affordability rather than fundamental differences in attitude towards Down syndrome. The findings of the study provide important information on how prenatal screening and diagnosis of fetal chromosomal abnormalities should be offered.

Alkaloids and chemical diversity of Stemona tuberosa.

Phytochemical investigation of the chemical components of Stemona tuberosa led to the isolation of two new alkaloids named tuberostemonine K (1) and tuberospironine (2), together with the known tuberostemonine (3). The new structures of 1 and 2 were elucidated through extensive spectroscopic analyses, while the molecular structure of 3 was confirmed by X-ray analysis. A gradient reversed-phase HPLC-ELSD method was established for the investigation of the chemical diversity of S. tuberosa from 13 localities, and four types of chemical variation featured by the major components 3, neotuberostemonine (4), croomine (5), and stemoninine (6), respectively, were observed.