Abstract
Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse ...of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFe
2
O
4
MNPs) offer distinct advantages in both purification and immobilization of enzymes. In this work, we demonstrate the preparation of NiFe
2
O
4
MNPs via a one-step solvothermal synthesis and their use in direct enzyme binding from cell lysates. These NiFe
2
O
4
MNPs have showed an average diameter of 8.9 ± 1.7 nm from TEM analysis and a magnetization at saturation (M
s
) value of 53.0 emu g
–1
from SQUID measurement. The nickel binding sites of the MNP surface allow direct binding of three his-tagged enzymes,
d
-phenylglycine aminotransferase (
d
-PhgAT),
Halomonas elongata
ω-transaminase (HeωT), and glucose dehydrogenase from
Bacillus subtilis
(
Bs
GDH). It was found that the enzymatic activities of all immobilized samples directly prepared from cell lysates were comparable to those prepared from the conventional immobilization method using purified enzymes. Remarkably,
d
-PhgAT supported on NiFe
2
O
4
MNPs also showed similar activity to the purified free enzyme. By comparing on both carrier preparation and enzyme immobilization protocols, use of NiFe
2
O
4
MNPs for direct enzyme immobilization from cell lysate can significantly reduce the number of steps, time, and use of chemicals. Therefore, NiFe
2
O
4
MNPs can offer considerable advantages for use in both enzyme immobilization and protein purification in pharmaceutical and other chemical industries.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing ...approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fetal DNA is present in the plasma of pregnant women. Massively parallel sequencing of maternal plasma DNA has been used to detect fetal trisomies 21, 18, 13 and selected sex chromosomal aneuploidies ...noninvasively. Case reports describing the detection of fetal microdeletions from maternal plasma using massively parallel sequencing have been reported. However, these previous reports were either polymorphism-dependent or used statistical analyses which were confined to one or a small number of selected parts of the genome. In this report, we reported a procedure for performing noninvasive prenatal karyotyping at 3 Mb resolution across the whole genome through the massively parallel sequencing of maternal plasma DNA. This method has been used to analyze the plasma obtained from 6 cases. In three cases, fetal microdeletions have been detected successfully from maternal plasma. In two cases, fetal microduplications have been detected successfully from maternal plasma. In the remaining case, the plasma DNA sequencing result was consistent with the pregnant mother being a carrier of a microduplication. Simulation analyses were performed for determining the number of plasma DNA molecules that would need to be sequenced and aligned for enhancing the diagnostic resolution of noninvasive prenatal karyotyping to 2 Mb and 1 Mb. In conclusion, noninvasive prenatal molecular karyotyping from maternal plasma by massively parallel sequencing is feasible and would enhance the diagnostic spectrum of noninvasive prenatal testing.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objectives To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically ...indicated for amniocentesis or chorionic villus sampling.Design Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples.Setting Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands.Participants 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Main outcome measures Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection.Results Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%.Conclusion Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.
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BFBNIB, CMK, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
To evaluate the clinical impact of chromosomal microarray (CMA) on the management of paediatric patients in Hong Kong.
We performed NimbleGen 135k oligonucleotide array on 327 children with ...intellectual disability (ID)/developmental delay (DD), autism spectrum disorders (ASD), and/or multiple congenital anomalies (MCAs) in a university-affiliated paediatric unit from January 2011 to May 2013. The medical records of patients were reviewed in September 2013, focusing on the pathogenic/likely pathogenic CMA findings and their "clinical actionability" based on established criteria.
Thirty-seven patients were reported to have pathogenic/likely pathogenic results, while 40 had findings of unknown significance. This gives a detection rate of 11% for clinically significant (pathogenic/likely pathogenic) findings. The significant findings have prompted clinical actions in 28 out of 37 patients (75.7%), while the findings with unknown significance have led to further management recommendation in only 1 patient (p < 0.001). Nineteen out of the 28 management recommendations are "evidence-based" on either practice guidelines endorsed by a professional society (n = 9, Level 1) or peer-reviewed publications making medical management recommendation (n = 10, Level 2). CMA results impact medical management by precipitating referral to a specialist (n = 24); diagnostic testing (n = 25), surveillance of complications (n = 19), interventional procedure (n = 7), medication (n = 15) or lifestyle modification (n = 12).
The application of CMA in children with ID/DD, ASD, and/or MCAs in Hong Kong results in a diagnostic yield of ∼ 11% for pathogenic/likely pathogenic results. Importantly the yield for clinically actionable results is 8.6%. We advocate using diagnostic yield of clinically actionable results to evaluate CMA as it provides information of both clinical validity and clinical utility. Furthermore, it incorporates evidence-based medicine into the practice of genomic medicine. The same framework can be applied to other genomic testing strategies enabled by next-generation sequencing.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To evaluate the effectiveness of whole-genome array comparative genomic hybridization (aCGH) in prenatal diagnosis in Hong Kong.
Array CGH was performed on 220 samples recruited prospectively as the ...first-tier test study. In addition 150 prenatal samples with abnormal fetal ultrasound findings found to have normal karyotypes were analyzed as a 'further-test' study using NimbleGen CGX-135K oligonucleotide arrays.
Array CGH findings were concordant with conventional cytogenetic results with the exception of one case of triploidy. It was found in the first-tier test study that aCGH detected 20% (44/220) clinically significant copy number variants (CNV), of which 21 were common aneuploidies and 23 had other chromosomal imbalances. There were 3.2% (7/220) samples with CNVs detected by aCGH but not by conventional cytogenetics. In the 'further-test' study, the additional diagnostic yield of detecting chromosome imbalance was 6% (9/150). The overall detection for CNVs of unclear clinical significance was 2.7% (10/370) with 0.9% found to be de novo. Eleven loci of common CNVs were found in the local population.
Whole-genome aCGH offered a higher resolution diagnostic capacity than conventional karyotyping for prenatal diagnosis either as a first-tier test or as a 'further-test' for pregnancies with fetal ultrasound anomalies. We propose replacing conventional cytogenetics with aCGH for all pregnancies undergoing invasive diagnostic procedures after excluding common aneuploidies and triploidies by quantitative fluorescent PCR. Conventional cytogenetics can be reserved for visualization of clinically significant CNVs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Collaborative research often combines findings across multiple, independent studies via meta-analysis. Ideally, all study estimates that contribute to the meta-analysis will be equally unbiased. Many ...meta-analyses require all studies to measure the same covariates. We explored whether differing minimally sufficient sets of confounders identified by a directed acyclic graph (DAG) ensures comparability of individual study estimates. Our analysis applied four statistical estimators to multiple minimally sufficient adjustment sets identified in a single DAG.
We compared estimates obtained via linear, log-binomial, and logistic regression and inverse probability weighting, and data were simulated based on a previously published DAG.
Our results show that linear, log-binomial, and inverse probability weighting estimators generally provide the same estimate of effect for different estimands that are equally sufficient to adjust confounding bias, with modest differences in random error. In contrast, logistic regression often performed poorly, with notable differences in effect estimates obtained from unique minimally sufficient adjustment sets, and larger standard errors than other estimators.
Our findings do not support the reliance of collaborative research on logistic regression results for meta-analyses. Use of DAGs to identify potentially differing minimally sufficient adjustment sets can allow meta-analyses without requiring the exact same covariates.
Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we ...demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively.
We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P=0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%.
Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Characterizing estradiol among women with HIV may have implications for breast cancer and cardiovascular disease risk but has not been adequately explored. We quantified differences in total (E2), ...free (FE2) estradiol, and sex hormone binding globulin (SHBG) by HIV and viral suppression status.
Women from a substudy (2003-2006) within the Women's Interagency HIV Study (IRB approved at each participating site) were included if they reported: a period in the last six months, were not pregnant/breastfeeding, no oophorectomy, and no exogenous hormone use in the prior year. Serum was collected on days 2-4 of the menstrual cycle. We assessed differences in biomarkers at 25th, 50th, and 75th percentiles by HIV and viral suppression status using weighted quantile regression.
Among 643 women (68% with HIV) median age was 37 years. All E2 percentiles were significantly (
< 0.05) lower in women with suppressed viral load versus women without HIV (4-10 pg/mL). The 25th and 50th percentile of E2 were 4-5 pg/mL lower in women with unsuppressed viral load compared to women without HIV (
< 0.05). The 25th and 50th percentile of SHBG was significantly higher in women with unsuppressed viral load compared to women without HIV (10 and 12 nmol/L, respectively). There were no consistent differences in estradiol or SHBG by suppression status.
There were no differences in FE2 but significantly lower E2 and higher SHBG among women with HIV versus without HIV. Further research is merited in a large contemporary sample to clarify the clinical implications of these findings.
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