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Objective: To systematically compare the endometrial microbiota in infertile women with and without chronic endometritis (CE), as diagnosed by a quantitative and reference range-based method., Design: Case-control observational study., Setting: University-affiliated hospital., Patient(s): One hundred and thirty infertile women., Intervention(s): Endometrial biopsy and fluid (uterine lavage, UL) collected precisely 7 days after LH surge, with plasma cell density (PCD) determined based on Syndecan-1 (CD138)-positive cells in the entire biopsy section and culture-independent massively parallel sequencing of the 16S ribosomal RNA gene performed on both the CE and non-CE endometrial fluid samples., Main Outcome Measure(s): Relative abundance of bacterial taxa., Result(s): Chronic endometritis was diagnosed if the PCD was above the 95th percentile (>5.15 cells per 10 mm 2 ) of the reference range in fertile control subjects. With this stringent diagnostic criterion, 12 women (9%) were diagnosed with CE. Sequencing was successfully performed on all endometrial samples obtained by UL) (CE, n = 12; non-CE, n = 118). The median relative abundance of Lactobacillus was 1.89% and 80.7% in the CE and non-CE microbiotas, respectively. Lactobacillus crispatus was less abundant in the CE microbiota (fold-change, range: 2.10-2.30). Eighteen non-Lactobacillus taxa including Dialister, Bifidobacterium, Prevotella, Gardnerella, and Anaerococcus were more abundant in the CE microbiota (fold-change, 2.10-18.9). Of these, Anaerococcus and Gardnerella were negatively correlated in relative abundance with Lactobacillus (SparCC correlation magnitude, range: 0.142-0.177)., Conclusion(s): Chronic endometritis was associated with a statistically significantly higher abundance of 18 bacterial taxa in the endometrial cavity., Clinical Trials Registry Number: ChiCTR-IOC-16007882., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: A recent study has reported that the microbiota in endometrial fluid of patients receiving in vitro fertilization and embryo transfer (IVF-ET) may predict implantation and pregnancy rates. However, studies are lacking that simultaneously compare the microbiota between endometrial fluid and tissue samples. Whether the microbiota composition in endometrial fluid reflects that in the endometrial tissue remains unclear., Methods: We systematically profiled the microbiota in endometrial fluid and tissue samples of IVF-ET patients using massively parallel sequencing. The bacterial 16S ribosomal RNA gene (V4 region) was PCR-amplified. Sequencing reads with >98% nucleotide identity were clustered as a bacterial taxon. To account for the different number of reads per sample, we normalized the read counts of each taxon before comparing its relative abundances across samples., Results: Thirteen taxa, including Verrucomicrobiaceae, Brevundimonas , Achromobacter , Exiguobacterium , and Flavobacterium , were consistently detected only in endometrial tissue samples but not fluid samples. Eight taxa were detected in fluid but not tissue. Twenty-two taxa were differentially abundant between fluid and tissue samples (adjusted P values, 4.1 × 10 -25 to 0.025). The numbers of taxa identified per 1000 sequencing reads, diversity, and evenness in fluid samples were smaller than those in tissue samples., Conclusions: Our data suggest that the microbiota composition in endometrial fluid does not fully reflect that in endometrial tissue. Sampling from both endometrial fluid and biopsy allows a more comprehensive view of microbial colonization. Further efforts are needed to identify the preanalytical effects, including sampling sites, methods, and sequencing depth, on profiling endometrial microbiota., (© 2018 American Association for Clinical Chemistry.)

Previously, we reported that proteomic fingerprints were present in sera of patients with severe acute respiratory syndrome (SARS), and could separate patients into subgroups with different prognoses. In the present study, we examined the prognostic values of the SARS-associated proteomic features by biostatistical analysis, and deciphered the identities of those with prognostic values. Data of 20 SARS-associated serum proteomic features and ten serological variables from 38 SARS adult patients before treatment were subjected to multivariate logistic regression. Proteomic features of m/z 6634, m/z 7769, m/z 8635, and m/z 8865 were identified as independent prognostic markers. After purification by cation-exchange chromatography and gel electrophoresis, proteomic features of m/z 7769 and m/z 8865 were found to be platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) by tandem mass spectrometry, respectively. The associations of decreased serum PF4 and increased serum beta-TG levels with poor prognosis were confirmed by Western blot. Previous studies suggest that PF4 and beta-TG are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) in a negative and positive way, respectively. Our results suggest that PF4 and beta-TG may also play similar roles in the development of ARDS in SARS patients., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Spontaneous preterm birth (SPB, before 37 gestational weeks) is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls., Principal Findings: Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB) placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the IL1RL1 mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60%) plasma samples collected during the presentation of preterm labor (≤32.9 weeks) in women eventually giving SPB, but was detected in only 1 of 27 (4%) samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056)., Conclusion: We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the IL1RL1 mRNA was more frequently detected in predelivery maternal plasma samples collected from women resulting in SPB than the gestational-age matched controls.

Background: The CHD5 gene located on 1p36 encodes a protein-chromodomain helicase DNA-binding protein 5. CHD5 has been shown to be a tumor suppressor gene candidate. This study investigated the involvement of CHD5 in ovarian cancer and its clinicopathological significance., Methods: CHD5 expression in ovarian cancer and its counterpart were determined by quantitative RT-PCR. The correlation of CHD5 expression to clinicopathological features of the tumor was analyzed., Results: CHD5 expression was downregulated by at least twofold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 controls in Hong Kong Chinese women. CHD5 downregulation was correlated to clinical status (p < 0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (p > 0.05). Survival analysis showed that patients with CHD5 downregulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 downregulation (p < 0.05). Cox proportional-hazards regression analysis indicated that downregulation of CHD5 is an independent adverse prognostic factor in ovarian cancer., Conclusion: This study shows that CHD5 is downregulated in a certain number of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival., (Copyright © 2011 S. Karger AG, Basel.)

Background: 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., Principal Findings: 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%., Conclusions: 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.

The relationship between DNA methylation, histone modifications and terminal differentiation in cardiomyocytes was investigated in this study. The upregulation of methylation-related proteins, including DNA methyltransferase (DNMT) 1, methyl-CpG binding domain proteins 1, 2 and 3, and the increase in global methylation during rat neonatal heart development were observed. Moreover, an increase in DNA synthesis and a delay in differentiation were found in 5-azacytidine (5-azaC)-treated cardiomyocytes. Increase in acetylation of H3-K9, H4-K5, H4-K8 and methylation of H3-K4 suggested a more accessible chromatin structure in 5-azaC-treated cells. Furthermore, methyl-CpG-binding protein 2 was found to be upregulated in differentiated cardiomyocytes. Overexpression of this protein resulted in an increase of global methylation levels. Therefore, we suggest that a hypermethylated genome and a more compact chromatin structure are formed during terminal differentiation of cardiomyocytes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: The term "transrenal DNA" was coined in 2000 to signify that DNA in urine may come from the passage of plasma DNA through the kidney barrier. Although DNA in the urine has the potential to provide a completely noninvasive source of nucleic acids for molecular diagnosis, its existence remains controversial., Methods: We obtained blood and urine samples from 22 hematopoietic stem cell transplant (HSCT) recipients and used fluorescence in situ hybridization, PCR for short tandem repeats, mass spectrometry, quantitative PCR, and immunofluorescence detection to study donor-derived DNA in the urine., Results: All HSCT recipients exhibited high amounts of donor-derived DNA in buffy coat and plasma samples. Male donor-derived DNA was detected in supernatants of urine samples from all 5 female sex-mismatched HSCT recipients. Surprisingly, the amount of DNA in urine supernatants was not correlated with the plasma value. Moreover, cell-free urine supernatants contained DNA fragments >350 bp that were absent in plasma. Donor-derived polymorphs were detected in urine by fluorescence in situ hybridization. Coincidentally, donor-derived cytokeratin-producing epithelial cells were discovered in urine samples from 3 of 10 sex-mismatched HSCT recipients as long as 14.2 years after transplantation., Conclusions: This report is the first to demonstrate the presence of donor-derived DNA in the urine of HSCT recipients; however, we show that much of this DNA originates from donor-derived cells, rather than from the transrenal passage of cell-free plasma DNA. Our discovery of donor-derived cytokeratin-producing epithelial cells raises interesting biological and therapeutic implications, e.g., the capacity of marrow stem cells to serve as an extrarenal source for renal tubule regeneration.

We investigated the role(s) of monoamine oxidases (MAOs) on the altered 5-hydroxytryptamine (5-HT, serotonin)-induced tension development of the isolated umbilical artery of preeclamptic pregnancy of Chinese women. An enhanced 5-HT-induced tension development of the umbilical artery of preeclamptic pregnancy was observed when compared with that of normal pregnancy. The enhanced component of 5-HT-induced tension development was eradicated by clorgyline (a MAO-A inhibitor). Blockade of eNOS (endothelial isoform nitric oxide synthase) (N(omega)-nitro-L-arginine methyl ester), 5-HT transporter (citalopram), 5-HT receptor subtypes (5HT2B, SB 204741; 5-HT2C, RS 102221; 5-HT7, SB 269970), and endothelium denudation of the umbilical artery of normal pregnancy mimicked the enhanced 5-HT-induced tension development as observed in the preeclamptic tissues. In contrast, no apparent changes in 5-HT-induced tension development of the umbilical artery of preeclamptic pregnancy were observed with the same pharmacological manipulations. A decreased protein expression levels of MAO-A and eNOS (no iNOS and MAO-B expression was detected) and no change in caveolin-1 and 5-HT transporter expression were demonstrated in the umbilical artery (endothelium intact) lysate of preeclamptic pregnancy, compared to that of the umbilical artery of normal pregnancy. Thus, in the umbilical artery of preeclamptic pregnancy, a decrease of MAO-A and eNOS protein expression levels are probably associated with, or responsible for, the exaggerated 5-HT-induced tension development.

Background: The discovery of circulating fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. MicroRNAs (miRNAs), a class of small RNAs, have been intensely investigated recently because of their important regulatory role in gene expression. Because nucleic acids of placental origin are released into maternal plasma, we hypothesized that miRNAs produced by the placenta would also be released into maternal plasma., Methods: We systematically searched for placental miRNAs in maternal plasma to identify miRNAs that were at high concentrations in placentas compared with maternal blood cells and then investigated the stability and filterability of this novel class of pregnancy-associated markers in maternal plasma., Results: In a panel of TaqMan MicroRNA Assays available for 157 well-established miRNAs, 17 occurred at concentrations >10-fold higher in the placentas than in maternal blood cells and were undetectable in postdelivery maternal plasma. The 4 most abundant of these placental miRNAs (miR-141, miR-149, miR-299-5p, and miR-135b) were detectable in maternal plasma during pregnancy and showed reduced detection rates in postdelivery plasma. The plasma concentration of miR-141 increased as pregnancy progressed into the third trimester. Compared with mRNA encoded by CSH1 [chorionic somatomammotropin hormone 1 (placental lactogen)], miR-141 was even more stable in maternal plasma, and its concentration did not decrease after filtration., Conclusion: We have demonstrated the existence of placental miRNAs in maternal plasma and provide some information on their stability and physical nature. These findings open up a new class of molecular markers for pregnancy monitoring.

Background: The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18., Methods: To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)-76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria-for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site., Results: We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22-0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00-0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance., Conclusion: Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

Objective: To study if quantitative aberrations in circulating placental-derived hypermethylated RASSF1A DNA in maternal plasma are associated with pre-eclamptic pregnancies., Method: Maternal plasma and placental tissues from third-trimester pre-eclamptic women and gestational-age matched normotensive controls were studied. Real-time PCR was performed to quantify RASSF1A concentrations before and after methylation-sensitive restriction digestion in a duplex assay, where ss-actin concentrations were quantified as an internal control to confirm complete enzyme digestion., Results: The median concentrations of hypermethylated RASSF1A were 4.3-fold higher in maternal plasma of pre-eclamptic subjects than in controls. There was no significant difference between the extent of RASSF1A hypermethylation in placental tissues obtained from pre-eclamptic and control pregnancies., Conclusion: This study demonstrated the potential utility of hypermethylated RASSF1A sequences in maternal plasma as a gender- and polymorphism-independent marker for pre-eclampsia., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Background and Purpose: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors) have been demonstrated to reduce cardiovascular mortality. It is unclear how the expression level of HMG CoA reductase in cardiovascular tissues compares with that in cells derived from the liver. We hypothesized that this enzyme exists in different cardiovascular tissues, and simvastatin modulates the vascular iberiotoxin-sensitive Ca2+-activated K(+) (BK(Ca)) channels., Experimental Approaches: Expression of HMG CoA reductase in different cardiovascular preparations was measured. Effects of simvastatin on BK(Ca) channel gatings of porcine coronary artery smooth muscle cells were evaluated., Key Results: Western immunoblots revealed the biochemical existence of HMG CoA reductase in human cardiovascular tissues and porcine coronary artery. In porcine coronary artery smooth muscle cells, extracellular simvastatin (1, 3 and 10 microM) (hydrophobic), but not simvastatin Na+ (hydrophilic), inhibited the BK(Ca) channels with a minimal recovery upon washout. Isopimaric acid (10 microM)-mediated enhancement of the BK(Ca) amplitude was reversed by external simvastatin. Simvastatin Na+ (10 microM, applied internally), markedly attenuated isopimaric acid (10 microM)-induced enhancement of the BK(Ca) amplitude. Reduced glutathione (5 mM; in the pipette solution) abolished simvastatin -elicited inhibition. Mevalonolactone (500 microM) and geranylgeranyl pyrophosphate (20 microM) only prevented simvastatin (1 and 3 microM)-induced responses. simvastatin (10 microM ) caused a rottlerin (1 microM)-sensitive (cycloheximide (10 microM)-insensitive) increase of PKC-delta protein expression., Conclusions and Implications: Our results demonstrated the biochemical presence of HMG CoA reductase in different cardiovascular tissues, and that simvastatin inhibited the BK(Ca) channels of the arterial smooth muscle cells through multiple intracellular pathways.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Background: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma., Methods: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses., Results: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence., Conclusion: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Background: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma., Methods: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection., Results: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses., Conclusions: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.

During cardiac muscle development, most cardiomyocytes permanently withdraw from the cell cycle. Previously, by suppressive subtractive hybridization, we identified calcyclin-binding protein/Siah-interacting protein (CacyBP/SIP) as one of the candidates being upregulated in the hyperplastic to hypertrophic switch, suggesting an important role of CacyBP/SIP in cardiac development. To show the importance of CacyBP/SIP during myoblast differentiation, we report here that CacyBP/SIP is developmentally regulated in postnatal rat hearts. The overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis of H9C2 cells and primary rat cardiomyocytes, as well as downregulates the expression of beta-catenin. Besides, CacyBP/SIP promotes the formation of myotubes and multinucleation upon differentiation. To investigate the cardioprotective role of CacyBP/SIP in cardiomyocytes, a hypoxia/reoxygenation model was employed. We found that CacyBP/SIP was upregulated during myocardial infarction (MI) and hypoxia/reoxygenation. As a conclusion, CacyBP/SIP may play a role in cardiomyogenic differentiation and possibly protection of cardiomyocytes during hypoxia/reoxygenation injury., ((c) 2005 Wiley-Liss, Inc.)

Background: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus strain, SARS-CoV. Specific proteomic patterns might be present in serum in response to the infection and could be useful for early detection of the disease., Methods: Using surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, we profiled and compared serum proteins of 39 patients with early-stage SARS infection and 39 non-SARS patients who were suspected cases during the SARS outbreak period. Proteomic patterns associated with SARS were identified by bioinformatic and biostatistical analyses. Features of interest were then purified and identified by tandem mass spectrometry., Results: Twenty proteomic features were significantly different between the 2 groups. Fifteen were increased in the SARS group, and 5 were decreased. Their concentrations were correlated with 2 or more clinical and/or biochemical variables. Two were correlated with the SARS-CoV viral load. Hierarchical clustering analysis showed that a majority of the SARS patients (95%) had similar serum proteomic profiles and identified 2 subgroups with poor prognosis. ROC curve analysis identified individual features as potential biomarkers for SARS diagnosis (areas under ROC curves, 0.733-0.995). ROC curve areas were largest for an N-terminal fragment of complement C3c alpha chain (m/z 28 119) and an internal fragment of fibrinogen alpha-E chain (m/z 5908). Immunoglobulin kappa light chain (m/z 24 505) positively correlated with viral load., Conclusions: Specific proteomic fingerprints in the sera of adult SARS patients could be used to identify SARS cases early during onset with high specificity and sensitivity.

The polymerase chain reaction (PCR), which can exponentially replicate a target DNA sequence, has formed the basis for the sensitive and direct examination of clinical samples for evidence of infection. During the epidemic of severe acute respiratory syndrome (SARS) in 2003, PCR not only offered a rapid way to diagnose SARS-coronavirus (SARS-CoV) infection, but also made the molecular analysis of its genomic sequence possible. Sequence variations were observed in the SAR-CoV obtained from different patients in this epidemic. These unique viral genetic signatures can be applied as a powerful molecular tool in tracing the route of transmission and in studying the genome evolution of SARS-CoV. To extract this wealth of information from the limited primary clinical specimens of SARS patients, we were presented with the challenge of efficiently amplifying fragments of the SARS-CoV genome for analysis. In this chapter, we will discuss how we managed to accomplish this task with our optimized protocols on reverse-transcription, nested PCR amplification, and DNA cycle sequencing. We will also discuss the sequence variations that typified some strains of SARS-CoV in the different phases during this epidemic. PCR amplification of the viral sequence and genomic sequencing of these critical sequence variations of re-emerging SARS-CoV strains would give us quick insights into the virus.

Background: The Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002-2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination., Methods: Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan). Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing., Results: Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort., Conclusion: We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

Background: It has been postulated that genetic predisposition may influence the susceptibility to SARS-coronavirus infection and disease outcomes. A recent study has suggested that the deletion allele (D allele) of the angiotensin converting enzyme (ACE) gene is associated with hypoxemia in SARS patients. Moreover, the ACE D allele has been shown to be more prevalent in patients suffering from adult respiratory distress syndrome (ARDS) in a previous study. Thus, we have investigated the association between ACE insertion/deletion (I/D) polymorphism and the progression to ARDS or requirement of intensive care in SARS patients., Method: One hundred and forty genetically unrelated Chinese SARS patients and 326 healthy volunteers were recruited. The ACE I/D genotypes were determined by polymerase chain reaction and agarose gel electrophoresis., Results: There is no significant difference in the genotypic distributions and the allelic frequencies of the ACE I/D polymorphism between the SARS patients and the healthy control subjects. Moreover, there is also no evidence that ACE I/D polymorphism is associated with the progression to ARDS or the requirement of intensive care in the SARS patients. In multivariate logistic analysis, age is the only factor associated with the development of ARDS while age and male sex are independent factors associated with the requirement of intensive care., Conclusion: The ACE I/D polymorphism is not directly related to increased susceptibility to SARS-coronavirus infection and is not associated with poor outcomes after SARS-coronavirus infection.

The epidemic of the severe acute respiratory syndrome (SARS) has swept through the globe with more than 8000 reported probable cases. In Hong Kong, the hardest hit areas included our teaching hospital and the Amoy Gardens apartment complex. A novel coronavirus, SARS-coronavirus (SARS-CoV), with a single-stranded plus sense RNA genome, was promptly implicated as the causative agent and subsequently fulfilled Koch's postulates. To aid the understanding of SARS-CoV, groups of investigators rapidly sequenced viral isolates around the world. We were the third group in the world to release the complete SARS-CoV genome sequence (isolate CUHK-W1) on the world-wide web. With other isolates from patients of distinct epidemiological backgrounds, we additionally sequenced four complete (CUHK-Su10, CUHK-AG01, CUHK-AG02, CUHK-AG03) and two partial SARS-CoV genomes. The reviewed data obtained from representative patients from the hospital and community outbreaks has documented the evolution of the virus in this epidemic. Their sequence variations also revealed a remarkable epidemiological correlation. We demonstrate that sequence variations in the SARS-CoV genome can be applied as a powerful molecular tool in tracing the route of transmission, when used adjunctively with standard epidemiology.

Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be associated with a new coronavirus (SARS-CoV). The clinical and epidemiological aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) analysis. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathology was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs., (Copyright 2004 John Wiley & Sons, Ltd.)

Background: The availability of an early diagnostic tool for severe acute respiratory syndrome (SARS) would have major public health implications. We investigated whether the SARS coronavirus (SARS-CoV) can be detected in serum and plasma samples during the early stages of SARS and studied the potential prognostic implications of such an approach., Methods: We developed two real-time quantitative reverse transcription-PCR (RT-PCR) assays, one for the polymerase and the other for the nucleocapsid region of the virus genome, for measuring the concentration of SARS-CoV RNA in serum/plasma samples from SARS patients. Plasma samples were obtained from 12 confirmed SARS patients on the day of hospital admission, as well as on days 7 and 14 after fever onset. Serum samples were also obtained from 23 confirmed SARS patients on the day of hospital admission, 11 of whom subsequently required intensive care. Viral RNA was extracted from the plasma/serum samples. The extracted RNA was subjected to analysis by the RT-PCR assays., Results: The RT-PCR system for the polymerase region detected SARS-CoV RNA in 50% of plasma and 78% of serum samples from SARS patients during the first week of illness. The detection rates for plasma dropped to 25% at day 14 after fever onset. The median serum SARS-CoV concentrations in patients who required and did not require intensive care unit admission during the course of hospitalization were 5800 and 140 copies/mL, respectively (Mann-Whitney test, P <0.005). These data were confirmed by the RT-PCR system for the nucleocapsid region, which showed an even higher detection rate of 87%. The correlation between the results obtained by the two RT-PCR systems was high (Pearson correlation analysis, r = 0.998; P <0.001)., Conclusion: Plasma/serum SARS-CoV quantification represents a potentially useful early diagnostic and prognostic tool for SARS.

Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.

Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases. The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed. This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids. The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes. In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte. The ANAPC11 gene is located at the human chromosome 17q25. ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected. Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment., (Copyright 2001 Wiley-Liss, Inc.)

Heart diseases have been one of the major killers among the human population worldwide. Because the vast majority of cardiomyocytes cannot regenerate once they cease to proliferate shortly after birth, functionally significant myocardial regeneration is not observed clinically. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle is controversial, but broadening our understanding of the rapid switch from hyperplastic to hypertrophic growth of cardiomyocytes during neonatal myocardial development may shed light on novel cardiovascular therapies. By suppression subtractive hybridization (SSH) and expressed sequence tag (EST) sequencing, we analyzed the differential gene expression of rat neonatal heart. SSH yielded subtracted and normalized cDNA libraries and enhanced the probability of detecting ESTs, which represent genes pertinent to signal transduction/cell regulation and replication/transcription/translation machinery, as compared to the traditional EST sequencing of heart cDNA libraries., (Copyright 2000 Wiley-Liss, Inc.)

During neonatal development, cardiac myocytes undergo a transition from hyperplastic to hypertrophic growth. Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle shortly after birth is controversial. Nevertheless, the clinical observation that functionally significant myocardial regeneration has not been documented in cardiovascular disease or injury during adulthood seems to support the notion that the vast majority of cardiac myocytes do not proliferate once they differentiate. Regardless of the controversy, the elucidation on how mitosis is blocked in cardiac myocytes may facilitate development of new cardiovascular therapies, based on the regeneration of the adult myocardium. To better understand postnatal myocardial development, we performed suppression subtractive hybridization to isolate genes that are differentially expressed in day one or day seven postnatal rat ventricular myocardium. Here we report the down-regulated mRNA expression of the 40-kDa subunit of replication factor C (RFC p40 or RFC2), which is an essential processive factor for proliferating cellular nuclear antigen-dependent DNA replication during neonatal myocardial development., (Copyright 2000 Wiley-Liss, Inc.)

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Four-and-a-half LIM domain proteins (FHL) possess four tandem repeats of LIM domain and an extra zinc finger. FHL family LIM proteins are unique when compared with other LIM-only proteins because they possess an odd number of zinc fingers. In this study, the tissue distribution and chromosomal mapping of skeletal muscle LIM protein FHL3 were reported. When the FHL3 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, and virtually no signal could be detected in heart, brain, placenta, lung, liver, kidney and pancreas. Using radiation hybrid technique, FHL3 gene was mapped to the distal end of the short arm of chromosome 1 (123.26 cR from the top of the Chr1 linkage group) and this region (near 1p34) is related to several human malignancies.

We report here on the mapping of a cDNA encoding for human cysteine-rich heart protein (HCRHP), a counterpart of the murine cysteine-rich intestinal protein CRIP. By somatic cell hybrid analysis and radiation hybrid mapping, we have located the gene CRIP1 (HGMW-approved symbol) on the subcentromeric region of the q arm of human chromosome 7, flanking a deletion associated with Williams syndrome., (Copyright 1998 Academic Press.)

We describe two pattern recognition algorithms for measuring half-micrometer focus-exposure dense photoresist line structures by using optical microscopes, such as a confocal scanning microscope and an interference microscope. The first approach depends on a data-clustering technique that allows us to measure resist lines down to 0.3 µm. The second method is an improved calibration procedure that utilizes multiple regression and moments of a cloud plot. Linearity between optical and scanning electron microscopy measurements is achieved down to 0.3 µm for nested focus-exposure resist structures with a standard deviation of 10 nm.

We describe an efficient algorithm based on the Hilbert transform for reconstructing cross-sectional or three-dimensional images from the input images acquired by an interference microscope. First the design of this filter is presented, and cross-sectional images of an integrated circuit constructed with this algorithm are demonstrated. It is shown that this Hilbert transform algorithm can be easily implemented with a low-cost frame grabber so that the computation time required for image reconstruction is drastically reduced.

A new algorithm for extracting amplitude and phase information from the data collected by the Mirau correlation microscope is presented. The accuracy of the phase measurements is first determined by measuring phase steps and vertical resolutions in the nanometer range. Experimental results with metal gratings show good agreement with Fourier theory. An inverse filter is then used to improve the resolution of the system. The filtered image has sharper edges and better phase contrast.

We have constructed a correlation microscope based on the Mirau interferometer configuration using a thin silicon nitride film beam splitter. This microscope provides the amplitude and phase information for the reflected signal from a sample located on the microscope-object plane. The device is remarkably insensitive to vibrations and is self-correcting for spherical and chromatic range aberrations of the objective. An imaging theory for the correlation microscope has been derived, which predicts accurately both the transverse resolution at a sharp edge and the range resolution for a perfect plane reflector. The range resolution is slightly better than that for a scanning optical microscope using a lens with the same aperture.

We have constructed a correlation microscope based on the Mirau interferometer configuration using a thin silicon nitride film beam splitter, and we have developed a method to extract the amplitude and phase information of the reflected signal from a sample located at the microscope object plane. An imaging theory for the interference microscope has been derived, which predicts accurately both the transverse response at a sharp edge and the range response to a perfect plane reflector.