Evolution of Feeding Shapes Swimming Kinematics of Barnacle Naupliar Larvae: A Comparison between Trophic Modes.

A central goal in evolutionary biology is connecting morphological features with ecological functions. For marine invertebrate larvae, appendage movement determines locomotion, feeding, and predator avoidance ability. Barnacle larvae are morphologically diverse, and the morphology of non-feeding lecithotrophic nauplii are distinct from those that are planktotrophic. Lecithotrophic larvae have a more globular body shape and simplified appendages when compared with planktotrophs. However, little is known about whether and how such morphological changes affect kinematics, hydrodynamics, and ecological functions. Here, we compared the nauplii kinematics and hydrodynamics of a lecithotrophic Rhizocephalan species, Polyascus planus, against that of the planktotrophic nauplii of an intertidal barnacle, Tetraclita japonica. High-speed, micro-particle image velocimetry analysis showed that the Polyascus nauplii swam faster and had higher amplitude and more synchronous appendage beating than the Tetraclita nauplii. This fast swimming was accompanied by a faster attenuation of induced flow with distance, suggesting reduced predation risk. Tetraclita nauplii had more efficient per beat cycles with less backward displacement during the recovery stroke. This "anchoring effect" resulted from the anti-phase beating of appendages. This movement, together with a high-drag body form, likely helps direct the suction flow toward the ventral food capturing area. In sum, the tradeoff between swimming speed and predation risks may have been an important factor in the evolution of the observed larval forms.

Correction: FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer.

In the published version of this article, the images for cytoplasmic and nuclear FGF7 in MDA-MB-231 cells were duplicated and mistaken for total FGF7 in SKBR-3 and MDA-MB-231 cells.

Factors associated with common and atypical chromosome abnormalities after positive combined first-trimester screening in Chinese women: a retrospective cohort study.

BACKGROUND: When cell-free DNA (cfDNA) testing is used as a secondary screening tool following combined first-trimester screening (cFTS), cFTS is used to estimate the prior risk for chromosome abnormalities. This study aimed to assess the factors that are associated with common and atypical abnormalities following cFTS, including cFTS risk, advanced maternal age, increased nuchal translucency (NT) ≥3.5 mm, and abnormal levels of serum markers. METHODS: We reviewed a historical cohort of 1855 Chinese women carrying singleton pregnancies with a positive cFTS [at a threshold of 1:250 for trisomy (T) 21 or 1:180 for T18] in one public hospital over a five-year period. All chromosome abnormalities were confirmed by invasive prenatal diagnosis (IPD) with karyotyping, with or without array comparative genomic hybridization. Using multivariable binary logistic regression analysis, we determined the parameters that were associated with common and atypical abnormalities. RESULTS: Overall, the prevalence of common and atypical abnormalities was 6.2 and 1.2%, respectively, and the prevalence increased with the risk of T21 by cFTS. In pregnancies with a risk of T21 > 1 in 100, a high risk of both T21 and T18, an increased NT, or a pregnancy-associated plasma A (PAPP-A) level <  0.2 multiple of medians (MoM), the prevalence of common abnormalities was 12.2, 64.7, 25.5 and 33.8%, respectively, while that of atypical abnormalities was 1.6, 3.9, 4.2, and 7.4%, respectively. In the multivariable binary logistic regression analysis, out of these four factors, only two (increased NT and PAPP_A <  0.2 MoM) were significant predictors of common and atypical abnormalities, respectively. Of all positive cFTS pregnancies, 50.4% did not have any of these four factors, and the prevalence of common and atypical abnormalities was 1.1 and 0.6%, respectively. There were three atypical abnormalities, all of which were mosaicism, and they were detected among women with IPD alone. The ages of these women were ≥ 35 years. All three pregnancies were continued after proper counseling. After giving birth, only one child had mild abnormalities, while the other two were phenotypically normal. CONCLUSIONS: Our study identified factors associated with common and atypical abnormalities after cFTS. These factors can be used to estimate the prior risk for these abnormalities to help with post-cFTS counseling in terms of choosing between cfDNA testing and IPD.

FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer.

Vascular endothelial growth factor (VEGF) has a central role in breast cancer development and progression, but the mechanisms that control its expression are poorly understood. Breast cancer tissue microarrays revealed an inverse correlation between the Forkhead transcription factor Forkhead box class O (FOXO)3a and VEGF expression. Using the lapatinib-sensitive breast cancer cell lines BT474 and SKBR3 as model systems, we tested the possibility that VEGF expression is negatively regulated by FOXO3a. Lapatinib treatment of BT474 or SKBR3 cells resulted in nuclear translocation and activation of FOXO3a, followed by a reduction in VEGF expression. Transient transfection and inducible expression experiments showed that FOXO3a represses the proximal VEGF promoter, whereas another Forkhead member, FOXM1, induces VEGF expression. Chromatin immunoprecipitation and oligonucleotide pull-down assays showed that both FOXO3a and FOXM1 bind a consensus Forkhead response element (FHRE) in the VEGF promoter. Upon lapatinib stimulation, activated FOXO3a displaces FOXM1 bound to the FHRE before recruiting histone deacetylase 2 (HDAC2) to the promoter, leading to decreased histones H3 and H4 acetylation, and concomitant transcriptional inhibition of VEGF. These results show that FOXO3a-dependent repression of target genes in breast cancer cells, such as VEGF, involves competitive displacement of DNA-bound FOXM1 and active recruitment of transcriptional repressor complexes.

Functional polymorphisms in the BRCA1 promoter influence transcription and are associated with decreased risk for breast cancer in Chinese women.

BACKGROUND: The BRCA1 gene is an important breast-cancer susceptibility gene. Promoter polymorphisms can alter the binding affinity of transcription factors, changing transcriptional activity and may affect susceptibility to disease. METHODS AND RESULTS: Using direct sequencing of the BRCA1 promoter region, we identified four polymorphisms c.-2804T-->C (rs799908:T-->C), c.-2265C-->T (rs11655505:C-->T), c.-2004A-->G (rs799906:A-->G) and c.-1896(ACA)(1)-->(ACA)(2) (rs8176071:(ACA)(1)-->(ACA)(2)) present in Hong Kong Chinese. Each polymorphism was studied independently and in combination by functional assays. Although all four variants significantly altered promoter activity, the c.-2265T allele had stronger binding than the C allele, and the most common mutant haplotype, which contains the c.-2265T allele, increased promoter activity by 70%. Risk association first tested in Hong Kong Chinese women with breast cancer and age-matched controls and replicated in a large population-based study of Shanghai Chinese, together totalling >3000 participants, showed that carriers of the c.-2265T allele had a reduced risk for breast cancer (combined odd ratio (OR) = 0.80, 95% CI 0.69 to 0.93; p = 0.003) which was more evident among women aged >or=45 years at first diagnosis of breast cancer and without a family history of breast cancer (combined OR = 0.75, 95% CI 0.61 to 0.91; p = 0.004). The most common haplotype containing the c.-2265T allele also showed significant risk association for women aged >or=45 years without a family history of breast cancer (OR = 0.64, 95% CI 0.46 to 0.89; p = 0.008). CONCLUSION: This comprehensive study of BRCA1 promoter polymorphisms found four variants that altered promoter activity and with the most significant contribution from c.-2265C-->T, which could affect susceptibility to breast cancer in the Chinese population. Its significance in other populations remains to be investigated.

Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indices.

AIMS: To assess the expression profile of the activated form of signal transducer and activator of transcription (Stat)3 in gestational trophoblastic disease (GTD) and correlate the findings with clinicopathological parameters. METHODS AND RESULTS: By immunohistochemistry, both cytoplasmic and nuclear expression of p-Stat3-Ser(727) was demonstrated in 88 trophoblastic tissues, including placentas and GTD. Nuclear immunoreactivity of p-Stat3-Ser(727) was significantly higher in hydatidiform mole (HM) (P < 0.001) and choriocarcinoma (P = 0.009) when compared with normal placentas. Placental site trophoblastic tumours (PSTT) and epithelioid trophoblastic tumours (ETT) also demonstrated higher nuclear p-Stat3-Ser(727) expression than their normal trophoblast counterparts. Higher p-Stat3-Ser(727) expression was confirmed in choriocarcinoma cell lines, JEG-3 and JAR, than in a normal trophoblast cell line, with both nuclear and cytoplasmic fractions demonstrated by immunoblotting. Spontaneously regressed HM showed significantly increased nuclear and cytoplasmic p-Stat3-Ser(727) immunoreactivity over those that developed gestational trophoblastic neoplasia (GTN) (P = 0.013, P = 0.039). There was a significant positive and inverse correlation between nuclear p-Stat3-Ser(727) immunoreactivity and apoptotic indices [terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling and M30 CytoDeath antibody] (P = 0.001, P < 0.001, Spearman's rho test) and Bcl-2 expression (P = 0.034), respectively. CONCLUSIONS: p-Stat3-Ser(727) plays a role in the pathogenesis of GTD, probably through the regulation of apoptosis. p-Stat3-Ser(727) immunoreactivity is a potential marker in predicting GTN in HM.

Oct4 is epigenetically regulated by methylation in normal placenta and gestational trophoblastic disease.

Oct4 is a transcription factor that plays a crucial role in maintaining pluripotency of embryonic stem cells. Down-regulation of Oct4 is associated with the differentiation of trophectoderm cell lineage, from which the normal placenta derives. We investigated the methylation and expression status of Oct4 in normal placenta and gestational trophoblastic disease (GTD) as attempts to investigate the role of Oct4 in the pathogenesis of GTD. By methylation-specific PCR, we observed both methylated and unmethylated Oct4 alleles in all 25 first trimester and 10 term placentas while 33% (18/54) of hydatidiform moles, and two choriocarcinoma cell line (JEG3 and JAR), only displayed methylated Oct4 allele. By quantitative TaqMan real-time PCR, Oct4 mRNA was significantly reduced in hydatidiform moles (P=0.04), JEG3 and JAR (P=0.024) when compared with normal placentas. Oct4 methylation was significantly correlated with Oct4 mRNA expression in placenta and GTD (P=0.012). Hypermethylation in minimal promoter and exon 1 region of Oct4 were confirmed in JEG3 and JAR by bisulfite genomic sequencing. The Oct4 mRNA expression in JEG3 and JAR increased after treatment with 5-aza-2'-deoxycytidine and/or trichostatin A. Our findings suggest that Oct4 is down-regulated by hypermethylation in normal placenta and GTD and such process is important in pathogenesis of GTD.

Characterization of the pathogenic mechanism of a novel BRCA2 variant in a Chinese family.

INTRODUCTION: Germline mutations of BRCA1 and BRCA2 account for the majority of hereditary breast cancers, many of which are classified as variants of unknown significance (VUS). We report the identification of a novel BRCA2 variant (c.7806-9T > G) in a Chinese family with multiple breast cancers and document it as a pathogenic mutation. METHODS: The proband in this family was diagnosed with breast cancer at age 50 with a strong family history of breast cancer. DNA and RNA were extracted from the blood of the proband and her family, and was used for BRCA gene mutation/deletion screening and RNA splicing analysis. RESULTS: BRCA2 c.7806-9T > G was identified in the proband, which was suggestive of a variant. This change was also found in two sisters of the proband with a history of breast cancer, as well as from the proband's maternal gastric cancer. The only sibling free of breast cancer did not carry the BRCA2 variant, thus demonstrating that the mutation segregates with the clinical phenotype in this family. RNA analysis on the proband blood sample revealed three aberrant splicing variants: c.7806_7874del, c.7806_7976del, and c.7806-8_7806-1ins. The latter causes a frameshift and creates a truncated protein, whilst the other two splicing variants resulted in shorter forms of the protein. CONCLUSIONS: The identified BRCA2 c.7806-9T > G [Genbank: DQ889340] was found to be pathogenic, based on aberrant splicing events resulting in the formation of truncated protein products. Thus, better understanding and classification of BRCA variants as neutral or disease causing has important implications for genetic counseling so that appropriate management can be given.

Single nucleotide polymorphisms of follicle-stimulating hormone receptor are associated with ovarian cancer susceptibility.

Epidemiological studies suggested that ovulation was associated with ovarian carcinogenesis. Follicle-stimulating hormone (FSH) played an important role in follicular development and was recently found to affect growth of ovarian epithelial cells. Single nucleotide polymorphisms (SNPs) Thr307Ala and Asn680Ser were two non-synonymous variations in the coding region of the FSH receptor (FSHR) gene. This hitherto first case-control study investigating the association between these two FSHR SNPs and the risk of ovarian cancer involved 202 histopathologically confirmed ovarian cancer patients and 266 age-matched cancer-free control subjects using restriction fragment length polymorphism assay and direct sequencing. Our results demonstrated that the 307Ala and 680Ser carriers were associated with significantly increased risk of developing serous and mucinous types of ovarian cancers (P < 0.0005, OR = 2.60, 95% CI = 1.56-4.34; and P < 0.0005, OR = 2.89, 95% CI = 1.73-4.84, adjusted for age, respectively) but not endometrioid and clear cell types. The two SNPs were found to be in modest linkage disequilibrium, D' = 0.804 and 0.701, r2 = 0.581 and 0.406 for the cancer and control groups, respectively. The major haplotype of 307Ala-680Ser was also associated with higher cancer risk (P = 0.033, OR = 1.39, 95% CI = 1.03-1.88), especially for the serous and mucinous carcinomas (P = 0.001, OR = 1.82, 95% CI = 1.27-2.60). Our results suggested that the two FSHR SNPs might affect the susceptibility of women to specific subtypes of ovarian cancer. Different types of ovarian cancer might adopt distinct carcinogenetic pathways. Such understanding may be important in selecting patients for ovulation induction therapy.

Caspase activity is downregulated in choriocarcinoma: a cDNA array differential expression study.

BACKGROUND: Placental trophoblast can be considered to be pseudomalignant tissue and the pathogenesis of gestational trophoblastic diseases remains to be clarified. AIMS: To examine the role of caspases 8 and 10, identified by differential expression, on trophoblast tumorigenesis. METHODS: cDNA array hybridisation was used to compare gene expression profiles in choriocarcinoma cell lines (JAR, JEG, and BeWo) and normal first trimester human placentas, followed by confirmation with quantitative real time polymerase chain reaction and immunohistochemistry. Caspase 10 and its closely related family member caspase 8 were analysed. RESULTS: Downregulation of caspase 10 in choriocarcinoma was detected by both Atlastrade mark human cDNA expression array and Atlastrade mark human 1.2 array. Caspase 10 mRNA expression was significantly lower in hydatidiform mole (p = 0.035) and chorioarcinoma (p = 0.002) compared with normal placenta. The caspase 8 and 10 proteins were expressed predominantly in the cytotrophoblast and syncytiotrophoblast, respectively, with significantly lower expression in choriocarcinomas than other trophoblastic tissues (p < 0.05). Immunoreactivity for both caspase 8 and 10 correlated with the apoptotic index previously assessed by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (p = 0.02 and p = 0.04, respectively) and M30 (p < 0.001 and p = 0.003, respectively) approaches. CONCLUSIONS: These results suggest that the downregulation of capases 8 and 10 might contribute to the pathogenesis of choriocarcinoma.

Id helix-loop-helix proteins are differentially expressed in gestational trophoblastic disease.

AIMS: To assess the expression of Id proteins in trophoblastic tissues and to correlate this with clinical parameters, proliferative and apoptotic indices as well as to related oncogene expression. METHODS AND RESULTS: Immunohistochemistry for Id1, Id2, Id3 and Id4 was performed on 83 trophoblastic tissues including 17 normal first-trimester placentas, seven term placentas, 47 hydatidiform moles (HM), and 12 spontaneous miscarriages. The four Id proteins were predominantly expressed in the villous and implantation site intermediate trophoblast. Expression of Id1 in HM was significantly higher than that in normal placenta (P = 0.0006) and spontaneous miscarriage (P = 0.0001) but did not correlate with subsequent development of gestational trophoblastic neoplasia (GTN). Id1 expression correlated with the proliferation index as assessed by MCM7 (P = 0.003) and Ki67 (P = 0.017) and with the apoptotic activity assessed by TUNEL (P = 0.001) and M30 CytoDeath antibody (P = 0.013). Moreover, the expression of Id1 correlated with the expression of p53 (P = 0.004), p21(WAF1) (/CIP1) (P = 0.003) but not with p16 (P = 0.107). CONCLUSIONS: Id proteins may play a role in the regulation of proliferative and apoptotic activity in trophoblastic tissue and are potentially useful in differentiating molar and non-molar gestation, but are not helpful in predicting GTN.

Replicative MCM7 protein as a proliferation marker in endometrial carcinoma: a tissue microarray and clinicopathological analysis.

AIMS: To assess, in tissue microarray (TMA), the proliferative activity of endometrial carcinoma using one of the minichromosome maintenance (MCM) proteins (MCM7), and to explore its potential value for prognosis. MCM proteins are essential for eukaryotic DNA replication and have recently been used to define the proliferative compartments in human tissues. METHODS AND RESULTS: Immunohistochemistry for MCM7 and Ki67 was performed on TMAs constructed from 212 cases of endometrial carcinoma. MCM7 and Ki67 expression was quantified according to the extent of nuclear staining. An analysis was carried out of the association between MCM7 expression and that of Ki67 and the clinicopathological characteristics of endometrial carcinoma. MCM7 and Ki67 immunoreactivity was clearly evident in the nuclei of tumour cells. MCM7 and Ki67 labelling indices in endometrial carcinomas correlated with each other (P < 0.001). A significant correlation existed between the MCM7 labelling index and histological grade (P = 0.008) and patients' age at diagnosis (P < 0.001). Well-differentiated carcinomas and younger patients had a lower MCM7 index. Poor survival was observed in patients with endometrial carcinoma with a high MCM7 index (P = 0.03) and MCM7 was found to be an independent prognostic factor by multivariate analysis (P = 0.04). The Ki67 labelling index correlated with histological grade (P = 0.01) but had no significant prognostic impact (P = 0.50). CONCLUSIONS: In this TMA study on endometrial carcinoma, MCM7 was found to be a more reliable and useful marker than Ki67 in assessing tumour proliferation and in the prognosis of patients.

Detection of hypermethylated genes in tumor and plasma of cervical cancer patients.

OBJECTIVE: The aim of this study is to investigate the prevalence of promotor CpG island methylation of the death-associated protein kinase (DAPK), p16, and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in both tumor and plasma samples of cervical cancers. METHODS: Methylation-specific PCR (MSP) was employed to detect promotor CpG island methylation of the DAPK, p16, and MGMT genes in 85 surgical tumor tissue samples and 40 pretreatment plasma samples from cervical cancers. RESULTS: Promotor CpG island methylation of DAPK, p16, and MGMT was detectable, respectively, in 60%, 28.2%, and 18.8% of cases of cervical tumor DNA; and in 40%, 10%, and 7.5% of cases of patients' plasma DNA. Moreover, at least one of the three methylated genes was detected in 75.3% (64/85) of cases of tumor and in 55% (22/40) of cases of plasma. Higher prevalence of methylation of DAPK was found in squamous cell carcinoma than in adenocarcinoma in both univariate and multivariate analysis. Methylation of p16 was significantly associated with that of MGMT in both univariate and multivariate analysis. The methylation pattern in primary tumor and plasma was found to be concordant in 23 patients with matched tissue and plasma samples. In cases positive for DAPK and p16 methylation in tumor, detection in the paired plasma sample was 64.3% (9/14) and 33.3% (3/9), respectively. CONCLUSIONS: Promotor CpG island methylation is a frequent event in cervical carcinogenesis. Detection of the methylated sequences in the circulation suggests that plasma DNA methylation warrants further study to determine its potential role in cancer management.

Methylation status and expression of E-cadherin and cadherin-11 in gestational trophoblastic diseases.

The clinical significance of cadherins in gestational trophoblastic diseases (GTD) is not fully understood. In this study, the expression of E-cadherin and cadherin-11 in 12 normal placentas, 32 cases of hydatidiform mole (HM) including 15 complete HMs and 17 partial HMs, and five choriocarcinomas was investigated by immunohistochemistry and correlated with follow-up of HMs. Cases with available frozen blocks were further analyzed by western blot and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Methylation of E-cadherin was investigated by methylation-specific PCR in six normal first trimester placentas, 19 HMs and their associated deciduas. E-cadherin expression was localized to cytotrophoblast and intermediate trophoblast whereas cadherin-11 was expressed in syncytiotrophoblast, intermediate trophoblast, and decidua. Immunoreactivity of E-cadherin was reduced in choriocarcinoma and complete HM when compared with that in normal first trimester placenta (P < 0.01, P = 0.04). Hypermethylation of E-cadherin was demonstrated in three complete HMs with the lowest level of E-cadherin. Compared with normal first trimester placenta, immunoreactivity of cadherin-11 was higher in complete HM (P = 0.02), but lower in choriocarcinoma (P = 0.02). Such differential expression was confirmed by western blot and semiquantitative RT-PCR. No obvious association was observed between the development of persistent trophoblastic disease with the expression of E-cadherin and cadherin-11.

Minichromosome maintenance protein 7 expression in gestational trophoblastic disease: correlation with Ki67, PCNA and clinicopathological parameters.

AIMS: To assess the proliferative activity of gestational trophoblastic disease (GTD) using one of the novel proliferation markers (MCM7) and to determine its prognostic value in hydatidiform mole (HM). METHODS AND RESULTS: Immunohistochemical staining for MCM7 was performed on 122 samples of paraffin-embedded trophoblastic tissues including 22 normal first-trimester placentas, 12 term placentas, 12 spontaneous miscarriages (SM), 21 partial moles (PM), 44 complete hydatidiform moles (CM), and 11 choriocarcinomas (CCA). The correlations between the proliferative indices assessed by MCM7, proliferating cell nuclear antigen (PCNA) and Ki67 (MIB1) immunoreactivity as well as clinical progress were assessed. MCM7 immunoreactivity was found predominantly in the nuclei of cytotrophoblast and intermediate trophoblast and decreased with placental maturation. MCM7 expression was highest in CCA, followed by CM, PM, normal first-trimester placenta, SM and term placenta. MCM7 index was significantly higher in PM and CM than in SM (P = 0.007, P < 0.001) but not between PM and CM themselves (P = 0.560). Eighteen of the 65 patients with HM developed persistent trophoblastic disease (PTD) requiring chemotherapy. There was no significant difference in MCM7 indices between the patients who developed PTD and those who did not (P = 0.312). MCM7 indices correlated well with Ki67 (P = 0.002) but not with PCNA (P = 0.054) indices. MCM7 indices demonstrated less variability than PCNA and Ki67 and may be a better proliferation marker than the latter two. CONCLUSIONS: We conclude that MCM7 is useful in differentiating molar and non-molar gestations but is not helpful in discriminating PM from CM or in predicting PTD.