Operating room-to-incision interval and neonatal outcome in emergency caesarean section: a retrospective 5-year cohort study.

We conducted a 5-year retrospective cohort study on women undergoing caesarean section to investigate factors influencing the operating room-to-incision interval. Time-to-event analysis was performed for category-1 caesarean section using a Cox proportional hazards regression model. Covariates included: anaesthetic technique; body mass index; age; parity; time of delivery; and gestational age. Binary logistic regression was performed for 5-min Apgar score ≥ 7. There were 677 women who underwent category-1 caesarean section and who met the entry criteria. Unadjusted median (IQR [range]) operating room-to-incision intervals were: epidural top-up 11 (7-17 [0-87]) min; general anaesthesia 6 (4-11 [0-69]) min; spinal 13 (10-20 [0-83]) min; and combined spinal-epidural 24 (13-35 [0-75]) min. Cox regression showed general anaesthesia to be the most rapid method with a hazard ratio (95%CI) of 1.97 (1.60-2.44; p < 0.0001), followed by epidural top-up (reference group), spinal anaesthesia 0.79 (0.65-0.96; p = 0.02) and combined spinal-epidural 0.48 (0.35-0.67; p < 0.0001). Underweight and overweight body mass indexes were associated with longer operating room-to-incision intervals. General anaesthesia was associated with fewer 5-min Apgar scores ≥ 7 with an odds ratio (95%CI) of 0.28 (0.11-0.68; p < 0.01). There was no difference in neonatal outcomes between the first and fifth quintiles for operating room-to-incision intervals. General anaesthesia is associated with the most rapid operating room-to-incision interval for category-1 caesarean section, but is also associated with worse short term neonatal outcomes. Longer operating room-to-incision intervals were not associated with worse neonatal outcomes.

Biallelic DICER1 mutations occur in Wilms tumours.

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.

Microanatomy of the male and female reproductive tracts in the long-tailed butterfly ray Gymnura poecilura, an elasmobranch with unusual characteristics.

The anatomy of the male and female reproductive systems was investigated in the long-tailed butterfly ray Gymnura poecilura using gross observation and light microscopy. The testes are highly asymmetrical, to the extent that only the left testis is functional and the right testis is completely absent. Both of the male genital ducts are present and symmetrical, although spermatozoa only occur in the left duct. The genital ducts are straight and unconvoluted, with regular incomplete internal partitions throughout. Females do not possess a right ovary, nor do the oviducal glands exhibit distinct club and papillary zones, and the baffle zone lacks baffle plates. In all sections of the gland, the tubules display different secretory activities depending on the proximity to the gland lumen. The gland produces a thin egg membrane that encases each egg individually, while the endometrium is formed into trophonemata.

Mitochondrial DNA deletions and depletion within paraspinal muscles.

AIMS: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age-related histopathological changes were previously reported. METHODS: Cervical and lumbar paraspinal muscles were obtained peri-operatively from 13 patients and from six post mortem control cases (age range 18-82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory-deficient muscle fibres (lacking complex IV with intact complex II activity). Real-time polymerase chain reaction, long-range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. RESULTS: The density of respiratory-deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory-deficient (range 0-10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory-deficient fibres with and without mtDNA deletions or mtDNA depletion. CONCLUSIONS: Paraspinal muscles appeared to be particularly susceptible to age-related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age-related postural abnormalities.

Mosaic and polymorphic imprinting of the WT1 gene in humans.

We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.

Constitutional relaxation of insulin-like growth factor II gene imprinting associated with Wilms' tumour and gigantism.

We have examined the imprinting of the insulin-like growth factor II gene (IGF2) in ten normal kidney samples from children with renal embryonal neoplasms. In kidney samples from nine children with normal growth profiles, IGF2 mRNA was transcribed monoallelically, consistent with normal imprinting of the gene. But in one child who had generalized somatic overgrowth, IGF2 was transcribed from both alleles in her kidney, peripheral blood leukocytes and Wilms' tumour. These findings suggest that a defect in genomic imprinting can occur constitutionally, leading to growth abnormalities and predisposition to Wilms' tumour.

Somatic overgrowth associated with overexpression of insulin-like growth factor II.

Overexpression of the normally imprinted fetal insulin-like growth factor II (IGF2) has been implicated in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). We have detected constitutional relaxation of imprinting of IGF2 in four children with somatic overgrowth who do not show diagnostic features of BWS. Three children showed constitutional abnormalities of H19 methylation. All four children showed nephromegaly and two developed Wilms' tumors. Gene methylation is known to be associated with gene silencing, and three children showed constitutional abnormalities of H19 gene methylation. Disruption of H19 methylation, and concomitant relaxation of IGF2 imprinting, provides another mechanism that can increase IGF2 expression in children with overgrowth. The accumulated data on normal and pathologic IGF2 expression are now sufficient to define an entity, "IGF2 overgrowth disorder," of which BWS may be one extreme manifestation. These findings have broad implications for the characterization of idiopathic overgrowth.

Slow proliferation as a biological feature of colorectal cancer metastasis.

BACKGROUND: We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential. METHODS: Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases. RESULTS: Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT-PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse. CONCLUSION: Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts.

Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.

Prey selection and functional response of juvenile lemon sharks Negaprion brevirostris.

Prey preference of juvenile lemon sharks Negaprion brevirostris was examined in experiments conducted in field-based pens where the fish were offered meals of live teleosts: grey snapper Lutjanus griseus and yellow fin majorra Gerres cinereus in ratios of L. griseus:G. cinereus varying from 10:0 to 2:8. Relative electivity indices for each treatment showed that N. brevirostris preyed on G. cinereus rather than L. griseus. They showed a prey preference relating to density of G. cinereus (type II functional response) but not to L. griseus.

Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilms' tumor cells.

Children with associated Wilms' tumor, aniridia, genitourinary malformations, and mental retardation (WAGR syndrome) frequently have a cytogenetically visible germ line deletion of chromosomal band 11p13. In accordance with the Knudson hypothesis of two-hit carcinogenesis, the absence of this chromosomal band suggests that loss of both alleles of a gene at 11p13 causes Wilms' tumor. Consistent with this model, chromosomes from sporadically occurring Wilms' tumor cells frequently show loss of allelic heterozygosity at polymorphic 11p15 loci, and therefore it has been assumed that allelic loss extends proximally to include 11p13. We report here that in samples from five sporadic Wilms' tumors, allelic loss occurred distal to the WAGR locus on 11p13. In cells from one tumor, mitotic recombination occurred distal to the gamma-globin gene on 11p15.5. Thus, allelic loss in sporadic Wilms' tumor cells may involve a second locus on 11p.

Genome wide expression profiling identifies genes associated with colorectal liver metastasis.

Tumour cells have to undergo gene expression changes in order to metastasise and adapt to a new site. We investigated these changes in liver metastases of colorectal cancer by using genome-wide microarray analysis to profile the expression of 48 primary tumours and 28 liver metastases. Statistical analysis of these expression profiles using the significance analysis of microarrays (SAM) method identified 778 genes differentially expressed between primary tumours and metastases. Gene ontology analysis revealed that genes associated with tissue remodelling and immune response were upregulated in metastases relative to primary tumours, whereas genes associated with proliferation and oxidative phosphorylation were downregulated. Quantitative real-time PCR confirmed the differential expression of selected genes, osteopontin, versican, ADAM17, CKS2, PRDX1, CXCR4, CXCL12, and LCN2. The upregulation of genes associated with tissue remodelling and immune response are likely to be involved in metastatic invasion and colonisation of the new site because these genes can promote tumour progression. However, downregulation of genes associated with proliferation suggests that proliferation in metastases was reduced relative to primary tumours.

Vitamin D deficiency in a multinational refugee population.

BACKGROUND: Populations with increased skin pigmentation who have migrated to countries of high latitude are at increased risk of low vitamin D. This study aimed to determine the prevalence of low vitamin D among the refugee population arriving in New Zealand. METHODS: An audit of all refugees arriving at the national refugee resettlement centre from May 2004 to May 2005 was carried out. Serum 25-hydroxyvitamin D3 levels were measured and defined as normal (50-150 nmol/L) or low, with low subdivided into insufficient (25 to <50 nmol/L) and deficient (<25 nmol/L). Whether vitamin D status varied with age and sex was determined. RESULTS: Vitamin D was measured in 869 (99%) of the refugees and was low in 470 (54%, 95% confidence interval (CI) 51-57%). It was insufficient in 323 (37%, 95%CI 34-41%) and deficient in 147 (17%, 95%CI 15-20%). Female sex was associated with at least a 10 times increased risk of vitamin D deficiency (relative ratio 13.93, 95%CI 10.15-17.96). Women aged between 17 and 45 years and men aged 46 years and more were at greatest risk. CONCLUSION: Poor vitamin D status is prevalent among refugees arriving in New Zealand. Women, particularly those of child-bearing age are at greatest risk. Screening and ongoing surveillance for vitamin D deficiency should be considered for all recent refugee immigrants to New Zealand.

Good outcome in HIV-infected refugees after resettlement in New Zealand: population study.

BACKGROUND: The aims of this study were to determine the clinical characteristics on arrival and the subsequent clinical outcome of HIV-infected UN quota refugees who settled in New Zealand during the last 11 years and to estimate their rate of HIV transmission. METHODS: A population study was conducted. Data were provided by the Mangere Refugee Resettlement Centre, the infectious disease physicians caring for the subjects, the New Zealand AIDS Epidemiology Group and laboratories carrying out HIV viral load assays. RESULTS: One hundred of 7732 (1.3%) UN quota refugees were HIV positive; mean age 30 years, 56% were men, median initial CD4 count was 320 (range 20-1358). HIV infection was most commonly acquired by heterosexual intercourse (74%). The median follow up was 5.0 years (range 1 month to 9.7 years). Five died and 15 subjects had 16 AIDS-defining illnesses, most commonly tuberculosis (n = 10). Sixty subjects commenced highly active antiretroviral therapy of whom 36/59 (61%) had an undetectable HIV viral load after 1 year of treatment. None of the six children born to HIV-infected women in New Zealand were infected. There were two known cases of horizontal transmission of HIV infection. CONCLUSION: Although HIV-infected quota refugees often have to overcome severe social, cultural and financial handicaps, their clinical outcome is generally very good, with response rates to highly active antiretroviral therapy that are similar to other patient groups. Furthermore, they have not been a significant source of transmission of HIV infection after resettlement in New Zealand.

Insulin-like growth factor II and WT1 transcript localization in human fetal kidney and Wilms' tumor.

In situ hybridization was used to examine, in parallel, the localization of insulin-like growth factor II (Igf2) and WT1 transcripts in normal fetal kidney and Wilms' tumor. The expression of Igf2 and WT1 transcripts in the fetal kidney is almost complementary in both the epithelial and stromal cell lineages derived from the undifferentiated metanephrogenic blastema. The patterns of transcription of Igf2 in three Wilms' tumors appeared to be perturbed as compared to the normal fetal kidney. In these tumors Igf2 transcripts were detected in structures that are developmentally equivalent to the renal vesicle, which in the normal kidney do not contain Igf2 transcripts. These results suggest that Wilms' tumors arise from an alteration in the regulation of Igf2 mRNA synthesis.

Nonlinkage of 16q markers to familial predisposition to Wilms' tumor.

Wilms' tumor (WT), a childhood cancer of the kidney, occurs in both familial and sporadic forms. Chromosome 11 genes have been implicated in the etiology of WT, and mutations in a gene at chromosomal band 11p13, WT1, have been identified in a few WT cases. However, 11p13 has been excluded as the site of the predisposition mutation segregating in several large WT families, which implies the existence of a non-11p familial predisposition gene. Recently, loss of heterozygosity for 16q markers located between chromosomal bands 16q13 and 16q22 has been reported in approximately 20% of sporadic Wilms' tumors. To determine if this region of 16q harbors the non-11p familial WT gene, a genetic linkage study of five WT families was undertaken. Using multipoint analyses, we ruled out genetic linkage of familial WT predisposition to 16q.

Human p57(KIP2) defines a new imprinted domain on chromosome 11p but is not a tumour suppressor gene in Wilms tumour.

Mouse p57(Kip2) arrests cells in G1 by functioning as a strong inhibitor of several G1 cyclin/Cdk complexes (Lee et al., 1995; Matsuoka et al., 1995; Sherr and Roberts, 1995). Human p57(KIP2) has been suggested to be a tumour suppressor gene because of its location at 11p15.5 which frequently undergoes maternal allele LOH in several types of cancer (Matsuoka et al., 1995; Sherr and Roberts, 1995; Hatada and Mukai, 1995). This suggestion was supported by the discovery that mouse p57(Kip2) is imprinted with expression from only the maternally inherited allele (Hatada and Mukai, 1995). Interestingly, p57(KIP2) is several hundred kilobases from the imprinted H19 and IGF2 genes which are involved in growth regulation (Hoovers et al., 1995). Here we show that human p57(KIP2) is imprinted with expression from the maternal allele. However, unlike the mouse, the imprinting is incomplete with significant expression from the paternal allele depending on the tissue examined. We have also shown that the imprinting of p57(KIP2) occurs independently of the H19/IGF2 domain and thus there must be at least two imprinted domains in 11p15.5. Finally, by examining Wilms tumours we have shown that following maternal 11p LOH, p57(KIP2) was expressed from the paternal allele. Therefore, p57(KIP2) cannot function as an imprinted tumour suppressor gene, at least in Wilms tumour.

Cloning of novel Wilms tumor gene (WT1) cDNAs; evidence for antisense transcription of WT1.

WT1 is a tumor suppressor gene that has been implicated in Wilms tumor, and is expressed in cells of mesodermal origin. The Wit-1 gene is located approximately 2 kb from the WT1 gene, and is expressed coordinately with WT1. WT1 and Wit-1 are bi-directionally transcribed from the same promoter region. We have screened a human fetal kidney cDNA library to identify novel WT1 cDNA clones. Here we report the cloning of cDNA clones which span part of intron 1 of WT1, exon 1, upstream sequences between WT1 and Wit-1 and part of the Wit-1 gene. Northern blot and RNAase protection analysis using subcloned fragments of the cDNAs corresponding to regions from within intron 1 of WT1 suggest that a 7-10 Kb RNA is expressed in human fetal kidney, which overlaps with WT1 and is transcribed in the same direction as Wit-1.

Exclusion of the Wilms tumour gene (WT1) promoter as a site of frequent mutation in Wilms tumour.

WT1 is a tumour suppressor gene expressed in a specific temporal and spatial pattern in the developing kidney. Up to 15% of Wilms tumours have point mutations in the WT1 gene coding sequence. We have now investigated whether mutations in the WT1 promoter could be associated with loss of control WT1 expression and subsequent Wilms tumour formation. Using single-strand conformational polymorphism (SSCP) analysis we analysed 39 sporadic Wilms tumours for WT1 promoter mutations. We found six linked common sequence polymorphisms and two unlinked less frequent polymorphisms which allowed us to identify four tumours with loss of heterozygosity but none with point mutations, small deletions, insertions or rearrangements. We therefore conclude that WT1 promoter mutations are unlikely to play an important role in Wilms tumorigenesis.

Relaxation of IGF2 imprinting in Wilms tumours associated with specific changes in IGF2 methylation.

Relaxation of IGF2 imprinting occurs in Wilms tumours and many other cancers, but the mechanism of loss of imprinting (LOI) remains unknown. To investigate the role of altered DNA methylation in LOI, we examined the pattern of methylation of the human insulin-IGF2 region in Wilms tumours and the normal kidney. The analysis included regions homologous to three 'differentially methylated regions' of the mouse Igf2 gene (dmrs 0, 1 and 2). In tumours displaying normal IGF2 imprinting, and in the normal kidney, maternal allele-specific DNA methylation was identified spanning exons 2 and 3. This region is homologous to dmr 0, a site of maternal-specific differential methylation in the mouse. In Wilms tumours with relaxed imprinting or 11p15.5 LOH this region was unmethylated. No other differential methylation was identified. In particular, two sites of paternal methylation in the mouse (dmrs 1 and 2), and all three imprinted IGF2 promoters were not methylated in the kidney or in Wilms tumours. We postulate that LOI in Wilms tumours is associated with loss of maternal allele-specific methylation from a region located upstream of the imprinted IGF2 promoters. This region may contain cis acting sequences that coordinately influence imprinting.