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.

Aggregated α-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons.

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson's disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.

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.

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.

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.

Detection of cytochrome c oxidase activity and mitochondrial proteins in single cells.

Cytochrome c oxidase or mitochondrial respiratory chain complex IV is where over 90% of oxygen is consumed. The relationship between complex IV activity and mitochondrial proteins, which provides a guide to understanding the mechanisms in primary mitochondrial disorders, has been determined by histochemistry (activity) and immunohistochemistry in serial sections. In the central nervous system (CNS), mitochondrial activity and immunoreactivity have been determined in populations of cells in serial sections as capturing cells in more than one section is difficult. In this report we describe a method to determine complex IV activity in relation to mitochondrial proteins at a single cell level in the CNS. We performed complex IV histochemistry and immunohistochemistry consecutively in snap frozen sections. Although the product of complex IV histochemistry reduces the sensitivity of standard immunohistochemistry (secondary antibody and ABC method) the biotin-free Menapath polymer detection system (A. Menarini Diagnostics, Wokingham, UK) enables mitochondrial proteins to be detected following complex IV histochemistry. The co-occurring chromogens may then be separately visualised and analysed using multi-spectral imaging (Nuance system CRi, Woburn, MA). Our technique is applicable for exploring mitochondrial defects within single cells in a variety of CNS disorders and animal models of those diseases.

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.

Canonical WNT signalling determines lineage specificity in Wilms tumour.

Wilms tumours (WTs) have two distinct types of histology with or without ectopic mesenchymal elements, suggesting that WTs arise from either the mesenchymal or epithelial nephrogenic lineages. Regardless of the presence or absence of CTNNB1 mutations, nuclear accumulation of beta-catenin is often observed in WTs with ectopic mesenchymal elements. Here, we addressed the relationship between the WNT-signalling pathway and lineage in WTs by examining CTNNB1 and WT1 mutations, nuclear accumulation of beta-catenin, tumour histology and gene expression profiles. In addition, we screened for mutations in WTX, which has been proposed to be a negative regulator of the canonical WNT-signalling pathway. Unsupervised clustering analysis identified two classes of tumours: mesenchymal lineage WNT-dependent tumours, and epithelial lineage WNT-independent tumours. In contrast to the mesenchymal lineage specificity of CTNNB1 mutations, WTX mutations were surprisingly observed in both lineages. WTX-mutant WTs with ectopic mesenchymal elements had nuclear accumulation of beta-catenin, upregulation of WNT target genes and an association with CTNNB1 mutations in exon 7 or 8. However, epithelial lineage WTs with WTX mutations had no indications of active WNT signalling, suggesting that the involvement of WTX in the WNT-signalling pathway may be lineage dependent, and that WTX may have an alternative function to its role in the canonical WNT-signalling pathway.

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.

Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes.

Current models of Wilms tumour development propose that histological features of the tumours are programmed by the underlying molecular aberrations. For example, tumours associated with WT1 mutations arise from intralobar nephrogenic rests (ILNR), concur with CTNNB1 mutations and have distinct histology, whereas tumours with IGF2 loss of imprinting (LOI) often arise from perilobar nephrogenic rests (PLNR). Intriguingly, ILNR and PLNR are found simultaneously in Wilms tumours in children with overgrowth who have constitutional IGF2 LOI. We therefore examined whether the precursor lesions or early epigenetic changes are the primary determinant of Wilms tumour histology. We examined the histological features and gene expression profiles of IGF2 LOI tumours and WT1-mutant tumours which are associated with PLNR and/or ILNR. Two distinct types of IGF2 LOI tumours were identified: the first type had a blastemal-predominant histology associated with PLNR, while the second subtype had a myogenic histology, increased expression of mesenchymal lineage genes and an association with ILNR, similar to WT1-mutant tumours. These ILNR-associated IGF2 LOI tumours also showed signatures of activation of the WNT signalling pathway: differential expression of beta-catenin targets (MMP2, RARG, DKK1) and WNT antagonist genes (DKK1, WIF1, SFRP4). Unexpectedly, the majority of these tumours had CTNNB1 mutations, which are normally only seen in WT1-mutant tumours. The absence of WT1 mutations in tumours with IGF2 LOI indicated that CTNNB1 mutations occur predominantly in tumours arising from ILNR independent of the presence or absence of WT1 mutations. Thus, even though these two classes of tumours with IGF2 LOI have the same underlying predisposing epigenetic error, the tumour histology and the gene expression profiles are determined by the nature of the precursor cells within the nephrogenic rests and subsequent CTNNB1 mutations.

Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer.

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P < 0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.

Do mitochondrial DNA mutations have a role in neurodegenerative disease?

A decline in mitochondrial function has long been shown to exist in neurodegenerative disease. Whether this decline is a secondary consequence of other factors or whether it causes the eventual death of a cell is unknown. In this review, we will discuss some of the major evidence surrounding mitochondrial DNA mutations leading to mitochondrial dysfunction in neurodegenerative disease and discuss their possible role in neurodegeneration.

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.

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.

Chronic secondary hypersensitivity of dorsal horn neurones following inflammation of the knee joint.

Intra-articular injection of Freund's complete adjuvant (FCA) into the rat knee joint produces a swelling of the joint and long lasting hypersensitivity. In this study we have used this model and in vivo electrophysiology to investigate the time course of spinal changes underlying chronic secondary hypersensitivity, by stimulating the ankle joint (an area outside the site of primary hypersensitivity), and have compared the results with behavioural data from the same population of animals at 4-8, 13-17 and 55-59 days following FCA injection. The magnitude of responses and the proportion of dorsal horn neurones receiving inputs from A beta- A delta- and C-fibre afferents were monitored. At all time points, there was a significant increase in the ongoing activity of deep dorsal horn neurones when compared to nai ve rats, correlating well with the behavioural hypersensitivity. Both the magnitude of neuronal responses, and the proportion of neurones responding to electrical or mechanical stimulation in an area of secondary hypersensitivity, were significantly increased 4-8 and 13-17 days following FCA injection. However, while there was still behavioural hypersensitivity at 55-59 days there was a substantial decline in the responses to mechanical stimulation and A-fibre responses to electrical stimulation, although the proportion of neurones responding in the C-fibre latency remained elevated. These results suggest that the behavioural hypersensitivity is due to hyperexcitability at the level of the dorsal horn reflected as an increase of both C-fibre responses and spontaneous activity.

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.

Imprinting, expression, and localisation of DLK1 in Wilms tumours.

BACKGROUND: Loss of imprinting (LOI) of the H19/IGF2 domain is a common feature of Wilms tumour. The GTL2/DLK1 domain is also imprinted and is structurally similar to H19/IGF2. The question arises as to whether DLK1 also undergoes LOI in Wilms tumour, or whether the LOI mechanism is restricted to the H19/IGF2 domain. AIM: To investigate the imprinting status of DLK1 in Wilms tumours with IGF2 LOI. The cellular localisation of DLK1 in the tumours was also examined. METHODS: DLK1 expression was measured by quantitative real time polymerase chain reaction (Q-PCR) in 30 Wilms tumours that had previously been classified according to whether they had IGF2 LOI, WT1 mutations, or 11p15.5 loss of heterozygosity. Allele specific expression of DLK1 was examined by direct sequencing using a DLK1 exon 5 polymorphism (rs1802710). Immunohistochemical analysis of DLK1 was performed on 13 tumours and two intralobar nephrogenic rests, in addition to two fetal kidneys and one fetal skeletal muscle sample. RESULTS: Ten of 30 tumours were heterozygous for rs1802710 and all tumours showed retention of imprinting of DLK1. Moderate to high expression of DLK1 was detected by Q-PCR in nine of 13 tumours with myogenic differentiation. Immunohistochemical expression of DLK1 was detected in the myogenic elements. CONCLUSION: LOI does not occur at the GTL2/DLK1 domain in Wilms tumour. This finding suggests that LOI at 11p15.5 does not reflect non-specific disruption of a shared imprinting mechanism. DLK1 expression in Wilms tumour might reflect the presence of myogenic differentiation, rather than an alteration of its imprinting status.