Molecular data reveal a cryptic species within the Culex pipiens mosquito complex.

The Culex pipiens mosquito complex is a group of evolutionarily closely related species including C. pipiens and Culex quinquefasciatus, both infected by the cytoplasmically inherited Wolbachia symbiont. A Wolbachia-uninfected population of C. pipiens was however described in South Africa and was recently proposed to represent a cryptic species. In this study, we reconsidered the existence of this species by undertaking an extensive screening for the presence of Wolbachia-uninfected C. pipiens specimens and by characterizing their genetic relatedness with known members of the complex. We first report on the presence of Wolbachia-uninfected specimens in several breeding sites. We next confirm that these uninfected specimens unambiguously belong to the C. pipiens complex. Remarkably, all uninfected specimens harbour mitochondrial haplotypes that are either novel or identical to those previously found in South Africa. In all cases, these mitochondrial haplotypes are closely related, but different, to those found in other C. pipiens complex members known to be infected by Wolbachia. Altogether, these results corroborate the presence of a widespread cryptic species within the C. pipiens species complex. The potential role of this cryptic C. pipiens species in the transmission of pathogens remains however to be determined. The designation 'Culex juppi nov. sp.' is proposed for this mosquito species.

Selective use of endoscopic ultrasound in the evaluation of carcinomas of the pancreatic head.

BACKGROUND: The aims of this study were to assess the role of endoscopic ultrasound (EUS) in the evaluation of adenocarcinoma of the head of the pancreas in cases of diagnostic dilemma and to determine the strength of agreement between perceived pre-operative stage as determined by computerised tomography (CT) and EUS and histopathological stage. METHODS: Patients undergoing pancreatic EUS were identified from a computerised radiology database. The strengths of agreement between the radiological and histopathological stages were determined by the weighted kappa (Kw) statistic. RESULTS: Fifty-eight patients were identified. Of 37 patients with a pancreatic head mass on prior imaging, 32 had a diagnosis of adenocarcinoma confirmed by EUS, as did 11 of 21 patients with suspicious pancreatic head lesions. Twenty-five of 43 patients were deemed to have resectable carcinomas, and 2 patients had resectable mucinous lesions. In comparing CT and EUS in the 25 patients undergoing resection, the Kw for T and N stages was 0.250 (p = 0.05) and -0.080 (p = 0.288), respectively, for CT, compared with 0.738 (p = 0.0001) and 0.606 (p = 0.0001), respectively, for EUS. CONCLUSIONS: EUS was effective in assessing the resectability of pancreatic head adenocarcinomas. Furthermore, EUS held a significant 3-fold advantage over CT with regard to T stage and an even higher significant advantage with regard to N stage.

Prospective multicentre randomised controlled trial of early enteral nutrition for patients undergoing major upper gastrointestinal surgical resection.

BACKGROUND & AIMS: The evidence in support of Early Enteral Nutrition (EEN) after upper gastrointestinal surgery is inconclusive. The aim of this study was to determine if EEN improved clinical outcomes and shortened length of hospital stay. METHODS: Open, prospective multicentre randomised controlled trial within a regional UK Cancer Network. One hundred and twenty-one patients with suspected operable upper gastrointestinal cancer (54 oesophageal, 38 gastric, 29 pancreatic) were studied. Patients were randomised to receive EEN (n = 64) or Control management postoperatively (nil by mouth and IV fluid, n = 57). Analysis was based on intention-to-treat and the primary outcome measure was length of hospital stay. RESULTS: Operative morbidity was less common after EEN (32.8%) than Control management (50.9%, p = 0.044), due to fewer wound infections (p = 0.017), chest infections (p = 0.036) and anastomotic leaks (p = 0.055). Median length of hospital stay was 16 days (IQ = 9) after EEN compared with 19 (IQ = 11) days after Control management (p = 0.023). CONCLUSIONS: EEN was associated with significantly shortened length of hospital stay and improved clinical outcomes. These findings reinforce the potential benefit of early oral nutrition in principle and as championed within enhanced recovery after surgery programmes, and such strategies deserve further research in the arena of upper GI surgery.

Initial impact of Australia's National Bowel Cancer Screening Program.

OBJECTIVE: To examine the initial impact of the National Bowel Cancer Screening Program (NBCSP), which was launched in May 2006 and offers faecal occult blood testing to Australians aged 55 or 65 years. DESIGN AND SETTING: Review of data on colorectal cancer (CRC) cases diagnosed between May 2006 and June 2008 from a prospective database used at 19 Australian hospitals, linked and analysed by BioGrid Australia. MAIN OUTCOME MEASURES: Number of CRC cases detected through the NBCSP or symptomatic presentation, and differences by sex, stage at diagnosis, tumour location and level of socioeconomic disadvantage. RESULTS: 1628 cases of CRC were identified; 1268 had information on the patients' test status as part of the NBCSP, and 40 of these (3.2%) were recorded as being detected by the NBCSP. Of 75 CRC cases in patients aged 55 or 65 at diagnosis, 22 were NBCSP-detected. Overall, there was no difference in NBCSP-detected cases by sex. The distribution of tumour locations was similar between NBCSP-detected cases and symptomatic cases, but NBCSP-detected cancers were diagnosed at an earlier stage than symptomatic cancers (stage I, 40% v 14%; stage IV, 3% v 15%, respectively). Of patients diagnosed through the NBCSP, 63% were from areas of least socioeconomic disadvantage (deciles 8-10) and 18% were from the most disadvantaged areas (deciles 1-4) (P=0.0375). CONCLUSION: Initiation of the Australian NBCSP has had a measurable impact on CRC stage at diagnosis, and an improvement in survival would be anticipated. The lower uptake among people from disadvantaged areas is of concern.

Endoscopic ultrasound reliably identifies chronic pancreatitis when other imaging modalities have been non-diagnostic.

CONTEXT: There are classical radiological features for the diagnosis of chronic pancreatitis when utilising endoscopic retrograde cholangiopancreatography (ERCP), magnetic resonance cholangiopancreatography (MRCP) or computed tomography (CT), however, not all patients exhibit these features despite convincing clinical histories, which may result in diagnostic delay. OBJECTIVE: The aim of this study was to assess the use of endoscopic ultrasound (EUS) in the diagnosis of chronic pancreatitis when other imaging modalities had not yielded a diagnosis. METHODS: All patients undergoing pancreatic EUS between January 1996 and December 2004 were identified from the radiology computerised database. Sixteen patients with a clinical diagnosis of chronic pancreatitis (10 males, 6 females; mean age 53+/-4 years) underwent EUS after normal conventional imaging. Patients were then followed clinically until December 2007. RESULTS: Thirteen patients exhibited features of chronic pancreatitis not identified by other modalities, which included duct dilatation (n=8), calcification (n=7); parenchymal change (n=6), irregular undilated ducts (n=2), pancreatic ductal calculi (n=1), and fine calcification (n=1). Of the remaining 3 patients, a diagnosis of autoimmune pancreatitis was made in one, in another there was a pancreatic duct stricture of uncertain origin that was stented, and in only one case was no diagnosis established. All 13 patients with an EUS diagnosis of chronic pancreatitis subsequently underwent a repeat CT scan for surveillance of their disease and in all cases, the CT scans subsequently demonstrated evidence of chronic pancreatitis indicating radiological progression. No new pancreaticobiliary diagnoses were established during this period. CONCLUSIONS: EUS is a useful diagnostic tool confirming the diagnosis of chronic pancreatitis in 13 of 16 cases where histories were suspicious of chronic pancreatitis, and providing an alternative diagnosis in another two cases. EUS should be considered an important tool for diagnosis of chronic pancreatitis and should be used when cross-sectional imaging is non-diagnostic.

Does endoscopic ultrasound have anything to offer in the diagnosis of idiopathic acute pancreatitis?

OBJECTIVE: The aim of this study was to investigate the role of EUS where other investigative techniques had failed to identify the cause of biochemically proven acute pancreatitis. SETTING: All biliary EUS examinations performed between January 2000 and December 2004 were identified from the radiology computerised database. PATIENTS: Forty-two patients (25 male, 17 female; mean age: 53+/-3.2 years) with negative prior radiological investigations underwent EUS. MAIN OUTCOME MEASURES: Prior and later radiological investigations, hospital readmission, and the need for further surgical intervention were also analysed. RESULTS: EUS was normal in 17 patients (40.5%) and demonstrated signs of recent acute pancreatitis but no other aetiological factor in 8 patients (19.0%). Cholelithiasis or microlithiasis was identified in 9 patients (21.4%), combined gallstones/microlithiasis and choledocholithiasis in was seen in 6 patients (14.3%). In one patient (2.4%), calculi were seen in the common bile duct but not the gallbladder. In a further case with recurrent acute pancreatitis (2.4%), chronic pancreatitis was diagnosed on EUS. All patients with common bile duct stones underwent ERCP and sphincterotomy, and stones were universally confirmed. One patient with gallbladder calculi alone required an ERCP after developing jaundice whilst awaiting cholecystectomy. CONCLUSIONS: EUS provided additional diagnostic information in 17 of the 42 patients (40.5%). Moreover, exclusion of gallstones/microlithiais is also important as it facilitates a search for other causes of pancreatitis. In conclusion, most cases of cholelithiasis can be diagnosed with standard imaging modalities but when these fail to identify a cause, EUS has an important role to play.

Upregulation of tumour endothelial marker-8 by interleukin-1beta and its impact in IL-1beta induced angiogenesis.

Tumour endothelial marker-8 (TEM-8) has been found to be selectively upregulated in tumour-associated endothelial cells and is implicated in tumour specific angiogenesis. Specific factors, indigenous to tissues and tumours that regulate the TEM-8 mechanism in angiogenesis are not defined. We report for the first time that interleukin-1beta induces the expression of TEM-8 in endothelial cells. Human vascular endothelial cells (HECV), which strongly express IL-1beta receptor (as revealed by RT-PCR, Western blotting), increased the level of TEM-8 expression following stimulation with IL-1beta (as revealed by conventional and quantitative RT-PCR). Using a newly developed antibody to human TEM-8, we have further demonstrated that IL-1beta significantly raised the level of TEM-8 at the protein level, as revealed by Western blotting. In vitro tubule forming assay, revealed that IL-1beta significantly induced the formation of capillary-like tubules from the HECV cells, accompanied by an increase in TEM-8 expression. It is concluded that IL-1beta is a powerful regulator of the expression of TEM-8 in vascular endothelial cells. Our results suggest an important pathway through which IL-1beta regulates tumour-associated angiogenesis.

Helicobacter pylori in children is strongly associated with poverty.

BACKGROUND AND AIMS: To examine the association between prevalence of H. pylori colonisation and social deprivation in a sample of children investigated in hospital. METHODS: A retrospective review of the hospital records of all children (n = 626) who underwent 13C-urea breath testing for suspected H. pylori infection at the Royal Hospital for Sick Children, Glasgow, Scotland between August 1995 to December 2002 was performed. Prevalence of H. pylori colonisation was measured by the 13C-urea breath test and socioeconomic status was measured by the Carstairs and Morris index of deprivation. RESULTS: The overall prevalence of H. pylori was 26%. There was a highly significant positive association between H. pylori colonisation and poor socioeconomic status (p < 0.000). The prevalence of colonisation was significantly higher in children from the most deprived areas (DepCat 6 and 7; 34%) compared to children from intermediate (DepCat 3 to 5; 22%) and the most affluent areas (DepCat 1 and 2; 16%) (p < 0.0001). CONCLUSIONS: Socioeconomic deprivation in childhood is associated with a high prevalence of H. pylori colonisation. While the incidence of H. pylori may be declining, it remains common in poor families. If the prevalence of H. pylori (26%) in this selected group reflects that of the population at large, then over 9000 (5%) of Glasgow's children are at risk of this preventable disease. In a city where the majority of adults are colonised with H. pylori, colonisation in early life adds to the burden of health risks to which deprived children are exposed.

Maternal docosahexaenoic acid supplementation during pregnancy and visual evoked potential development in term infants: a double blind, prospective, randomised trial.

AIM: To test the hypothesis that maternal docosahexaenoic acid (DHA) supplementation during pregnancy enhances maturation of the visual evoked potential (VEP) in healthy term infants. METHODS: One hundred women were supplemented with either fish oil capsules rich in DHA (n = 50) or placebo capsules (n = 50) from week 15 of pregnancy until delivery. Total fatty acids in red blood cells and plasma were measured at weeks 15, 28, and 40 of pregnancy and at delivery in umbilical cord blood. Infant visual pathway development was assessed using VEPs recorded to flash stimuli shortly after birth and to both flash and pattern-reversal stimuli at 50 and 66 weeks post-conceptional age (PCA). RESULTS: Maternal supplementation did not significantly elevate the level of DHA in umbilical cord blood. Moreover, there were no significant differences in any of the VEP measures observed between supplementation groups. However, maturity of the pattern-reversal VEP at 50 and 66 weeks PCA was associated with DHA status of the infants at birth. Infants with higher DHA status, both as a concentration and as a percentage of total fatty acids, showed shorter P100 peak latencies of the pattern-reversal VEP than those with lower DHA status. CONCLUSIONS: Maternal DHA supplementation during pregnancy did not enhance VEP maturation in healthy term infants. However, these results show an association between the DHA status of infants at term and early postnatal development of the pattern-reversal VEP, suggesting that DHA status itself may influence maturation of the central visual pathways.

Extent of mesorectal invasion is a prognostic indicator in T3 rectal carcinoma.

BACKGROUND: The aim of this study was to determine if local recurrence (LR) rates in patients with minimally invasive and advanced T3 rectal cancer are different. This may influence the use of adjuvant therapy. METHODS: Consecutive patients with T3 rectal cancer undergoing curative surgery were classified into minimally invasive or advanced groups. Minimally invasive T3 was defined as a tumour that had invaded beyond the muscularis propria on microscopic examination only, whereas advanced T3 tumours had invasion beyond the muscularis propria that was obvious on macroscopic examination and confirmed histologically. Local recurrence rates of the two groups were compared by construction of Kaplan-Meier curves. The log-rank test was used to determine equivalence, and Cox regression to estimate the hazard ratio. The Grambsch- Therneau test and graphical comparison of predicted and observed Kaplan-Meier curves was used to test the proportional hazards assumption. RESULTS: There were 222 patients in total, 74 in the minimally invasive group and 148 in the advanced. The overall LR rate was 11.2%. The LR rates in the minimally invasive and advanced groups were 5.4% and 14.2%, respectively. The log-rank test gives a P value of 0.042 for equivalence, with the minimally invasive patients doing significantly better. The hazard ratio estimated by Cox regression was 0.35 (early relative to advanced), 95% confidence intervals (0.12, 1.0). There was no evidence of confounding by age at surgery, pathology type, gender or postoperative adjuvant therapy. CONCLUSIONS: The extent of invasion into the mesorectum appears to be an independent prognostic variable. If oncologically sound surgical techniques are employed, the LR rate of patients with minimal invasion is low. Adjuvant therapy may not confer additional benefit in this group.

Pattern-reversal visual evoked potentials in infants: gender differences during early visual maturation.

This paper investigates gender differences in the peak latency and amplitude of the P1 component of the pattern-reversal visual evoked potential (pattern-reversal VEP) recorded in healthy term infants. Pattern-reversal VEPs in response to a series of high contrast black and white checks (check widths 120', 60', 30', 24', 12', 6') were recorded in 50 infants (20 males, 30 females) at 50 weeks post-conceptional age (PCA) and in 49 infants (22 males, 27 females) at 66 weeks PCA. Peak latency of the major component, P1, was considerably shorter in female compared with male infants. Differences in head circumference do not entirely account for the gender differences in peak latency reported here. A gender difference in P1 amplitude was not detected. These findings stress the importance of considering gender norms as well as age-matched norms when utilizing the pattern-reversal VEP in clinical investigations. Studies including a wider range of ages are clearly necessary in order to establish whether the earlier peak latencies in female infants represents a difference in the onset or rate of visual maturation.

Dramatic developmental changes in larval knockdown response enhance genetic sexing based on DDT resistance in Anopheles stephensi (Diptera: Culicidae).

Genetic sexing systems based on a conditional lethal require good discrimination between the different phenotypes. DDT resistance in the early instars of Anopheles stephensi Liston is not a good candidate when based on mortality, but this study shows that the knockdown response gives exceptional discrimination between heterozygous resistant and homozygous susceptible individuals. One- and two-day-old larvae of the DlDDT strain showed high (417-fold) resistance to knockdown by DDT, but very low resistance to mortality (3.3-fold). This changes with the onset of the third instar, so that by the fourth instar, mortality resistance is high (108-fold) and knockdown resistance is low (6.5-fold). Susceptibility to DDT decreases from first to fourth instar in the susceptible strain by 443-fold for knockdown and 15-fold for mortality and in the resistant strain by 8.5-fold for knockdown and 491-fold for mortality. The DDT knockdown response in young larvae was successfully used to identify two Y-autosome translocations linked to the resistance gene, DDT. T(Y-3)69 and T(Y-3)72 gave recombination values between the translocation breakpoint and the DDT locus of 4.1 and 10.1 crossover units, respectively. T(Y-3)69 proved to be an adequate genetic sexing system for laboratory studies.

Anopheles stephensi Dox-A2 shares common ancestry with genes from distant groups of eukaryotes encoding a 26S proteasome subunit and is in a conserved gene cluster.

The sequence of a cloned Anopheles stephensi gene showed 72% inferred amino acid identity with Drosophila melanogaster Dox-A2 and 93% with its putative ortholog in Anopheles gambiae. Dox-A2 is the reported but herein disputed structural locus for diphenol oxidase A2. Database searches identified Dox-A2 related gene sequences from 15 non-insect species from diverse groups. Phylogenetic trees based on alignments of inferred protein sequences, DNA, and protein motif searches and protein secondary structure predictions produced results consistent with expectations for genes that are orthologous. The only inconsistency was that the C-terminus appears to be more primitive in the yeasts than in plants. In mammals, plants, and yeast these genes have been shown to code for a non-ATPase subunit of the PA700 (19S) regulatory complex of 26S proteasome. The analyses indicated that the insect genes contain no divergent structural features, which taken within an appraisal of all available data, makes the reported alternative function highly improbable. A plausible additional role, in which the 26S proteasome is implicated in regulation of phenol oxidase, would also apply to at least the mammalian genes. No function has yet been reported for the other included sequences. These were from genome projects and included Caenorhabiditus elegans, Arabidopsis thaliana, Fugu rubripes, and Toxoplasma gondii. A consensus of the results predicts a protein containing exceptionally long stretches of helix with a hydrophilic C-terminus. Phosphorylation site motifs were identified at two conserved positions. Possible SRY and GATA-1 binding motifs were found at conserved positions upstream of the mosquito genes. The location of A. stephensi Dox-A2 was determined by in situ hybridization at 34D on chromosome arm 3R. It is in a conserved gene cluster with respect to the other insects. However, the A. stephensi cluster contains a gene showing significant sequence identity to human and pigeon carnitine acetyltransferase genes, therefore showing divergence with the distal end of the D. melanogaster cluster.

A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens.

An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2-5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum-eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.

Genetic differentiation between and within strains of the saw-toothed grain beetle, Oryzaephilus surinamensis (Coleoptera: Silvanidae) at RAPD loci.

Nine strains of Oryzaephilus surinamensis have been kept in laboratory culture for periods ranging from 5 to 30 years (30-180 generations). Two RAPD primers provided sufficient information to separate the strains reliably and unambiguously. The strains are maintained at a population size of 200 breeding adults. The marked divergence between strains is consistent with the small population size, which for the older strains, according to population genetics theory, implies that roughly half the original genetic variation should now be lost from within strains. However, there is no indication that the older strains have less inter-strain variation. The results demonstrate RAPD loci can reliably detect population subdivision, which in field populations of pest species is of fundamental importance in understanding the population genetics of insecticide resistance.

Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera:Culicidae).

Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens. The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 x 10(-4) M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 x 10(-4) M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 x 10(-4) M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.

The selection and genetic analysis of esterase electromorphs in an organophosphate-resistant strain of Culex pipiens from Italy.

An Italian organophosphate-resistant strain of Culex pipiens (Lucca) was found to be polymorphic for elevated and nonelevated esterases. Selection for high esterase activity produced a strain homozygous for elevated esterases A2 and B2. Selection for low activity produced a strain homozygous for nonelevated esterases, A4i and B1i. Crossing experiments showed that A2 and B2 are coded by separate but closely linked genes, as are A4i and B1i. Results indicate that elevated A2 and nonelevated A4i are alleles of a single gene (Est-3 locus), as are elevated B2 and nonelevated B1i (Est-2 locus). Selection for electromorph variants gave four elevated A variants and three elevated B variants. These esterases were not selected in the field. In Lucca, A2 and B2 replaced A1, suggesting a selective advantage to the former over the latter in the presence of chlorpyrifos. It is hypothesized that the degree of amplification is an important factor in the selection of a particular esterase electromorph as a resistance mechanism and that migrating individuals with amplified genes could have an advantage when invading a population under selection.

The acetylcholinesterase gene of Anopheles stephensi.

1. The acetylcholinesterase (AChE) gene from the important malaria vector Anopheles stephensi has been isolated by homology to the Drosophila acetylcholinesterase gene. 2. The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity to Drosophila AChE and 38 and 36% identity to Torpedo AChE and human butyrylcholinesterase, respectively.

Location of a gene conferring DDT resistance but no pyrethroid cross-resistance in larvae of Anopheles stephensi.

In larvae of Anopheles stephensi, DDT resistance of 30 to 40-fold, involving no cross-resistance to pyrethroids, showed fully dominant monofactorial inheritance. The gene, termed DDT, is located 36.6 cross-over units from the morphological mutant, black larvae (Bl), on chromosome III. A polygenic system, which confers a 17-fold reduction in susceptibility to knockdown by the pyrethroid, permethrin, also makes a minor contribution to DDT resistance. It was not possible to block DDT resistance with the dehydrochlorinase inhibitor DMC.