Comparison of the metabolism of 10 chemicals in human and pig skin explants.

Skin metabolism is important to consider when assessing local toxicity and/or penetration of chemicals and their metabolites. If human skin supply is limited, pig skin can be used as an alternative. To identify any species differences, we have investigated the metabolism of 10 chemicals in a pig and human skin explant model. Phase I metabolic pathways in skin from both species included those known to occur via cytochrome P450s, esterases, alcohol dehydrogenases and aldehyde dehydrogenases. Common Phase II pathways were glucuronidation and sulfation but other conjugation pathways were also identified. Chemicals not metabolized by pig skin (caffeine, IQ and 4-chloroaniline) were also not metabolized by human skin. Six chemicals metabolized by pig skin were metabolized to a similar extent (percentage parent remaining) by human skin. Human skin metabolites were also detected in pig skin incubations, except for one unidentified minor vanillin metabolite. Three cinnamyl alcohol metabolites were unique to pig skin but represented minor metabolites. There were notable species differences in the relative amounts of common metabolites. The difference in the abundance of the sulfate conjugates of resorcinol and 4-amino-3-nitrophenol was in accordance with the known lack of aryl sulfotransferase activity in pigs. In conclusion, while qualitative comparisons of metabolic profiles were consistent between pig and human skin, there were some quantitative differences in the percentage of metabolites formed. This preliminary assessment suggests that pig skin is metabolically competent and could be a useful tool for evaluating potential first-pass metabolism before testing in human-derived tissues.

Connectivity of neuronal populations within and between areas of primate somatosensory cortex.

Functions of the cerebral cortex emerge via interactions of horizontally distributed neuronal populations within and across areas. However, the connectional underpinning of these interactions is not well understood. The present study explores the circuitry of column-size cortical domains within the hierarchically organized somatosensory cortical areas 3b and 1 using tract tracing and optical intrinsic signal imaging (OIS). The anatomical findings reveal that feedforward connections exhibit high topographic specificity, while intrinsic and feedback connections have a more widespread distribution. Both intrinsic and inter-areal connections are topographically oriented across the finger representations. Compared to area 3b, the low clustering of connections and small cortical magnification factor supports that the circuitry of area 1 scaffolds a sparse functional representation that integrates peripheral information from a large area that is fed back to area 3b. Fast information exchange between areas is ensured by thick axons forming a topographically organized, reciprocal pathway. Moreover, the highest density of projecting neurons and groups of axon arborization patches corresponds well with the size and locations of the functional population response reported by OIS. The findings establish connectional motifs at the mesoscopic level that underpin the functional organization of the cerebral cortex.

Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice.

Earlier diagnosis and treatment of Alzheimer's disease would greatly benefit from the identification of biomarkers at the prodromal stage. Using a prominent animal model of aspects of the disease, we here show using clinically relevant methodologies that very young, pre-pathological PDAPP mice, which overexpress mutant human amyloid precursor protein in the brain, exhibit two cryptic deficits that are normally undetected using standard methods of assessment. Despite learning a spatial memory task normally and displaying normal brain glucose uptake, they display faster forgetting after a long delay following performance to a criterion, together with a strong impairment of brain glucose uptake at the time of attempted memory retrieval. Preliminary observations suggest that these deficits, likely caused by an impairment in systems consolidation, could be rescued by immunotherapy with an anti-ß-amyloid antibody. Our data suggest a biomarker strategy for the early detection of ß-amyloid-related abnormalities.

Polygamy and an absence of fine-scale structure in Dendroctonus ponderosae (Hopk.) (Coleoptera: Curcilionidae) confirmed using molecular markers.

An understanding of mating systems and fine-scale spatial genetic structure is required to effectively manage forest pest species such as Dendroctonus ponderosae (mountain pine beetle). Here we used genome-wide single-nucleotide polymorphisms to assess the fine-scale genetic structure and mating system of D. ponderosae collected from a single stand in Alberta, Canada. Fine-scale spatial genetic structure was absent within the stand and the majority of genetic variation was best explained at the individual level. Relatedness estimates support previous reports of pre-emergence mating. Parentage assignment tests indicate that a polygamous mating system better explains the relationships among individuals within a gallery than the previously reported female monogamous/male polygynous system. Furthermore, there is some evidence to suggest that females may exploit the galleries of other females, at least under epidemic conditions. Our results suggest that current management models are likely to be effective across large geographic areas based on the absence of fine-scale genetic structure.

Epidemiology of vaginal prolapse in mixed-age ewes in New Zealand.

AIMS: Identify environmental, animal, and management factors associated with risk of vaginal prolapse in ewes, to enable farmers and advisors to make pragmatic decisions based on empirical observations for control of the condition. METHODS: Two longitudinal studies conducted over 2 years to identify factors associated with incidence of prolapse in (i) cohorts of 200 individually identified mixed-age (MA) ewes, and (ii) all MA ewes, on voluntarily participating sheep-breeding farms in Hawkes Bay (HB) and Southland regions of New Zealand. RESULTS: The overall annual incidences of prolapse on 113 farms in 2000 and 88 in 2001 were 1.21 and 0.82 per 100 MA ewes, respectively, and 1.05 for both years combined. A total of 406 prolapses were recorded among 36,695 individually identified cohort ewes. Individual farm incidences for both years varied from 0-5.9 (mean=1.56, median=1.39) on Southland and 0-3.9 (mean=0.75, median=0.54) per 100 ewes on HB farms. The crude relative risk of a prolapse occurring in a MA ewe was 5.31 times higher for ewes carrying twins and 11.3 times higher for ewes carrying triplets, than single lambs. Flocks made up of predominantly pure or crossbred Perendale ewes appeared to be at lower risk than flocks with other breeds. Shearing in the 3 months leading up to mating appeared to be protective, as was shearing in the second half of pregnancy. The risk was higher on farms with moderate to steep terrain than on farms with flat terrain. The identified risk factors in the individually identified cohorts were: access to salt and feeding of swedes in the latter part of pregnancy, moderate to steep lambing paddocks, multiple lambs detected at scanning, and weight gain between start of mating and scanning. The condition recurred in 2001 in six (35%) of 17 study ewes that had prolapsed during 2000. Culling policies for female offspring of affected ewes did not influence incidence at the farm level; nor did feeding hay or grain in late pregnancy. Furthermore, there was no association between incidence and body condition scores measured prior to and after mating, at scanning, or at time of set stocking. CLINICAL SIGNIFICANCE: Vaginal prolapse is an inevitable consequence of sheep reproduction and its incidence is expected to increase as reproductive rates increase. This study provides some firm leads as to the relative importance of risk factors and gives guidance for risk reduction, e.g. by identification and separate management of ewes carrying twins or triplets, using flat paddocks for lambing, and guarding against gain in weight between the start of mating and scanning.

Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Selecting patients with faecal incontinence for anal sphincter surgery: the influence of irritable bowel syndrome.

AIM: This was a prospective study to determine the prevalence of irritable bowel syndrome (IBS) in patients with faecal incontinence and to investigate the association of IBS positivity with sphincter dysfunction. METHOD: Patients with faecal incontinence were referred for physiological assessment and further management. Those with an acute obstetric injury, rectal prolapse or previous anorectal surgery were excluded. One hundred and seventy-seven women were studied. Symptom questionnaires were used prospectively to identify IBS positivity using Rome 11 criteria and the Cleveland Clinic incontinence score. RESULTS: The prevalence of IBS was 44%. IBS patients had higher incontinence scores than non-IBS patients (11.0 vs 9.0, P < 0.01). A normal or high maximum resting anal pressure in an incontinent woman increased the chance of her having IBS by a factor of 2.6. CONCLUSION: The prevalence of IBS positivity in patients presenting with faecal incontinence was high. A normal or high anal resting pressure and a high incontinence score should raise the suspicion of IBS.

A rapid topographic mapping and eye alignment method using optical imaging in Macaque visual cortex.

In optical imaging experiments, it is often advantageous to map the field of view and to converge the eyes without electrophysiological recording. This occurs when limited space precludes placement of an electrode or in chronic optical chambers in which one may not want to introduce an electrode each session or for determining eye position in studies of ocular disparity response in visual cortex of anesthetized animals. For these purposes, we have developed a spot imaging method that can be conducted rapidly and repeatedly throughout an experiment. Using small 0.2 degrees -0.5 degrees spots, the extent of the imaged field of view is mapped by imaging cortical response to single spots, placed at different positions (0.2 degrees steps) in either the horizontal or vertical axes. By shifting the relative positions of two spots, one presented to each eye, eye convergence can be assessed to within 0.1 degrees resolution. Once appropriate eye alignment is determined, stimuli for further optical imaging procedures (e.g. imaging random dot stimuli for study of disparity responses) can then be confidently placed. This procedure can be quickly repeated throughout the experiment to ensure maintained eye alignment.

The fate of anastomotic sinuses after total mesorectal excision for rectal cancer.

OBJECTIVE: Chronic anastomotic sinus is a recognized complication of total mesorectal excision (TME) surgery. We observed two patients who developed new tumours within chronic anastomotic sinuses 6 and 19 years after initial surgery. The aim of this study was therefore to report the incidence and outcome of anastomotic sinuses, thus identifying those at potential risk of malignant change. METHOD: We retrospectively reviewed patient records and radiology reports to identify potentially curative rectal cancer cases between 1998 and 2005. RESULTS: In a consecutive series of 100 TMEs with ileostomy, there were 70 males and 30 females, aged 66 (33-88) years. Anastomosis was by double staple technique. A policy of instant enema was used prior to ileostomy closure. Eighty-six patients had instant enemas. Of the 14 that did not, four died prior to enema, one returned to theatre for sepsis, three had their anastomoses assessed by sigmoidoscopy alone. Six had incomplete records. Of the 86 patients, eight presacral sinuses were identified. Three sinuses closed spontaneously. Five persisted of whom two required further surgery. CONCLUSION: Persistent anastomotic sinuses occurred in 5% after curative TME. Malignant transformation can occur. Active treatment for chronic sinuses should, therefore, be considered.

Isolation and characterization of 16 microsatellite loci in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae).

We isolated 16 polymorphic microsatellite loci in the mountain pine beetle (Dendroctonus ponderosae Hopkins) and developed conditions for amplifying these markers in four multiplex reactions. Three to 14 alleles were detected per locus across two sampled populations. Observed and expected heterozygosities ranged from 0.000 to 0.902 and from 0.100 to 0.830, respectively. Three loci deviated from Hardy-Weinberg equilibrium in one sampled population. One of these loci may be sex linked. These markers will be useful in the study of population structure in this important pest species.

Enhancement of bacterial competitive fitness by apramycin resistance plasmids from non-pathogenic Escherichia coli.

The study of antibiotic resistance has in the past focused on organisms that are pathogenic to humans or animals. However, the development of resistance in commensal organisms is of concern because of possible transfer of resistance genes to zoonotic pathogens. Conjugative plasmids are genetic elements capable of such transfer and are traditionally thought to engender a fitness burden on host bacteria. In this study, conjugative apramycin resistance plasmids isolated from newborn calves were characterized. Calves were raised on a farm that had not used apramycin or related aminoglycoside antibiotics for at least 20 months prior to sampling. Of three apramycin resistance plasmids, one was capable of transfer at very high rates and two were found to confer fitness advantages on new Escherichia coli hosts. This is the first identification of natural plasmids isolated from commensal organisms that are able to confer a fitness advantage on a new host. This work indicates that reservoirs of antibiotic resistance genes in commensal organisms might not decrease if antibiotic usage is halted.

Short course preoperative radiotherapy is the single most important risk factor for perineal wound complications after abdominoperineal excision of the rectum.

AIM: To determine factors associated with perineal wound complications following abdominoperineal excision of the rectum (APER) for rectal adenocarcinoma and their effects on time to healing. PATIENTS AND METHODS: We studied all cases of APER performed in our unit by four consultants over 7 years. Seven out of nine factors considered important in wound healing were analysed using logistic regression and a multivariate model was built to examine interactions. Wound persistence was calculated using the Kaplan-Meier method. RESULTS: Data were available for 94 of 96 patients [male:female, 3:2, median age 72.5 (IQR: 64-78)]. Thirty-nine (41%) patients had 25 Gray, 3-portal, fractionated 5-day short course preoperative radiotherapy (SCPRT). Dukes stages were A (34%), B (26%), C (40%). Perineal wound complications occurred in 44 (47%), 16% of these requiring return to theatre. Local recurrences occurred in 13 (15%). There was no evidence to suggest that either patient gender, age, smoking status, preoperative albumin or haemoglobin level, or T stage were associated with the development of wound complications. The odds of wound complications for a patient who had SCPRT was over 10 times that for a patient who did not have preoperative radiotherapy (odds ratio 10.15, 95% CI: 3.80-27.05, n = 94). Seventy-four per cent of SCPRT and 96% of non-SCPRT wounds had healed by 1 year. Estimated failed wound healing rates at 30 and 90 days were 64% (95% CI: 46-78) and 48% (95% CI: 30-64) in SCPRT patients compared with 23% (95% CI: 12-35) and 9% (95% CI: 3-20) in non-SCPRT patients (log rank test P < 0.0001). CONCLUSION: Patients who have an APER are over 10 times more likely to have a perineal wound complication if they have SCPRT than not. Two-thirds of these will not have healed by 1 month, half by 3 months and over a quarter will still remain unhealed at 1 year. This has important implications for patient management decisions. Large prospective studies are needed to evaluate the effects of a selective policy for radiotherapy administered to patients requiring APER.

The positive predictive value of diagnostic ultrasound for occult herniae.

INTRODUCTION: The aim of this study is to ascertain the accuracy of diagnostic ultrasound in the assessment of the occult abdominal and groin herniae. The authors have previously demonstrated its efficacy in diagnosing the type of clinical groin herniae but occult herniae provide a further diagnostic problem. PATIENTS AND METHODS: A total of 113 consecutive patients were referred prospectively for ultrasound examinations with clinically suspected occult herniae. All positive scans were offered surgery whilst the negative results were offered further imaging or other diagnostic tests depending on the clinical criteria. The end point for negative scans was based on 18-month follow-up or resolution of symptoms. RESULTS: Overall, 59 scans showed positive results for herniae and 56 of these had surgery. In the other three patients, two refused an operation, and one had no hernia detected at operation. In the remaining 57 scans, ultrasound offered alternative soft tissue diagnoses in 23 patients and surgical/endoscopic diagnoses accounted for a further 8 patients. CONCLUSIONS: Ultrasound offered a diagnosis for the symptomology in 82 patients (70.6%) of which 59 were herniae. The positive predictive value for hernia is 98.3%. Twenty-six patients with no diagnosis or confirmation of herniae on follow-up showed symptom resolution in 22 cases, and four patients were treated by the pain clinic.

Congenital polycystic kidney disease in lambs.

AIM: To describe the pathology and inheritance of a congenital polycystic kidney disease (PKD) of sheep. METHODS: Mode of inheritance of PKD was investigated by evaluation of results of the disorder from planned matings in two consecutive years within subsets of a flock that had a high prevalence of PKD in lambs. Gross pathological and histopathological studies were based on tissues derived from this study. Haematoxylin and eosin (H&E)-stained paraffin sections of kidney, liver, extrahepatic biliary and pancreatic ducts, pancreas and epididymis were used to describe the lesions. RESULTS: Twenty-five lambs affected by PKD, of both sexes, were born, numbers in accord with those expected for an autosomal recessive disorder in the population studied. In all cases for which tissues were available, the renal, bile ductal (intrahepatic and extrahepatic), pancreatic and epididymal tissues had widespread dysplastic changes and associated cyst formation. CONCLUSIONS: The findings of renal cysts in conjunction with cysts in other organs are unifying features in many of the human and animal forms of PKD and suggest a related pathogenic and genetic base consistent with an autosomal recessive disorder.

The metabolic profile of azimilide in man: in vivo and in vitro evaluations.

The metabolic fate of azimilide in man is unusual as it undergoes a cleavage in vivo resulting in the formation of two classes of structurally distinct metabolites. During a metabolite profiling study conducted in human volunteers to assess the contribution of all pathways to the clearance of (14)C-azimilide, greater than 82% of radioactivity was recovered in urine (49%-58%) and feces (33%). Urine, feces, and plasma were profiled for metabolites. A cleaved metabolite, 4-chloro-2-phenyl furoic acid was present at high concentration in plasma (metabolite/parent AUC ratio approx. 4), while other plasma metabolites, azimilide N-oxide (metabolite/parent AUC ratio 0.001), and a cleaved hydantoin metabolite (metabolite/parent AUC ratio = 0.3) were present at lower concentrations than azimilide. In urine, the cleaved metabolites were the major metabolites, (> 35% of the dose) along with phenols (as conjugates, 7%-8%), azimilide N-oxide (4%-10%), a butanoic acid metabolite (2%-3%), and desmethyl azimilide (2%). A limited investigation of fecal metabolites indicated that azimilide (3%-5%), desmethyl azimilide (1%-3%), and the butanoic acid metabolite (< 1%) were present. Contributing pathways for metabolism of azimilide, identified through in vitro and in-vivo studies, were CYPs 1A1 (est. 28%), 3A4/5 (est. 20%), 2D6 (< 1%), FMO (est. 14%), and cleavage (35%). Enzyme(s) involved in the cleavage of azimilide were not identified.

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence.

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.

The groin hernia - an ultrasound diagnosis?

AIMS: Ultrasound is a non-invasive, non-ionising radiation modality which is highly successful at soft tissue imaging. Groin pain from an occult hernia can be a difficult clinical diagnosis made easier by good imaging. The aim of this study was to demonstrate the accuracy of ultrasound in diagnosing and typing the groin hernia, which could lead to its justification of use in the imaging of the occult hernia. PATIENTS AND METHODS: A total of 118 patients with a clinical diagnosis of a groin hernia, prospectively underwent a blinded, ultrasound examination of the groin. All patients underwent surgery and these findings were compared with the ultrasound results. RESULTS: 120 symptomatic groins in 118 patients underwent surgery. Ultrasound diagnosed 118 hernias and there were two normal ultrasound examinations. Surgery confirmed the same two patients to be normal - ultrasound sensitivity for all hernias was 100% with 100% specificity. Two femoral hernias were seen in this study, both correctly identified at ultrasound. Of the inguinal hernias, ultrasound identified 36 of 42 direct hernias (sensitivity 86%, specificity 97%) and 72 of 74 indirect hernias (sensitivity 97%, specificity 87%) CONCLUSIONS: This study confirms that ultrasound can accurately diagnose groin hernias and this may justify its use in the assessment of the occult hernia.

Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 causes gastrointestinal disease with the potential for life-threatening sequelae. Although Shiga-like toxins are responsible for much of the serious pathology in humans, the bacterium also possesses a type III protein secretion system that is responsible for intimate attachment to host intestinal mucosa. This sophisticated interaction requires co-ordination that is governed by environmental and genetic factors. Ongoing research supports the following model for how EHEC enables and controls this process: (i) specific environmental cues that are present in the host result in the expression of a number of adhesins, including fimbriae, which allow the initial binding to the mucosal surface. The same conditions support the expression of the basal type III secretion apparatus; (ii) targeting and assembly of the translocon requires both an mRNA signal and chaperones, with coupled translation and secretion of translocon proteins, EspA, B and D; (iii) opening up of a conduit between the bacterium and host cell releases a cytoplasmic pool of effector proteins. A consequence of this is increased expression of particular effector proteins. Potentially, different proteins could be released into the cell at different times or have activities modulated with time; (iv) intimate contact between the translocated intimin receptor (Tir) and the bacterial surface factor intimin requires translocon expression to be down-regulated and translocon filaments to be lost. Fluorescent protein fusions allow contact-mediated regulation and protein targeting through the type III secretion system to be studied in detail.

Intra-operative injection of Patent Blue V dye to facilitate nodal staging in colorectal cancer.

OBJECTIVES: Routine histopathology may understage colorectal cancer by failing to detect involved lymph nodes. This study examined the feasibility of dye staining those lymph nodes most likely to harbour metastases. PATIENTS AND METHODS: Patent Blue V dye 2.5% was injected intra-operatively into left-sided colorectal carcinomas prior to resection in 19 patients. RESULTS: Blue-stained nodes were found in 12/19 patients (63%). Examination of blue-stained nodes alone correctly identified overall nodal status in 11 (92%) of these 12 patients. CONCLUSION: The technique needs to be refined further. Nonetheless, intra-operative lymph node staining using blue dye offers the prospect of improving the ease and accuracy of nodal staging in colorectal cancer.