Investigation into ethylene oxide treatment and residuals on DNA and downstream DNA analysis.

Recent years have seen a significant increase in the sensitivity of DNA testing, enabling the determination of DNA profiles from low levels of cellular material. However, the increased sensitivity is in many ways a double-edged sword as background contaminating DNA generated during the manufacture of consumables and sampling devices is now being detected and may compromise the interpretation of the DNA profile results. This study initially demonstrated the effectiveness of ethylene oxide (EO) as a post-production treatment to eliminate DNA on swabs, used as a sampling device for the recovery of cellular material. Subsequently, the potential adverse effects of any residual EO remaining on the swabs on the downstream DNA analysis on both rayon and cotton swabs were investigated and the levels of remaining EO measured. Two main variables were tested: the amount of time elapsed since EO treatment of the swabs prior to use, and the time elapsed between cellular material collection and DNA analysis. Residual levels of EO were found to be below quantitation levels and therefore also international standards. The results indicated that while there was a negligible effect of EO treatment on DNA recovered from rayon swabs, there was however an adverse effect on the DNA profiles recovered from cotton swabs. The adverse effect was negatively correlated with time since EO treatment and positively correlated with time to DNA analysis.

Evaluation of a mucoadhesive fenretinide patch for local intraoral delivery: a strategy to reintroduce fenretinide for oral cancer chemoprevention.

Systemic delivery of fenretinide in oral cancer chemoprevention trials has been largely unsuccessful due to dose-limiting toxicities and subtherapeutic intraoral drug levels. Local drug delivery, however, provides site-specific therapeutically relevant levels while minimizing systemic exposure. These studies evaluated the pharmacokinetic and growth-modulatory parameters of fenretinide mucoadhesive patch application on rabbit buccal mucosa. Fenretinide and blank-control patches were placed on right/left buccal mucosa, respectively, in eight rabbits (30 min, q.d., 10 days). No clinical or histological deleterious effects occurred. LC-MS/MS analyses of post-treatment samples revealed a delivery gradient with highest fenretinide levels achieved at the patch-mucosal interface (no metabolites), pharmacologically active levels in fenretinide-treated oral mucosa (mean: 5.65 µM; trace amounts of 4-oxo-4-HPR) and undetectable sera levels. Epithelial markers for cell proliferation (Ki-67), terminal differentiation (transglutaminase 1-TGase1) and glucuronidation (UDP-glucuronosyltransferase1A1-UGT1A1) exhibited fenretinide concentration-specific relationships (elevated TGase1 and UGT1A1 levels <5 µM, reduced Ki-67 indices >5 µM) relative to blank-treated epithelium. All fenretinide-treated tissues showed significantly increased intraepithelial apoptosis (TUNEL) positivity, implying activation of intersecting apoptotic and differentiation pathways. Human oral mucosal correlative studies showed substantial interdonor variations in levels of the enzyme (cytochrome P450 3A4-CYP3A4) responsible for conversion of fenretinide to its highly active metabolite, 4-oxo-4-HPR. Complementary in vitro assays in human oral keratinocytes revealed fenretinide and 4-oxo-4-HPR's preferential suppression of DNA synthesis in dysplastic as opposed to normal oral keratinocytes. Collectively, these data showed that mucoadhesive patch-mediated fenretinide delivery is a viable strategy to reintroduce a compound known to induce keratinocyte differentiation to human oral cancer chemoprevention trials.

Mucoadhesive fenretinide patches for site-specific chemoprevention of oral cancer: enhancement of oral mucosal permeation of fenretinide by coincorporation of propylene glycol and menthol.

The objective of this study was to enhance oral mucosal permeation of fenretinide by coincorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Coincorporation of 5-10 wt % PG (mean J(s) = 16-23 µg cm⁻² h⁻¹; 158-171 µg of fenretinide/g of tissue) or 1-10 wt % PG + 5 wt % menthol (mean J(s) = 18-40 µg cm⁻² h⁻¹; 172-241 µg of fenretinide/g of tissue) in fenretinide/Eudragit RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt % in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (∼43 µg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt % PG + 5 wt % menthol) relative to permeation obtained from enhancer-free patch (∼17 µg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by coincorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch coincorporated with 2.5 wt % PG + 5 wt % menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide.

Molecular epidemiology of Mycobacterium tuberculosis in East Lancashire 2001-2009.

BACKGROUND: East Lancashire has had high rates of tuberculosis for 40 years. The ethnically diverse population is predominantly of South Asian and white origin. Drug resistance data from 1960 to 1999 indirectly suggest that no significant inter-ethnic transmission has occurred. This study used mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) fingerprinting to assess clustering within and between ethnic groups. METHODS: All isolates of Mycobacterium tuberculosis from January 2001 to July 2009 from East Lancashire postcode areas were MIRU-VNTR fingerprinted. Clusters of strains with indistinguishable profiles were also assessed epidemiologically, and their MIRU-VNTR profiles compared with the UK M tuberculosis Strain Typing Database. RESULTS: 332 strains were typed (63 white patients, and 269 non-white patients). 198 MIRU-VNTR profiles were identified, with 144 profiles occurring only once. The typing clustered 187 strains into 53 clusters indistinguishable at all 12 loci and these were further characterised using the exact tandem repeat loci A, B, and C. The 15 loci clustered 32/63 (50.8%) of white and 110/269 (40.9%) of non-white cases and all but nine clusters were of the same ethnicity. The nine inter-racial clusters were further assessed from an epidemiological and clinical perspective and fingerprinting using nine additional loci. Isolates within two of the clusters were further discriminated using the additional nine loci. However, the additional loci did not further discriminate the isolates in the other seven inter-racial clusters. CONCLUSIONS: MIRU-VNTR fingerprinting indicates that although there is evidence of a high rate of transmission within the South Asian sub-population, the data suggest that there is little inter-ethnic transmission.

A textile fibre survey as an aid to the interpretation of fibre evidence in the Sydney region.

Frequency figures of the fibre population on textile cinema seats were measured in Sydney, Australia, in winter. Sixteen seats were analysed from a very popular cinema complex, with 3025 fibres classified according to colour, generic class and fluorescence properties (100 grey-black cotton fibres only). The recovered fibres were mostly natural fibres (84%) with cotton the most common generic type (70%). On the contrary, man made fibres were relatively rare (15%) with rayon constituting the majority of these (51%). The most common colour/generic class combinations were grey-black cotton (33%) and blue cotton (30%) accounting for 63% of the total population. All other frequencies were below 5%, most below 1% using only the two properties of colour and generic class. Fluorescence properties were found to be very discriminating as far as grey-black cotton fibres were concerned. These features are considered and discussed and in particular, to emphasise the significance of fibres as evidence of contact.

Adhesive tape analysis: establishing the evidential value of specific techniques.

This study investigated the evidential value of specific methods of analysis for packaging tapes and clear adhesive tapes available in Australia. Fifty-eight adhesive tapes were analyzed using a wide range of optical, physical, and chemical techniques. The results were collated for the purpose of creating an Australian database of adhesive tapes, which would be of assistance in criminal investigation. Each technique was evaluated for its discriminating power, both for comparative purposes and for the identification of adhesive tapes by comparing unknown samples with the database. The combined discriminating power of the techniques applied is very high. It is possible to individually identify the source of an unknown adhesive tape sample in many instances by searching the database. It is also possible to form an opinion on the significance of a failure-to-discriminate result in comparative casework. Further work is still needed to expand and update the database, as well as compiling data on the relative market share of various products.

A protocol for the forensic analysis of condom and personal lubricants found in sexual assault cases.

In sexual assault cases, lubricant trace evidence may supplement biological evidence, or may be the primary physical evidence where biological evidence is unavailable. This study considered a total of 50 lubricants from condoms and personal lubricant products available in Australia. Differentiation of the samples was attempted using fluorescence examination, Fourier transform infrared spectroscopy (DRIFTS), gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and pyrolysis gas chromatography-mass spectrometry. Eleven of the samples were uniquely identified by the analysis scheme, while the remainder of the samples were separated into nine groups. As a result of this study, a recommended protocol for the detection and analysis of an "unknown" biological swab was produced.

Venous blood pressure in broilers during acute inhalation of five percent carbon dioxide or unilateral pulmonary artery occlusion.

We evaluated the hypothesis that venous congestion (increased venous volume), as reflected by venous hypertension (increased venous pressure), can arise when the right ventricle is unable to elevate the pulmonary arterial pressure sufficiently to propel the cardiac output through an anatomically inadequate or inappropriately constricted pulmonary vasculature. Changes in venous pressure were evaluated in clinically healthy broilers during modest increases in pulmonary vascular resistance induced by inhalation of 5% CO2 and during large increases in pulmonary vascular resistance accomplished by acutely tightening a snare around one pulmonary artery. Inhalation of 5% CO2 induced a pronounced respiratory acidosis, as reflected by increases the partial pressure of CO2 and the hydrogen ion concentration in arterial blood. Inhalation of 5% CO2 also increased pulmonary arterial pressure by approximately 3 mm Hg and increased venous pressure by approximately 1 mm Hg when compared with the pre-inhalation venous pressure. Tightening the pulmonary artery snare increased the pulmonary arterial pressure by approximately 10 mm Hg, and this degree of pulmonary hypertension was sustained until the snare was released. When compared with the pre- and post-snare intervals, tightening of the pulmonary artery snare induced a sustained increase in venous pressure of > or = 1 mm Hg. Veins have highly compliant walls that permit an approximate doubling in volume with only small (4 to 6 mm Hg) increases in central venous pressure. Presumably the apparently modest 1 mm Hg increase in venous pressure measured after CO2 inhalation or unilateral pulmonary artery occlusion reflects a large increase in venous volume and, thus, substantial venous congestion. These observations support the hypothesis that increases in pulmonary vascular resistance can initiate increases in venous pressure by challenging the capacity of the right ventricle to propel all of the returning venous blood through the lungs. Central venous congestion predisposes broilers to the onset of cirrhosis and ascites by impeding the outflow of hepatic venous blood and increasing the hydrostatic pressure within hepatic sinusoids.

Thromboxane mimics the pulmonary but not systemic vascular responses to bolus HCl injections in broiler chickens.

Bolus i.v. injections of 1.2 N HCl elicit a rapid but transient pulmonary vasoconstriction in broiler chickens. In mammals, the pulmonary vasoconstrictive response to bolus acid injection depends on increased synthesis of thromboxane A2; however, the vascular responsiveness of domestic fowl to thromboxane previously had not been evaluated. In the present study, we tested the hypothesis that, if HCl triggers pulmonary vasoconstriction by stimulating thromboxane A2 synthesis in broilers, then bolus i.v. injections of the potent thromboxane A2 mimetic U44069 (9,11-dideoxy-9alpha,11alpha-epoxy-methanoprostaglandin++ + F2alpha; 1 micromol/mL; 0.5 mL injected volume) should trigger hemodynamic responses similar to those elicited by HCl (1.2 N; 1.5 mL injected volume). Both HCl and the thromboxane mimetic elicited twofold or greater increases in pulmonary vascular resistance, which in turn increased pulmonary arterial pressure by 50% despite concurrent reductions in cardiac output. The reductions in cardiac output were associated with reductions in stroke volume but not heart rate. The thromboxane mimetic also increased the total peripheral resistance, which minimized the reduction in mean systemic arterial pressure associated with the decrease in cardiac output. In contrast, HCl injections did not increase total peripheral resistance; consequently, the reduction in cardiac output caused the mean systemic arterial pressure to decrease by 30 mm Hg. Mannitol (2.5%; 1.5 mL) was injected i.v. as a volume control, and had no influence on any of the variables. This study provides the first direct evidence that thromboxane is a potent pulmonary vasoconstrictor in broilers, and provides support for the hypothesis that thromboxane mediates the pulmonary vasoconstrictive response to bolus i.v. injections of HCl. The differential response of the systemic vasculature to the thromboxane mimetic and HCl may indicate that cardiopulmonary responses to HCl injections are not mediated solely via thromboxane production. Alternatively, a direct dilatory effect of elevated hydrogen ion concentrations on the systemic vasculature may have counteracted any tendency for simultaneously evolved endogenous thromboxane to elicit systemic vasoconstriction.

Effect of Santoquin and oxidized fat on liver and intestinal glutathione in broilers.

Experiments were conducted to determine effects of Santoquin (ethoxyquin) and oxidized fat on liver and intestinal reduced (GSH) and oxidized (GSSG) glutathione, and pulmonary hypertension syndrome (PHS) mortality. Male broilers were randomly assigned in a 2 x 2 factorial consisting of 3.5% normal (NF) or oxidized (OxF) fat with or without ethoxyquin (E). Body weights and feed intake were monitored weekly, and tissues obtained at 3 and 7 wk for GSH and GSSG analysis. Compared to the NF group, NF/E gained more weight during the starter (0 to 3 wk), but not the grower (4 to 7 wk) period. Birds fed NF/E or NF exhibited greater feed efficiency in the starter period and greater gains during the starter and grower periods than birds fed OxF or OxF/E. No differences in PHS mortality between treatments were observed. Birds fed OxF exhibited lower liver GSSG at 3 wk than the other groups, but there were no differences in liver GSH. Duodenal GSH was higher in birds fed OxF/E than in birds of NF group at 3 and 7 wk. Ileal GSH was higher at 3 wk in OxF/E birds than in OxF birds, but no differences were observed at 7 wk. All tissues exhibited higher GSH levels at 7 wk than at 3 wk. Birds fed ethoxyquin, regardless of fat source, exhibited higher duodenal GSH at 3 and 7 wk and higher ileal GSH at 3 wk than birds that did not receive ethoxyquin. Higher GSH would be beneficial by enhancing protection of intestinal cells to deleterious effects of toxins or other forms of oxidative stress.

The effect of social deprivation on birthweight, excluding physiological and pathological effects.

OBJECTIVE: To study the effect of social deprivation on birthweight, excluding the effect of known physiological factors and exploring the effect of possible pathological factors. DESIGN: Retrospective analysis of computerised obstetric database. SETTING: Two teaching hospitals and an associated district general hospital which provided a defined catchment area in the East Midlands. SUBJECTS: The final analysis included 7493 women with complete datasets and gestations of between 259 and 300 days at delivery, dated by ultrasound scan. MAIN OUTCOME MEASURES: Smoking habit, alcohol consumption, weight gain during pregnancy, systolic and diastolic blood pressures at booking, bleeding during pregnancy and Jarman score; also, the effect of these variables on birthweight, adjusted for the effects of physiological factors using the individualised birthweight ratio. RESULTS: Smoking during pregnancy reduced birthweight but the effect is not linear, becoming less marked as the number of cigarettes smoked increases. Alcohol intake, diastolic and systolic blood pressures at the booking visit and vaginal bleeding during early pregnancy were not significantly related to birthweight. Pregnancy weight gain was significantly positively related to birthweight especially in the normal weight range (60-99 kg). A multivariate analysis including physiological and pathological factors found increasing Jarman score to be negatively related to birthweight. CONCLUSIONS: In this central British population social deprivation is correlated negatively with birthweight: the most socially deprived mothers have the smallest babies. This association cannot be explained in terms of physiological differences in the population nor in a higher prevalence of known pathological factors.

The individualised birthweight ratio: a more logical outcome measure of pregnancy than birthweight alone.

OBJECTIVE: To provide a new outcome measure for pregnancy specifically related to the individual. DESIGN: Computer analysis of physiological factors affecting birthweight. SETTING: Two provincial teaching hospitals (University and City Hospitals, Nottingham) and an associated district general hospital (Derby City Hospital) serving a defined catchment area in the East Midlands. SUBJECTS: All women delivering in the above hospitals since the start of computerised obstetric records: 31,561 women with gestational age verified by early pregnancy ultrasound scan data. MAIN OUTCOME MEASURES: Calculation of the predicted birthweight taking into account maternal and fetal physiological factors. Derivation of the individualised birthweight ratio (actual birthweight divided by predicted birthweight expressed as a percentage) for each individual baby. RESULTS: The individualised birthweight ratio redefines as normally grown 41% of babies below the 10th centile of crude birthweight for gestation. Other babies previously regarded as normal are redefined as growth retarded. At the upper end of the distribution 46% of those above the 90th centile of birthweight for gestation are redefined as normally grown. CONCLUSIONS: The predicted birthweight can be calculated for an individual pregnancy at a given gestation. The standardised comparison between this predicted birthweight and the actual birthweight is a more logical reflection of the normality of intrauterine growth and therefore more logical as an outcome measure for pregnancy than crude birthweight for gestation.

Response of the avian kidney to acute changes in arterial perfusion pressure and portal blood supply.

Domestic fowl kidneys autoregulate total renal blood flow and glomerular filtration rate (GFR) over a wide range of renal arterial perfusion pressure (RAPP). Sustained (approximately 2-4 h) restriction of renal portal blood flow attenuates the autoregulatory responses. The present study was designed to assess the effects of acute (approximately 10 min) alterations of renal portal blood flow on renal function, and to dissociate the renal responses to altered renal portal blood flow from the renal responses to reductions in RAPP. The thermal pulse decay (TPD) technique and p-aminohippuric acid clearance (CPAH) were used to measure blood flow. During acute increases and decreases in renal portal blood flow, regional renal blood flow as measured by the TPD system (RBFTPD) was significantly positively correlated with total kidney blood flow represented by CPAH (RBFPAH). These results indicate that changes in total kidney blood flow induced by alteration of portal perfusion were reflected in the regional measurement of renal blood flow. Changes in renal portal blood flow did not affect the urine flow rate (UFR), GFR, or fractional excretion of sodium (FENa). Reducing RAPP from 120 to 50 mmHg significantly reduced UFR, GFR, and FENa. Overall, these results indicate that large acute changes in renal portal blood flow can significantly alter total renal blood flow without significantly affecting parameters (UFR, GFR, and FENa) primarily influenced by the renal arterial vasculature.

A case-control study of congenital hip dislocation.

In this study we have investigated various epidemiological factors which may be related to congenital dislocation of the hip (CDH). Eighty-one cases born during the period 1st January 1988 to 31st August 1990, with four matched controls per case, were identified from consultants' records held at the Queen's Medical Centre and City Hospital, Nottingham. One hundred and twenty-four subjects who were referred to a new Hip Instability Clinic with suspected CDH, but not diagnosed or treated for CDH, were also included as a third group. Information about the mother's pregnancy, previous medical history and family history was collected from obstetric records kept at the two hospitals. Multigravidae and similarly multiparous women had a statistically significantly reduced risk of having a baby with CDH. The relative risks were 0.55 (95% confidence interval 0.33, 0.93) and 0.53 (95% confidence interval 0.31, 0.91) respectively. The method of delivery was also of importance. Babies born by Caesarean section or in breech position had an increased risk of CDH which was statistically significant. Using addition clinical information obtained from subjects attending the Hip Instability Clinic we also found that cases were more likely to have a family history of CDH than subjects who were screened for CDH but found to be normal.

Effect of glutathione depletion on tissue and plasma prostacyclin and thromboxane in rats.

Experiments were designed to determine the effects of glutathione (GSH) depletion with L-buthionine sulfoximine (BSO) or diethyl maleate (DEM) on tissue and plasma prostacyclin (6-keto-PGF1 alpha) and thromboxane (TxB2) levels in male Sprague-Dawley rats. Despite depleting hepatic GSH to as much as 34% of control, BSO at various levels (0.4, 0.8 and 1.2 g/kg body wt) had no effect on hepatic, renal, pulmonary or cardiac tissue levels of 6-keto-PGF1 alpha and TxB2 or circulating levels of 6-keto-PGF1 alpha in portal or arterial plasma. When rats were pretreated with 3-methylcholanthrene (3-MC) to induce cytochrome P450, BSO (0.8 g/kg body wt) also had no effect on tissue or plasma prostanoid levels with the exception of a slight, but significant, increase in hepatic 6-keto-PGF1 alpha in non-induced rats. In contrast, depletions of hepatic, renal and pulmonary tissue GSH by DEM (1 mL/kg body wt) to 12, 50 and 30% of control, respectively, were associated with elevations of 6-keto-PGF1 alpha in these tissues and in plasma obtained by right ventricular heart puncture. Pretreatment of rats with 3-MC had no significant effect on tissue GSH or prostanoid levels in controls or DEM-treated rats but plasma levels of 6-keto-PGF1 alpha were lower in comparison to non-induced rats. DEM with or without 3-MC pretreatment was associated with increased TxB2 in renal tissue, whereas DEM elevated TxB2 only in pulmonary tissue from non-induced rats. It appears that factors besides GSH depletion may be required to raise plasma and/or tissue 6-keto-PGF1 alpha levels in vivo.

Biosensors.

This review introduces biosensors as analytical devices that respond selectively to analytes in appropriate samples and convert their concentrations into electrical signals via a combination of a biological recognition system and a suitable transducer. The last decade has seen dramatic advances in the design of sensor configurations, the marriage of biological systems with modern monolithic silicon and optical technologies, the development of effective electron-exchange systems and the introduction of direct immunosensors.

Endometrial oxytocin binding sites in normal women and in subfertile patients.

Specific binding of oxytocin to high affinity sites in endometrial membrane preparations has previously been shown in sheep. Endometrial tissue preparations from 27 'normal' women of proven fertility were incubated with tritiated oxytocin and the existence of significant binding sites in human endometrium was shown. Furthermore, the level of binding sites underwent a cyclical variation with the highest concentration of binding at midcycle. A cyclical pattern of binding site concentration not unlike that found in the normal women was shown in 19 subfertile patients taking clomiphene. However, in 20 subfertile patients not taking clomiphene, no cyclical pattern emerged with significantly lower levels of binding sites in the mid-portion of the cycle (P less than 0.02) and significantly higher levels in the mid-late luteal phase (P less than 0.01), as compared to the normal women. In the mid-portion of the cycle levels were significantly lower in the subfertile patients not taking clomiphene (P less than 0.03) as compared to those taking clomiphene. No significant differences were shown between the normal women and those patients taking clomiphene.

A microelectronic conductimetric biosensor.

The fabrication and operation of a microelectronic conductimetric biosensor is described. The device monitors the change in solution conductance occasioned by the catalytic action of enzymes immobilised over a planar conductance cell comprising serpentined and interdigitated metal conductor tracks. The output of the instrument was linear over a 3 min period on addition of urea to a sample cell overlaid with immobilised urease. The responses to any given urea concentration were reproducible to within approximately +/- 1%. The device responds to urea present in serum samples.