G-protein estrogen receptor as a regulator of low-density lipoprotein cholesterol metabolism: cellular and population genetic studies.

OBJECTIVE: Estrogen deficiency is linked with increased low-density lipoprotein (LDL) cholesterol. The hormone receptor mediating this effect is unknown. G-protein estrogen receptor (GPER) is a recently recognized G-protein-coupled receptor that is activated by estrogens. We recently identified a common hypofunctional missense variant of GPER, namely P16L. However, the role of GPER in LDL metabolism is unknown. Therefore, we examined the association of the P16L genotype with plasma LDL cholesterol level. Furthermore, we studied the role of GPER in regulating expression of the LDL receptor and proprotein convertase subtilisin kexin type 9. APPROACH AND RESULTS: Our discovery cohort was a genetically isolated population of Northern European descent, and our validation cohort consisted of normal, healthy women aged 18 to 56 years from London, Ontario. In addition, we examined the effect of GPER on the regulation of proprotein convertase subtilisin kexin type 9 and LDL receptor expression by the treatment with the GPER agonist, G1. In the discovery cohort, GPER P16L genotype was associated with a significant increase in LDL cholesterol (mean±SEM): 3.18±0.05, 3.25±0.08, and 4.25±0.33 mmol/L, respectively, in subjects with CC (homozygous for P16), CT (heterozygotes), and TT (homozygous for L16) genotypes (P<0.05). In the validation cohort (n=339), the GPER P16L genotype was associated with a similar increase in LDL cholesterol: 2.17±0.05, 2.34±0.06, and 2.42±0.16 mmol/L, respectively, in subjects with CC, CT, and TT genotypes (P<0.05). In the human hepatic carcinoma cell line, the GPER agonist, G1, mediated a concentration-dependent increase in LDL receptor expression, blocked by either pretreatment with the GPER antagonist G15 or by shRNA-mediated GPER downregulation. G1 also mediated a GPER- and concentration-dependent decrease in proprotein convertase subtilisin kexin type 9 expression. CONCLUSIONS: GPER activation upregulates LDL receptor expression, probably at least, in part, via proprotein convertase subtilisin kexin type 9 downregulation. Furthermore, humans carrying the hypofunctional P16L genetic variant of GPER have increased plasma LDL cholesterol. In aggregate, these data suggest an important role of GPER in the regulation of LDL receptor expression and consequently LDL metabolism.

Spore germination of the psychrotolerant, red meat spoiler, Clostridium frigidicarnis.

AIMS: To determine germination triggers of Clostridium frigidicarnis, an important spoilage bacterium of chilled vacuum-packed meat. METHODS AND RESULTS: Germination of Cl. frigidicarnis spores in the presence of a range of potential nutrient and non-nutrient germinants was tested by monitoring the fall in optical density and by phase-contrast microscopy. The amino acid L-valine induced strong germination when paired with L-lactate in sodium phosphate under anaerobic conditions. Several other amino acids promoted germination when paired with L-lactate in sodium phosphate and the co-germinants NaHCO3 and L-cysteine. Heat activation, while not necessary for germination, increased the rate of germination. Spore germination was not observed when spores were incubated aerobically. CONCLUSIONS: Spores of psychrotolerant Cl. frigidicarnis germinated in the presence of L-valine in combination with L-lactate in sodium phosphate buffer under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Anaerobic conditions, L-valine and L-lactate, have been identified as triggering germination in Cl. frigidicarnis, and are all present in packs of fresh, vacuum-packaged, red meat. This new information adds to what is known about red meat spoilage by cold tolerant clostridia and can be used to develop intervention strategies to prevent meat spoilage.

Computed tomography-magnetic resonance image registration in radiotherapy treatment planning.

Magnetic resonance imaging (MRI) is being increasingly used in radiotherapy treatment planning (RTP). MRI has the potential to provide improved localisation of target volumes, leading to better tumour control rates and reduced normal tissue complications, due to capabilities including excellent soft-tissue discrimination and the ability to provide scans in which the image contrast is weighted according to different tissue properties. When computed tomography (CT)-MRI image registration is deployed, MR's advantages are combined with CT's geometrical security and its ability to provide electron density information. The quality of CT-MRI image registration can be favourably influenced by aspects of scan acquisition, including patient positioning/immobilisation and scan protocols. Appropriate protocols can ameliorate the possible presence of MR spatial distortions and other artefacts, but quality assurance of scanning remains essential. Here, the methods and quality assurance of CT-MR image registration are discussed. Developments in MRI scanner technology are progressively offering advantages for RTP, in terms of the possibility of better matching of patient positioning versus CT in a greater range of anatomical regions, while allowing thinner slices for better image quality in reformatted orthogonal planes.

MRI image-based FE modelling of the pelvis system and bladder filling.

In this study, high-resolution magnetic resonance imaging was performed in the transaxial, coronal and sagittal planes to provide comprehensive structural details of the bladder and surrounding systems. Detailed finite-element (FE) models that were specific to each participant were developed by rendering the images, and the process of bladder filling was simulated. The overall model of bladder deformation was compared with repeated images of the filled bladder that were obtained using computed tomography to validate the FE models. The relationship between the changes in the key dimensions of the bladder and the increase in bladder volume during the filling process was also investigated. The numerical results showed that the bladder dimensions increased linearly with its volume during the filling process and the predicted coefficients are comparable to some of the published clinical results.

Monte Carlo simulation and polymer gel dosimetry of 60 MeV clinical proton beams for the treatment of ocular tumours.

Monte Carlo (MCNPX) simulations of a clinical proton beam-line under a range of beam conditions have been compared with MR analysis of irradiated polymer gel (BANG-1). Gel results were found to under-estimate the height of the full energy Bragg peak relative to simulation by the order of 30%, due to increased LET in this region, which has been reported elsewhere. Comparison of narrow-beam lateral profiles suggests a slight over-prediction of lateral proton scatter in MCNPX, which has been reported previously.

Determination of lipoprotein(a) kringle repeat number from genomic DNA: copy number variation genotyping using qPCR.

Plasma lipoprotein(a) [Lp(a)] concentration is related to risk of cardiovascular disease. The defining protein component of Lp(a) particles, apolipoprotein(a) [apo(a)], is encoded by the LPA gene. Apo(a) is extremely heterogeneous in size due to a common copy number variation, leading to a variable number of kringle-IV type 2 (KIV2)-like domains. Alleles with fewer KIV2 repeats, encoding smaller apo(a) isoforms, are associated with higher plasma Lp(a) concentrations. Two principal methods to detect variation in KIV2 repeat number are electrophoresis with immunoblotting to detect apo(a) protein isoforms or pulse-field electrophoresis of unamplified genomic DNA to detect the variation of the LPA gene. Both methods are technically challenging, laborious, and time consuming. Here, we report a rapid method to determine the number of KIV2 repeats in LPA from genomic DNA using quantitative real-time polymerase chain reaction (qPCR). With qPCR, we found KIV2 repeat number was correlated with both apo(a) isoform size as determined by immunoblotting (r(s) = 0.50, P < 1 x 10(-6)) and with plasma Lp(a) concentration (r(s) = 0.30, P < 1 x 10(-6)). The qPCR technique permits rapid evaluation of apo(a) size from genomic DNA, and thus would provide an adjunctive genomic variable, in addition to LPA single nucleotide polymorphisms, for evaluating the genetic determinants of plasma Lp(a) concentration in genetic epidemiology studies of cardiovascular disease outcomes.

Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi.

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (approximately 93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi. Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of approximately 93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.

Efficacy of in-feed probiotics against Aeromonas bestiarum and Ichthyophthirius multifiliis skin infections in rainbow trout (Oncorhynchus mykiss, Walbaum).

AIMS: The aim of this study was to assess the efficacy of in-feed probiotics as a preventive measure against skin infections caused by Aeromonas bestiarum and Ichthyophthirius multifiliis (Ich) in rainbow trout. METHODS AND RESULTS: Fin rot was induced in fish by intradermal injection with 0.1 ml volumes containing 10(5) cells per ml A. bestiarum at the base of the dorsal fin. Ich infections resulted from immersion in Ich-contaminated water. Each probiotic was administered orally [10(8) cells per g feed for GC2 (Aeromonas sobria) and 10(10) cells per g feed for BA211 (Brochothrix thermosphacta)] for 14 days. Results showed that, after challenge with A. bestiarum, probiotics GC2 and BA211 led to 76% and 88% survival, respectively, in contrast to 22% survival for controls. Fish fed with probiotic GC2 had 100% survival after challenge with Ich compared with 2% for probiotic BA211 and 0% for controls. Analysis of innate immune responses revealed that probiotic GC2 promoted higher phagocytic activity, whereas probiotic BA211 led to enhanced respiratory burst activity. CONCLUSION: Of the two probiotics examined, GC2 was more effective in protecting against both fin rot and Ich. Each probiotic appeared to stimulate different pathways within the innate immune system. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first demonstration that probiotics can protect fish against surface infections. Furthermore, this is the first time a probiotic has been shown to protect against a eucaryotic pathogen, namely I. multifiliis.

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum).

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.

The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum).

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 x 10(8) cells g(-1) of feed, JB-1 led to a reduction in mortalities to 0-13% after challenge with a range of bacterial pathogens compared to 80-100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0-16% following the challenge compared to 80-100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss).

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum).

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.

Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum).

From a comparison of 125 bacterial isolates recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus sp., a culture was obtained which was effective at preventing clinical disease caused by Lactococcus garvieae and Streptococcus iniae when used as a feed additive. The culture, Aeromonas sobria GC2, was incorporated into the feed and fed to rainbow trout (average weight = 20 g) for 14 days at a dose equivalent to 5 x 10(7) cells g(-1) of feed. Whereas the untreated controls experienced losses of 75-100% when challenged intraperitoneally with L. garvieae and S. iniae, the probiotic-treated groups remained healthy with total mortalities of only 0-6%. Formalized and sonicated preparations of GC2 and cell-free supernatant fared less well. The mode of action reflected stimulation of innate immunity, namely an increased number of leucocytes and enhanced phagocytic and respiratory burst activity.

Modelling concepts of proton eye radiotherapy.

Information concerning the application of proton beams in radiotherapy of ocular tumours is provided using conventional website technology by a number of treatment facilities around the world. We hypothesized, however, that many of the key concepts would be better conveyed by an interactive computer model utilizing virtual reality technology. We describe the implementation, using Virtual Reality Modelling Language (VRML), of such a model, the Proton Therapy Concepts Demonstrator (PTCD). The World-Wide-Web-accessible PTCD is intended to provide information useful both to trainee and to qualified clinical staff and also to patients. A model was created of the radiotherapy room that was linked to an interactive model depicting a simple explanation of the process of proton eye radiotherapy. This model also allows the user to explore specific elements of the treatment planning and delivery process, such as beam collimation and range modulation. A further level of detail has been provided by a dynamic model demonstrating how a specific modulated dose distribution is achieved as the time integration of discrete modulated Bragg peaks. We believe this VR-based model has the potential of enabling users to gain more rapid insight into the proton therapy process. Its development as an educational tool is continuing, following feedback collected from clinical staff and patients.

Variable association between genetic variation in the CYP7 gene promoter and plasma lipoproteins in three Canadian populations.

The promoter sequence variant -278A in the CYP7 gene, which encodes cholesterol 7-alpha hydroxylase, was previously reported to be associated with reduced plasma low density lipoprotein (LDL) cholesterol concentration. We tested for association of CYP7-278A with plasma lipoprotein traits in samples taken from three distinct Canadian populations: 594 Alberta Hutterites, 325 Ontario Oji-Cree and 190 Keewatin Inuit. The CYP7-278A allele frequencies in these three groups were 0.708, 0.466 and 0.490, respectively. The frequencies of CYP7-278A/A homozygotes were 0.481, 0.215 and 0.247, respectively. In the Hutterites, CYP7-278A was associated with reduced plasma HDL-cholesterol and apolipoprotein AI concentration. In the Oji-Cree, CYP7-278A was not significantly associated with any plasma lipoprotein trait. In the Inuit CYP7-278A was associated with elevated plasma total and LDL-cholesterol. There was no consistent relationship between the population mean plasma LDL-cholesterol concentration and the population CYP7-278A frequency. Our findings suggest that the common -278A promoter variant of CYP7 was inconsistently associated with variation in plasma LDL- and HDL-cholesterol in samples from three independent populations. The inconsistencies could be due to differences in genetic background or to unspecified environmental or genetic factors.

Absence of association between genetic variation in the LIPC gene promoter and plasma lipoproteins in three Canadian populations.

The promoter sequence variant -480T in the hepatic lipase gene (LIPC) has been shown to be significantly associated with low post-heparin hepatic lipase activity. Some studies have also found that the -480T variant is associated with elevation in plasma HDL cholesterol. We tested for associations of LIPC -480T with plasma lipoprotein traits in samples taken from three distinct Canadian populations: 657 Alberta Hutterites, 328 Ontario Oji-Cree and 210 Keewatin Inuit. Plasma HL activity was not available for analyses. The LIPC -480T allele frequencies in these three groups, respectively, were 0.219, 0.527 and 0.383, and the prevalence of LIPC -480T/T homozygotes was, respectively, 0.042, 0.274 and 0.167. No significant association was found between LIPC -480T and plasma HDL cholesterol or apolipoprotein AI concentration, after adjusting for covariates including gender and body mass index. There was no consistent relationship between the population mean plasma HDL cholesterol concentration and the population LIPC -480T frequency. Our findings are consistent with the idea that the common promoter variation in LIPC, which has been reported to be associated with variation in post heparin HL activity and HDL triglyceride concentration, is not always associated with variation in plasma HDL cholesterol concentration, possibly due to yet unspecified environmental or genetic factors.

Determination of tumour regression rates during radiotherapy for cervical carcinoma by serial MRI: comparison of two measurement techniques and examination of intraobserver and interobserver variability.

Tumour regression rates of 11 patients with cervical carcinoma were estimated during external beam radiotherapy (EBRT) using serial MRI (average time interval 7 days; range 3-15 days). An average of five investigations (range 4-8) was performed per subject. Tumour volume was measured by two observers using the Cavalieri method of modern design stereology in combination with (a) planimetry and (b) point counting. The mean precision of all the volume estimates obtained by manually tracing the outline of the tumour was 6.6%. The mean precision obtained by counting an average of 176 points per investigation on the same transects was 6.7%. The intraobserver repeatability of planimetry, interobserver reproducibility of planimetry and point counting were excellent with no significant difference between the volume estimates obtained using either technique. Based on the planimetry measurements, initial tumour volumes ranged from 6.5 to 222 cm3 (mean 63 cm3, median 44 cm3). Based on the point counting measurements, initial tumour volumes ranged from 7.2 to 235 cm3 (mean 68 cm3, median 46 cm3). Tumour regression began within a few days of commencing EBRT and showed an exponential relationship with time (p < 0.01). There was good agreement between the regression rates obtained by planimetry and those obtained by point counting. No significant correlation was found between initial tumour volume and tumour regression rate for either planimetry or point counting. Planimetry measurements were, on average, obtained in about half the time taken for point counting (i.e. 30 min and 50 min, respectively). Although point counting is generally likely to be the more efficient approach, planimetry may be the preferred approach for estimating tumour volume when a purpose built track ball is available and the tumour morphology is relatively simple. Volume measurement should be obtained using the Cavalieri method to ensure that the estimates are unbiased and that their precision can be predicted. The measured tumour regression rates may have important implications for improving local tumour control, optimum timing of brachytherapy and minimizing the risk of radiation damage.

Teaching quantitative and qualitative analysis to undergraduate students.

Data analysis preparation for undergraduate nursing students conventionally has consisted of a required statistics course and a brief discussion of analysis strategies within an introductory research course. For students to view inquiry as integral to their practice, it is imperative that data analysis be taught in a manner whereby it may be incorporated into their repertoire of skills for nursing inquiry. This article describes an interdisciplinary, introductory course in quantitative and qualitative analysis taught in the context of three requisite inquiry courses: knowledge development, nursing inquiry, and nursing research.