Eastern Canadian Colorectal Cancer Consensus Conference: standards of care for the treatment of patients with rectal, pancreatic, and gastrointestinal stromal tumours and pancreatic neuroendocrine tumours.

The annual Eastern Canadian Colorectal Cancer Consensus Conference was held in Halifax, Nova Scotia, October 20-22, 2011. Health care professionals involved in the care of patients with colorectal cancer participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management of rectal cancer, including pathology reporting, neoadjuvant systemic and radiation therapy, surgical techniques, and palliative care of rectal cancer patients. Other topics discussed include multidisciplinary cancer conferences, treatment of gastrointestinal stromal tumours and pancreatic neuroendocrine tumours, the use of folfirinox in pancreatic cancer, and treatment of stage ii colon cancer.

Coomassie blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection.

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.

Alterations in protein and gene expression within the barrel cortices of ZnT3 knockout mice: experience-independent and dependent changes.

Much evidence exists for the involvement of vesicular zinc in neurotransmission and cortical plasticity. Recent studies have reported that mice deficient in zinc transporter-3 protein (ZnT3) and thus, vesicular zinc, have significant behavioural and biochemical deficits. Here, we examined whether phenotypic differences existed in the barrel cortices of ZnT3 KO mice using functional proteomics and quantitative PCR. Additionally, by manipulating whisker input, we also investigated experience-dependent changes in protein and gene expression, thereby assaying how cortical plasticity is different in the absence of vesicular zinc. The GABA metabolizing protein ABAT was observed in lower abundances consistently in KO mice. Several presynaptic proteins were identified that were abundant in differing amounts between the WT and KO groups in an experience-dependent manner. At baseline, we observed a decrease in the relative expression of Dlg4, Grin2a, Mt3, and Ntrkb genes in KO mice. The reduced expression of Nrtkb persisted with whisker plucking. These data demonstrate that fundamental changes in the expression of proteins and genes important in neurotransmission occur in the absence of vesicular zinc. Furthermore, the complement of experience-dependent changes were different between WT and KO mice, indicating that the lack of vesicular zinc affects the process of cortical plasticity.

Enabling coupled quantitative genomics and proteomics analyses from rat spinal cord samples.

Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.

Assessing detection methods for gel-based proteomic analyses.

Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.

An initial proteomic analysis of human preterm labor: placental membranes.

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.

Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses.

Here we quantitatively characterize two common homogenization strategies in the analysis of tissue proteomes: classical manual homogenization (MH) and an automated frozen disruption (AFD) technique. In a variety of tissues, many proteins were more efficiently extracted, resolved and detected, with high reproducibility after AFD, amounting to as much as 2% of the total resolved proteome. The benefits of AFD over MH are 2-fold: (1) AFD yields a much more thorough homogenate than MH; and (2) as a deep frozen alternative, AFD maintains a level of biological complexity that is not retained during MH. Thus, AFD coupled with refined 2DE protocols and Sypro Ruby staining yields quantitative proteomic analyses.

Actin is not an essential component in the mechanism of calcium-triggered vesicle fusion.

Actin has been suggested as an essential component in the membrane fusion stage of exocytosis. In some model systems disruption of the actin filament network associated with exocytotic membranes results in a decrease in secretion. Here we analyze the fast Ca2+-triggered membrane fusion steps of regulated exocytosis using a stage-specific preparation of native secretory vesicles (SV) to directly test whether actin plays an essential role in this mechanism. Although present on secretory vesicles, selective pharmacological inhibition of actin did not affect the Ca2+-sensitivity, extent, or kinetics of membrane fusion, nor did the addition of exogenous actin or an anti-actin antibody. There was also no discernable affect on inter-vesicle contact (docking). Overall, the results do not support a direct role for actin in the fast, Ca2+-triggered steps of regulated membrane fusion. It would appear that actin acts elsewhere within the exocytotic cycle.

Postfractionation for enhanced proteomic analyses: routine electrophoretic methods increase the resolution of standard 2D-PAGE.

Here we have addressed common issues of resolution in two-dimensional polyacrylamide gel electrophoresis (2DE) experiments including proteins 'stacked' at pH extremes, unresolved peptides migrating at the front of separation, and areas of the 2D gel obscured by high abundance proteins. Postfractionation, by selective application of well-established electrophoretic separations immediately following standard 2DE, yields markedly improved resolution in these traditional problem areas using no more specialized equipment or techniques than SDS-PAGE itself.

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis.

BACKGROUND: The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. RESULTS: After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. CONCLUSION: This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Nimesulide, a COX-2 inhibitor, does not reduce lesion size or number in a nude mouse model of endometriosis.

BACKGROUND: Women with endometriosis have elevated levels of cyclooxygenase-2 (COX-2) in peritoneal macrophages and endometriotic tissue. Inhibition of COX-2 has been shown to reduce inflammation, angiogenesis and cellular proliferation. It may also downregulate aromatase activity in ectopic endometrial lesions. Ectopic endometrial establishment and growth are therefore likely to be suppressed in the presence of COX-2 inhibitors. We hypothesized that COX-2 inhibition would reduce the size and number of ectopic human endometrial lesions in a nude mouse model of endometriosis. METHODS: The selective COX-2 inhibitor, nimesulide, was administered to estrogen-supplemented nude mice implanted with human endometrial tissue. Ten days after implantation, the number and size of ectopic endometrial lesions were evaluated and compared with lesions from a control group. Immunohistochemical assessment of vascular development and macrophage and myofibroblast infiltration in control and treated lesions was performed. RESULTS: There was no difference in the number or size of ectopic endometrial lesions in control and nimesulide-treated nude mice. Nimesulide did not induce a visually identifiable difference in blood vessel development or macrophage or myofibroblast infiltration in nude mouse explants. CONCLUSION: The hypothesized biological properties of COX-2 inhibition did not influence lesion number or size in the nude mouse model of endometriosis.

Unexpected inhibition of fusion in nucleus-nucleus collisions.

Unstable heavy atomic nuclei not found in nature can be created by fusing two stable nuclei, in a process analogous to colliding charged droplets of liquid. Recently, the formation of a handful of super-heavy nuclei with atomic numbers 114 (ref. 1) and 116 (ref. 2) has been achieved by fusion of heavy nuclei. The electrostatic energy of such systems is very large (which is the reason super-heavy nuclei are unstable), so although the two nuclei may initially be captured by the nuclear potential, rather than fusing, they almost always separate after transfer of mass to the lighter nucleus. This process, called quasi-fission, can inhibit fusion by many orders of magnitude. Understanding this inhibition may hold the key to forming more super-heavy elements. Theoretically, inhibition is predicted (ref. 5 and references therein) when the product Z1Z2 of the charges of the projectile and target nuclei is larger than about 1,600. Here we report measurements of three fusion reactions with Z1Z2 around half this value, each forming 216 88Ra. We find convincing model-independent evidence both of inhibition of fusion, and of the presence of quasi-fission. These results defy interpretation within the standard picture of nuclear fusion and fission.

How soon is too soon? Addiction recovery and family reunification.

This article describes the addiction recovery process and its impact on parenting behaviors-information needed by child welfare workers involved in decisionmaking regarding family reunification. Two models of recovery-from alcoholism and from cocaine addiction-are reviewed, along with issues encountered in recovery, particularly for women. Case examples and discussion demonstrate how child welfare workers can apply these models in determining the appropriateness of reunification.

ARCTIC: assessment of haemodynamic response in patients with congestive heart failure to telmisartan: a multicentre dose-ranging study in Canada.

BACKGROUND: The aim of this study was to examine the acute hemodynamic and neurohormonal effects of the angiotensin II antagonist telmisartan relative to placebo in patients with chronic symptomatic (New York Heart Association class II to III) congestive heart failure and to explore the dose-response relation for these effects. METHODS AND RESULTS: After baseline hemodynamic and neurohormonal measurements made with the use of a pulmonary artery and radial arterial catheter, 82 patients were randomly assigned to placebo or 10, 20, 40, or 80 mg of telmisartan in a double-blind fashion. Hemodynamic and neurohormonal measurements were carried out over 24 hours. Telmisartan caused significant decreases in systemic arterial, pulmonary arterial, and pulmonary capillary wedge pressures with evidence of a dose-response relation for each of these parameters. The drug had no significant effects on heart rate, cardiac index, or systemic vascular resistance. Telmisartan did not have consistent effects on either plasma norepinephrine or plasma atrial natriuretic peptide levels, although it did cause significant increases in both plasma renin activity and angiotensin II levels at higher doses. CONCLUSIONS: The acute administration of the angiotensin II antagonist telmisartan was associated with significant dose-dependent reductions in systemic arterial blood pressure and pulmonary pressures. Long-term follow-up studies are required to translate changes in hemodynamic parameters into a clinical benefit.

Mechanical load enhances the stimulatory effect of serum growth factors on cardiac fibroblast procollagen synthesis.

The mechanical environment is a key determinant of cellular activity in many tissues. In the cardiovascular system it plays a role in tissue remodelling during both development and disease. In the heart changes in mechanical tension stimulate myocyte hypertrophy and fibroblast collagen synthesis. To elucidate the mechanisms for the latter response, we determined the direct effect of mechanical load on cardiac fibroblast activity. Primary cultures of fetal rat cardiac fibroblasts were mechanically loaded in the presence or absence of fetal calf serum or growth factors, and the effects on fibroblast replication and procollagen metabolism and gene expression determined. Procollagen synthesis was increased by 99.7 +/- 4.3% in response to mechanical load and 10% fetal calf serum, compared to 10% fetal calf serum control (P<0.01) after 48 h. Procollagen alpha1(I) steady-state mRNA levels were increased two-fold. No effect was observed in the absence of serum. Transforming growth factor beta1 and insulin-like growth factor 1 have been demonstrated to stimulate procollagen metabolism by these cells. Mechanical load enhanced the response to these growth factors, stimulating alpha1(I) mRNA levels by 4.3 and three-fold, respectively, above growth factor alone controls. These results demonstrate a synergistic effect on procollagen gene expression and metabolism by mechanical load and profibrotic growth factors. Since these factors are released during the development of cardiac hypertrophy, interactions between the mechanical environment and these polypeptides may provide a mechanism for enhanced collagen deposition in the heart.

Vascular innervation in atherogenesis.

Although collar-induced atherosclerosis continues to be used as an investigative tool, the underlying mechanism has not been established. Two primary mechanisms suggested are adventitial ischemia due to reduction in vasa vasorum, and perivascular denervation. We have examined the effect of injuring the common carotid artery in the pattern produced by the ends of a silastic collar, and have correlated the effect on innervation with change in intima/media ratios in normal and cholesterol-fed rabbits. The serum cholesterol of cholesterol-fed rabbits was significantly elevated by 10 days following initiation of cholesterol feeding, and further elevated at 21 days. No structural difference was detected between the uninjured carotid arteries of control and cholesterol-fed rabbits. At the site of injury in freeze injured carotid arteries there was a thickening of the intima which was increased in cholesterol-fed rabbits. The intima at the site of injury was composed of lipid-laden cells embedded in a matrix of collagen and elastin fibres. In carotid artery segments, between two sites of freeze injury, denervation was established by immunohistochemistry and electron microscopy. The denervated segments were not morphologically different from uninjured carotid arteries in either control or cholesterol-fed rabbits. While injury induced intimal thickening and foam cell development, denervation did not. It is concluded that perivascular denervation is a consequence of silastic collar application and is not involved in the induction of atherosclerosis.

Stereotactic biopsy of brain tumours.

Computerized tomography assisted Stereotactic biopsy technique using Leksell stereotactic frame was performed on 27 patients with small, multiple and deep seated brain tumours. There were 19 men and 8 women with an age range from 17 to 65 years. Histological diagnosis of 18 glial tumours, 9 non-glial tumours (5 colloid cysts, 4 metastatic lesions) was obtained. There was no mortality and minimal morbidity of 3.7%, histological diagnosis provided the information regarding differentiation from infectious and vascular lesions and grading of malignancy leading to logical guidance for therapeutic management of each lesion, confirming the value of stereotactic biopsy in brain tumours.

Collagen production and replication by cardiac fibroblasts is enhanced in response to diverse classes of growth factors.

The tissue distribution and cellular effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGF beta 1) and insulin-like growth factor 1 (IGF-1) suggest a potential role for these factors in cardiovascular matrix deposition. The objective of this study was to assess the capacity of these growth factors to promote cardiac fibroblast collagen production and replication in vitro which will lead to studies identifying their role in vivo during cardiac development and disease. Fibroblasts were isolated from fetal rat hearts by explant culture, and their response to growth factors was assessed with respect to fibroblast replication and collagen synthesis. Fibroblast replication was stimulated by PDGF and by bFGF.IGF-1 and TGF beta 1 had no effect on fibroblast replication. Collagen production was stimulated by all of the growth factors tested in order of potency TGF beta 1 > PDGF, IGF > bFGF. None of the growth factors affected the proportion of newly synthesized collagen rapidly degraded. We have shown that TGF beta 1, PDGF, bFGF and IGF-1 are all capable of increasing collagen deposition by cardiac fibroblasts by either stimulating fibroblast replication or collagen synthesis or both. The sensitivity of cardiac fibroblasts to these factors is consistent with their playing a role in the rapid changes in cardiac collagen deposition seen during development and disease.