Activating K-Ras mutations outwith 'hotspot' codons in sporadic colorectal tumours - implications for personalised cancer medicine.

BACKGROUND: Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras 'hotspot' codons 12 and 13. METHODS: Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification. RESULTS: Four additional K-Ras mutations (Leu(19)Phe (1 out of 106 tumours), Lys(117)Asn (1 out of 106), Ala(146)Thr (7 out of 106) and Arg(164)Gln (1 out of 106)) were identified. Lys(117)Asn and Ala(146)Thr had phenotypes similar to the hotspot mutations, whereas Leu(19)Phe had an attenuated phenotype and the Arg(164)Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours. CONCLUSIONS: The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

Evaluation of Disease Thresholds and Predictors for Managing Late Blight in Celery.

Late blight of celery, incited by Septoria apiicola, results in necrotic lesions on leaves and petioles and reduces yield when it occurs on marketable petioles. Michigan celery growers typically begin applying fungicides 1 to 3 weeks after transplanting and reapply fungicides at 7- to 10-day intervals to manage late blight. Using disease thresholds or predictors to prompt fungicide sprays may reduce the number of applications needed for effective disease control. Weekly treatments of azoxystrobin alternated with chlorothalonil were initiated early (1 week after transplanting), preventively (4 weeks after transplanting), or when disease symptoms were detected at a trace, 5%, or 10% level on 'Dutchess' celery plants in 2003 and 2004. The early and preventive programs were equally effective in preventing petiole blight and yield loss. The preventive program required three fewer applications, at a savings of up to $134/ha, when compared with an early fungicide program initiated 1 week after transplanting. Delaying the initial fungicide application until disease symptoms were detected resulted in high disease levels at harvest that were often similar to untreated control plants. Additional field trials were established in 2004 and 2005 at a research farm where S. apiicola inoculum was applied and at a commercial field where early blight, caused by Cercospora apii, developed from naturally occurring inoculum. A fungicide program that alternated chlorothalonil with azoxystrobin was initiated preventively and reapplied weekly or according to the Septoria predictor or TOM-CAST 10-disease severity value (DSV) and was compared with the standard, weekly application program initiated early. Combining the use of preventive initial applications with the Septoria predictor or TOM-CAST 10-DSV reduced the number of sprays by two to six while providing disease control that was comparable with the standard weekly fungicide program initiated early. These programs reduced fungicide expenditures by $71 to $213/ha compared with the weekly fungicide program initiated early.

Integrating Disease Thresholds with TOM-CAST for Carrot Foliar Blight Management.

Cercospora carotae and Alternaria dauci cause foliar blight on carrot (Daucus carota subsp. sativus) and are managed with fungicides to prevent yield loss. Sprays are initiated prior to disease symptoms and reapplied frequently, but some of these applications may not be necessary when the threat of disease is low. Delaying the initial fungicide spray until disease symptoms are observed and applying subsequent sprays according to the TOM-CAST disease forecaster may reduce fungicide inputs. The objective of this 2-year field study was to compare preventive, calendar-based application schedules with an integrated management approach that incorporates disease scouting to initiate fungicide application and the TOM-CAST system for timing subsequent fungicide sprays to manage foliar blight on processing, fresh market, and cut-and-peel carrot cultivars in Michigan. Applications of the fungicides chlorothalonil alternated with azoxystrobin were made prior to disease symptoms (0% blight) or when the foliage became blighted at a trace, 5%, or 10% severity level. Fungicides were reapplied every 7 or 10 days or according to TOM-CAST using disease severity value (DSV) thresholds of 15, 20, or 25. Initiating fungicide treatment at a trace level of disease and timing subsequent sprays according to the TOM-CAST 15-DSV forecaster was comparable to the preventive, calendar-based fungicide regime. One to five fewer applications were needed, while fungicide costs were reduced by $21 to $141 per hectare, when spraying at the trace disease threshold and reapplying according to the TOM-CAST 15-DSV program compared with the 7- or 10-day intervals initiated at 0% blight. Fungicide programs initiated at 5 or 10% leaf blight often provided less control than programs initiated at 0% and trace disease. This study highlights the importance of initiating a fungicide program prior to advanced foliar blight and validates the TOM-CAST 15-DSV forecaster for managing Cercospora leaf spot and Alternaria leaf blight in three carrot production systems.

Comparing Disease Predictors and Fungicide Programs for Late Blight Management in Celery.

Late blight, incited by the fungus Septoria apiicola, is the most important foliar disease of celery in Michigan and results in necrotic lesions on leaves and petioles that can reduce yield up to 80%. Most celery growers apply the fungicide chlorothalonil as frequently as every 7 days to protect the crop; however, some of these applications may not be needed when environmental conditions do not favor disease development. The purpose of this study was to evaluate disease predictors using a standard (chlorothalonil) and a reduced-risk (azoxystrobin alternated with chlorothalonil) fungicide program for managing late blight on 'Dutchess' celery in 2003 to 2005. Fungicides were initiated 1 week after transplanting and reapplied every 7 days or according to the Septoria, Cercospora, or TOM-CAST predictors. The TOM-CAST predictor was tested using 10, 15, and 20 disease severity values (DSV) as thresholds for fungicide application. In each year of this study, timing sprays according to the TOM-CAST 10-DSV predictor resulted in disease control comparable with the 7-day interval, but required up to five fewer sprays and reduced fungicide costs up to $215/ha. The Septoria, Cercospora, and TOM-CAST 15-DSV predictors often provided control similar to the 7-day interval, but these predictors were somewhat inconsistent compared with TOM-CAST 10-DSV. The TOM-CAST 20-DSV predictor required the fewest number of sprays but unacceptable levels of disease resulted. The standard and reduced-risk fungicide programs frequently provided similar control; however, azoxystrobin alternated with chlorothalonil was more effective than chlorothalonil alone when disease pressure was high.

Comparing Disease Forecasters for Timing Fungicide Sprays to Control Foliar Blight on Carrot.

Alternaria dauci and Cercospora carotae cause foliar blight on carrot and can reduce yield in severely blighted fields. Historically, fungicides are applied every 7 to 14 days even though applications may be made when environmental conditions do not favor blight development. The purpose of this study was to compare a calendar-based application schedule with three disease forecasting systems for timing fungicide sprays to limit foliar blight, and included (i) an A. dauci disease forecaster, (ii) TOM-CAST, using a threshold of 15 disease severity values, and (iii) a disease forecaster developed to control C. apii on celery. Chlorothalonil was applied weekly or according to the forecasting systems to blight-susceptible 'Cellobunch' carrot plants in 2001 and 2002. Overall petiole health was poor ≥8.3; 10 = 100% petiole necrosis) when fungicides were not used. Although all disease forecasters maintained petiole health (≤5.3; 1 = healthy and vigorous), the TOM-CAST program had the best petiole health rating each year (≤2.8). TOM-CAST prompted 38 to 54% fewer applications than the weekly application schedule, resulting in a fungicide savings of $105 and $147/ha in 2001 and 2002, respectively, while providing similar blight control. The number of sprays also was reduced when fungicides were applied according to the A. dauci and C. apii forecasters, but acceptable blight control was not always achieved.

Combination of estrogen replacement and exercise protects against HDL oxidation in post-menopausal women.

The incidence of atherosclerosis and cardiovascular disease (CVD) in women increases following menopause and has been associated with a reduction in circulating estrogen. Increased CVD risk is also perpetuated by sedentary lifestyle. Growing evidence indicates that oxidation of lipoproteins leads to a powerful immune response, disruption of normal lipoprotein function, and deposition of atherosclerotic plaques. For example, once high-density lipoproteins (HDL) are oxidized, they lose the ability to a) participate in reverse transport of cholesterol to the liver, and b) protect low-density lipoproteins (LDL) against oxidation. The purpose of this study was to determine the effects of combining estrogen replacement and exercise upon lipid peroxidation of the HDL fraction (HDL-ox). Blood samples were drawn from 34 post-menopausal women from four groups: women who were not receiving estrogen replacement and who were sedentary (NSD) (n = 9); women who were not receiving estrogen replacement and who were participating in regular exercise (NEX) (n = 8); women who were receiving estrogen replacement and who were sedentary (ESD) (n = 8); and women who were receiving estrogen replacement and who were participating in regular exercise (EEX) (n = 9). Total-HDL cholesterol was significantly higher (p<0.05) in EEX when compared with NEX, NSD, and ESD. HDL-ox was assessed via malondialdehyde (MDA). Mean (+/- SEM) values for HDL MDA expressed in nM are as follows: NSD = 903.3 +/- 118.4; NEX = 1226.7 +/- 247.7; ESD = 876.7 +/- 116.3; EEX = 537.4 +/- 74.8. EEX lipid peroxidation was significantly (p = 0.02) lower than NEX. Lipid peroxidation tended to be lower in EEX than in NSD and ESD (p = 0.07). These data indicate that the combination of estrogen replacement and regular exercise in post-menopausal women may be most effective in reducing oxidation of HDL in vivo.

Diet and short term plasma lipoprotein-lipid changes after exercise in trained Men.

To test the effect of diet on the short-term lipid response to exercise, fourteen moderately trained (VO2max: 50.2 +/- 6.7 ml/kg/min), healthy men (mean age: 28 +/- 4 years) were alternately fed a high fat (60 +/- 6.7% fat) and a high carbohydrate (63 +/- 3.2% carbohydrate) isoenergetic diet for 2 weeks in a randomized crossover design. During the last 4 days of the treatments, fasting total cholesterol, triglyceride, HDL-cholesterol, and HDL3-cholesterol were measured the day before, and again immediately, 24 hr, and 48 hr after exercise (4190 kJ, 70% VO2max). LDL-cholesterol and HDL2-cholesterol were calculated. Lipid concentrations were adjusted for plasma volume changes after exercise. A 2 (diet) x 4 (time) ANOVA with repeated measures revealed no significant interaction between the diet and exercise treatments. Furthermore, diet alone did not influence lipid concentrations in these trained men. Exercise resulted in an increase in HDL-C (10.7%) and HDL3-C (8.5%) concentrations and a concomitant fall in triglyceride (-25%) and total cholesterol (-3.5%). Thus, we conclude that diet composition does not affect the short-term changes in blood lipids and lipoproteins that accompany a single session of aerobic exercise in moderately trained men.