Contribution of usage to endoscope working channel damage and bacterial contamination.

BACKGROUND: Biofilm formation has been shown to be associated with damaged areas of endoscope channels. It was hypothesized that the passage of instruments and brushes through endoscope channels during procedures and cleaning contributes to channel damage, bacterial attachment and biofilm formation. AIM: To compare surface roughness and bacterial attachment in used and new endoscope channels in vivo and in vitro. METHODS: Surface roughness of 10 clinically used (retired) and seven new colonoscope biopsy channels was analysed by a surface profiler. For the in-vitro study, a flexible endoscope biopsy forceps was passed repeatedly through a curved 3.0-mm-diameter Teflon tube 100, 200 and 500 times. Atomic force microscopy was used to determine the degree of inner surface damage. The number of Escherichia coli or Enterococcus faecium attached to the inner surface of the new Teflon tube and the tube with 500 forceps passes in 1 h at 37oC was determined by culture. RESULTS: The average surface roughness of the used biopsy channels was found to be 1.5 times greater than that of the new biopsy channels (P=0.03). Surface roughness of Teflon tubes with 100, 200 and 500 forceps passes was 1.05-, 1.12- and 3.2-fold (P=0.025) greater than the roughness of the new Teflon tubes, respectively. The number of E. coli and E. faecium attached to Teflon tubes with 500 forceps passes was 2.9-fold (P=0.021) and 4.3-fold (P=0.004) higher compared with the number of E. coli and E. faecium attached to the new Teflon tubes, respectively. CONCLUSION: An association was found between endoscope usage with damage to the biopsy channel and increased bacterial attachment.

The simultaneous use of three ventilatory techniques to maintain oxygenation in a patient undergoing tracheal laser resection of tumour.

Patients undergoing subglottic airway surgery present a challenge to both anaesthetist and surgeon, and often a balance between surgical access and method of ventilation has to be struck. We report a case in which a 38-year-old female with a large mediastinal mass causing distal tracheal obstruction underwent tracheal laser resection. In order to maintain oxygenation throughout she required simultaneous transnasal humidified rapid-insufflation ventilatory exchange, supraglottic high-frequency jet ventilation and suprastomal manual jet ventilation through her tracheostomy stoma. Where the use of one technique alone failed, the simultaneous use of all three maintained oxygenation and facilitated surgical access for the duration of the procedure.

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations.

BACKGROUND: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. OBJECTIVE: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. METHODS: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 µg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. RESULTS AND CONCLUSIONS: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 µg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies.

Osmoadaptation of wine yeast (Saccharomyces cerevisiae) during Icewine fermentation leads to high levels of acetic acid.

AIMS: Volatile acidity (VA) production along with gene expression patterns, encoding enzymes involved in both acetic acid production and utilization, were investigated to relate gene expression patterns to the production of undesired VA during Icewine fermentation. METHODS AND RESULTS: Icewine juice and diluted Icewine juice were fermented using the Saccharomyces cerevisiae wine yeast K1-V1116. Acetic acid production increased sixfold during the Icewine fermentation vs the diluted juice condition, while ethyl acetate production increased 2·4-fold in the diluted fermentation relative to the Icewine. Microarray analysis profiled the transcriptional response of K1-V1116 under both conditions. ACS1 and ACS2 were downregulated 19·0-fold and 11·2-fold, respectively, in cells fermenting Icewine juice compared to diluted juice. ALD3 expression was upregulated 14·6-fold, and gene expressions involved in lipid and ergosterol synthesis decreased during Icewine fermentation. CONCLUSIONS: Decreased expression of ACS1 and ACS2 together with increased ALD3 expression contributes to the higher acetic acid and lower ethyl acetate levels generated by K1-V1116 fermenting under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents a more comprehensive understanding of how and why commercial wine yeast respond at the transcriptional and metabolic level during fermentation of Icewine juice, and how these responses contribute to increased acetic acid and decreased ethyl acetate production.

The Effect of Open-Ended High Tunnels in Western Washington on Late Blight and Physiological Leaf Roll Among Five Tomato Cultivars.

A 3-year study in western Washington from 2010 to 2012 evaluated five tomato cultivars for tomato disease development and yield in open-ended high-tunnel versus open-field settings. Findings in 2010 revealed that severity of late blight, caused by Phytophthora infestans (US-11), was significantly (P = 0.002) lower in high-tunnel compared with open-field experimental plots based on area under disease progress curve (AUDPC) values of 0.02 versus 321, respectively. In spite of rescue foliar fungicide applications to open-field plots in 2011 and 2012, the mean number of late blight infections across cultivars was 1.8 to 30.8 compared with only 0 to 6.5 in high tunnels for these years. Furthermore, accumulated hours of leaf wetness were fewer in high tunnels than the open field each year (857 versus 1,060 in 2010, 598 versus 998 in 2011, and 885 versus 923 in 2012). Cultivar susceptibility to late blight could not be differentiated in high tunnels due to low disease pressure. However, all five cultivars proved susceptible in the open field, with 'Oregon Spring' consistently having the most lesions. In contrast, high-tunnel production contributed to an increased severity of physiological leaf roll compared with open-field production each year, and these values differed significantly (P = 0.0335 and 0.0252) in 2011 and 2012, respectively. AUDPC values for physiological leaf roll showed that Oregon Spring was significantly (P = <0.0001) less susceptible than other cultivars each year. Physiological leaf roll correlated positively (r values of 0.758 to 0. 960) and significantly (P < 0.05) with leaf wetness and air temperature in all years in both high-tunnel and open-field settings but the same was not true for relative humidity. Even with severe physiological leaf roll, high-tunnel production in 2010 resulted in significantly (P < 0.0001) greater total tomato yield than open-field production (35.0 versus 10.6 t ha-1). Although a significant interaction between production system and cultivar occurred in 2011 and 2012, tomato yield always was greater in high-tunnel than open-field plots. Open-ended high tunnels offer tomato growers a potential tool for managing late blight in western Washington while also increasing yield, and could be especially useful in organic production.

First Report of Tomato Pith Necrosis (Pseudomonas corrugata) on Tomato (Solanum lycopersicum) in Washington.

Tomato pith necrosis was observed on 2.7% of tomatoes grown in rows covered with black polyethylene, various biodegradable plastics, and an experimental spunbond poly(lactic) acid agricultural mulch in high tunnel and open field experimental plots, in western Washington in 2011. Symptoms developed on 3-month-old plants and progressed acropetally until night temperatures dropped to 10°C. Affected plants had chlorotic leaves, produced adventitious roots, and pith tissue was brown and either corrugated or rotted. Similar symptoms were observed again in 2012 on 2.0% of plants, but only in experimental plots with black polyethylene mulch. Diseased stem tissue was homogenized with a mortar and pestle in sterile water and the extract was streaked onto King's medium B (KMB) agar. Colonies were white and smooth initially, and after 5 days had an irregular surface and margin and produced a tan diffuse pigment. One isolate, Pc.Sl.2011, was gram-negative, grew at 37°C on nutrient broth yeast (NBY) agar, did not fluoresce on KMB (3), and was arginine dihydrolase positive. A partial 16S fragment, 1,387 bp, was obtained via PCR with universal 27f and 1492f primers. The resulting sequence exhibited 99% identity to Pseudomonas corrugata Roberts & Scarlett, and has been assigned GenBank Accession KC812729. Pathogenicity of Pc.Sl.2011 was tested in two greenhouse trials with five replications of one tomato plant per treatment. Seeds of 'Celebrity' were surface sterilized by soaking in 70% EtOH for 30 s and then 10% NaOCl for 30 s, then rinsed with sterile water and sown into 14 cm diameter pots filled with non-sterile Sunshine Mix #1 (SunGro Horticulture Distribution Inc., Bellevue, WA). Seedlings were inoculated at the four leaf stage using 5 ml NBY broth cultures of Pc.Sl.2011 grown at 28°C for 12 h with agitation. A sterile needle was used to inject 10 µl of either sterile water or a bacterial suspension of 1.0 × 1010 CFU/ml into the axil of the second true leaf. Inoculum concentration was confirmed by NBY dilution plate counts. The plants were incubated in clear polyethylene bags for 4 days and placed in a greenhouse at 21.1 ± 1.2°C with a 14-h photoperiod. The first and second trials were sampled at 8 and 9 weeks after inoculation, respectively. Plants inoculated with sterile water had green pith tissue. However, 60 and 40% of inoculated plants had brown pith tissue around the inoculation site in the first and second trial, respectively, but wilting and adventitious roots were not observed. Stem tissue from the inoculation site of symptomatic plants was homogenized as above, and the extract streaked onto NBY agar plates. Three isolates recovered from inoculated plants from both trials had the same characteristics as the original isolate, including similar colony morphology, ability to grow on NBY at 37°C, and lack of fluorescence on KMB. To our knowledge, this is the first documented report of tomato pith necrosis in Washington. Pith necrosis has been reported previously in high tunnel tomato production (4), where excess nitrogen fertilization occurs with cool evening temperatures (3), and when plastic mulch is utilized (2). In the cool climate of western Washington, successful tomato production requires the use of agricultural mulches and covers that trap heat. Since P. corrugata has been isolated from soil and the tomato seeds of inoculated plants (1), local growers attempting to manage pith necrosis need to select tomato seed lots carefully and avoid applying excess nitrogen, especially when using plastic mulch. References: (1) V. Catara. Mol. Plant Pathol. 8:233, 2007. (2) E. J. Sikora and W. S. Gazaway. Online. ACES.edu ANR-0797, 2009. (3) C. M. Scarlett and J. T. Fletcher. Ann. Appl. Biol. 88:105, 1978. (4) X. Xu et al. Plant Dis. 97:988, 2013.

First Report of Verticillium Wilt on Lettuce (Lactuca sativa) in Washington Caused by Verticillium tricorpus.

Symptoms of Verticillium wilt were observed on lettuce (Lactuca sativa L.) harvested from high tunnel and open field experimental plots in annual, consecutive spring plantings in western Washington from 2010 to 2012. Leaves had v-shaped, chlorotic lesions, and yellow or brown vascular tissue was noted in the crowns. Total disease incidence increased from 0.2% in 2010 to 1.9% in 2011 and to 14.4% in 2012. Verticillium spp. obtained from infected crown tissues and cultured on half-strength potato dextrose agar medium produced yellow pigment, black microsclerotia, white mycelia, tan chlamydospores, and uniseptate conidia averaging 10.6 × 3.7 µm. Isolates were identified tentatively as Verticillium tricorpus I. (3). Three isolates, Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2, were evaluated for pathogenicity on 4-week-old 'Coastal Star' seedlings in two greenhouse trials. In Trial I, four replicates of two duplicate plants per each isolate, and in Trial II, five replicates of one plant per each isolate were inoculated with conidial suspensions adjusted to 2.0 × 106 and 5.0 × 106 conidia/ml, respectively. Additionally, in each trial, two sets of control treatments of five plants each were inoculated with either an isolate of V. dahliae at the same conidial concentration or with sterile water. Root tips were cut and exposed to the suspensions for 5 s, then seedlings were transplanted into Sunshine Mix #1 (SunGro Horticulture Distribution Inc., Bellevue, WA), and kept in a greenhouse at 17.7 ± 3.4°C. Plants were harvested 8 to 9 weeks post-inoculation, and symptoms were rated visually. Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2 caused chlorosis and vascular discoloration on 25, 13, and 13% of the plants in Trial I; and 40, 60, and 20% of plants in Trial II, respectively. V. dahliae caused similar symptoms on 25 and 40% of the plants in the two trials, respectively, but these plants had greater intensity and length of vascular discoloration compared with the three test isolates. None of the water control plants were symptomatic. All V. tricorpus isolates were recovered from inoculated plants, and colony morphologies were similar to the original isolates. The internal transcribed spacer (ITS) rDNA of isolate Vt.Ls.2010 was amplified with ITS4 and ITS6 primer sets. ITS rDNA sequences between Vt.Ls.2010 and two isolates of V. tricorpus in GenBank (Accession Nos. FJ900211 and AB353343) were 100% identical. V. tricorpus is considered a weak pathogen of lettuce crops in California (2), but authors in Japan recently reported pathogenic isolates of V. tricorpus on lettuce (4). To our knowledge, this is the first report of Verticillium wilt caused by V. tricorpus in Washington. Lettuce is the number two crop grown in high tunnels in the United States (1), and cropping lettuce continuously in them can increase the risk of this and other soilborne pathogens. References: (1) E. E. Carey et al. HortTechnology 19:37, 2009. (2) Q.-M. Qin et al. Plant Dis. 92:69, 2008. (3) H. C. Smith. N. Z. J. Agric. Res. 8:450, 1965. (4) T. Usami et al. J. Gen. Plant Pathol. 77:17, 2010.

First Report of Verticillium Wilt Caused by V. dahliae on Grafted Solanum aethiopicum in Washington.

Solanum aethiopicum L., previously S. integrifolium Poir. (4), has been used as a rootstock for commercial, grafted eggplant production throughout Asia (3). In August 2010 and 2011, symptoms of Verticillium wilt were observed on 'Epic' eggplant (S. melongena L.) grafted onto S. aethiopicum at two sites with a history of the disease: one in the irrigated, dryland Columbia Basin of eastern Washington near Eltopia, and the other in maritime western Washington near Mount Vernon. Interveinal chlorosis, V-shaped necrotic lesions, and wilting were evident at both sites in both years. Each year, stems of 20 symptomatic plants from each field site were cut at the soil line to a 20-cm length, surface sterilized for 5 min in a 10% bleach solution, rinsed in tap water for 30 s, cut longitudinally, and incubated in moisture chambers for 4 weeks at room temperature in the dark. Microsclerotia that formed in the stems were typical of those produced by V. dahliae. One isolate, 'MVEgg301', from an infected stem at Mount Vernon, developed dark microsclerotia and verticillate conidiophores in a radiating pattern, typical of Verticillium on half-strength potato dextrose agar (1/2 PDA) medium. The internal transcribed spacer (ITS) region of rDNA amplified by PCR assay using primers ITS6 and ITS4 from mycelia sampled directly from 1/2 PDA media revealed a 100% match with ITS rDNA sequences of >50 V. dahliae accessions in GenBank (1). Pathogenicity of 'MVEgg301' was assessed in two tests. In both, 12 each of non-grafted and grafted (with cv. Epic) S. aethiopicum plants were inoculated with 'MVEgg301' by cutting approximately 5 mm off the root tips and dipping the remaining roots in a suspension of 106 conidia/ml for 5 s. Similarly, 12 each of non-grafted and grafted S. aethiopicum plants were cut and dipped similarly in sterile water as controls. Chlorosis, necrosis, and wilting were observed in 11 of the 12 inoculated, non-grafted plants and 8 of the 12 inoculated, grafted plants in Test I. The same symptoms were observed in 10 of the 12 inoculated, non-grafted plants and 10 of the 12 inoculated, grafted plants in Test II. V. dahliae was reisolated and confirmed from symptomatic, inoculated non-grafted and grafted plants using the stem assay and direct PCR assay described above. Chlorosis, necrosis, and wilting were observed in one non-grafted control plant in Test I, and two non-grafted and four grafted control plants in Test II. The symptoms were mild and likely due to nutritional deficiencies; microsclerotia were not observed in any assayed water-inoculated plant stems. Although there are several reports of V. albo-atrum infecting S. aethiopicum in the United States (2), to our knowledge, this is the first report of V. dahliae causing Verticillium wilt on this eggplant species. This finding is significant because S. aethiopicum is used as a rootstock for control of soilborne diseases like Verticillium wilt in commercial grafted eggplant production (3). References: (1) G. Calmin et al. Biotechnol. Biotechnol. Equ. 21:40, 2007. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN, 1989. (3) M. Oda. Food Fert. Technol. Ctr. Ext. Bul. 480:1, 1999. (4) PBI Solanum Project. 2012. Solanaceae Source. Accessed at http://www.nhm.ac.uk/solanaceaesource/ . Natural History Museum, London, 29 Aug. 2012.

Volume measurement of bone erosions in magnetic resonance images of patients with rheumatoid arthritis.

The volume of bone erosions in the metacarpophalangeal joints is a radiological feature that can be used to track the progression of rheumatoid arthritis. We introduce a hybrid segmentation algorithm that combines region growing and level-set segmentation algorithms to semiautomatically measure the volume of bone erosions in magnetic resonance images. A total of 40 rheumatoid arthritis patients were included in the study. The scans of eight patients were used for training, whereas the remaining 32 scans were used to determine the accuracy, precision, and speed of the technique. The reproducibility of the semiautomated technique and that of manual segmentation was defined in terms of intraclass correlation coefficients. Both techniques were equally precise with intraclass correlation coefficient values greater than 0.9. The hybrid algorithm was highly accurate: the least squares fit between the semiautomated segmentations to those manually traced by a musculoskeletal radiologist resulted in a slope of 1.030 with an x-intercept of 1.385 mm(3) and an R(2) value of 0.923. The semiautomated technique was significantly faster than manual segmentation, which took two to four times longer to complete. Our hybrid algorithm shows promise in the quantitative assessment of radiological features of rheumatoid arthritis in a clinical setting.

Quantitative analysis of subchondral sclerosis of the tibia by bone texture parameters in knee radiographs: site-specific relationships with joint space width.

OBJECTIVES: To determine the ability of radiographic bone texture (BTX) parameters to quantify subchondral tibia sclerosis and to examine clinical relevance for assessing osteoarthritis (OA) progression. We examined the relationship between BTX parameters and each of (1) location-specific joint space width (JSW) [JSW(x)] and minimum JSW (mJSW) of the affected compartment, and (2) knee alignment (KA) angle in knee radiographs of participants undergoing total knee arthroplasty (TKA). DESIGN: Digitized fixed-flexion knee radiographs were analyzed for run-length and topological BTX parameters in a subchondral region using an algorithm. Medial JSW(x) was computed at x=0.200, 0.225, 0.250 and 0.275 according to a coordinate system defined by anatomic landmarks. mJSW was determined for medial and lateral compartment lesions. KA angles were determined from radiographs using an anatomic landmark-guided algorithm. JSW measures and the magnitude of knee malalignment were each correlated with BTX parameters. Reproducibility of BTX parameters was measured by root-mean square coefficients of variation (RMSCV%). RESULTS: Run-length BTX parameters were highly reproducible (RMSCV%<1%) while topological parameters showed poorer reproducibility (>5%). In TKA participants (17 women, 13 men; age: 66+/-9 years; body mass index (BMI): 31+/-6 kg m(-2); WOMAC: 41.5+/-16.1; Kellgren-Lawrence score mode: 4), reduced trabecular spacing (Tb.Sp) and increased free ends (FE) were correlated with decreased JSW after accounting for BMI, gender and knee malalignment. These relationships were dependent on site of JSW measurement. CONCLUSION: High reproducibility in quantifying bone sclerosis using Tb.Sp and its significant relationship with JSW demonstrated potential for assessing OA progression. Increased trabecular FE and reduced porosity observed with smaller JSW suggest collapsing subchondral bone or trabecular plate perforation in advanced knee OA.

Reproducibility of computer-assisted joint alignment measurement in OA knee radiographs.

OBJECTIVES: (1) To investigate the reproducibility of computer-assisted measurements of knee alignment angle (KA) from digitized radiographs of osteoarthritis (OA) participants requiring total knee arthroplasty (TKA) and (2) to determine whether landmark choice affects the precision of KA measurements on radiographs. METHODS: Using a custom algorithm, femoral, central, and tibial measurement-guiding rules were interactively placed on digitized posteroanterior fixed-flexion knee radiographs by mouse control and positioned according to different anatomic landmarks. The angle subtended by lines connecting these guiding rules was measured by three readers to assess interobserver, intraobserver and experience-inexperience reproducibility. Test-retest reproducibility was evaluated with duplicate radiographs from a healthy cohort. Reproducibility was assessed using root-mean square coefficients of variation (RMSCV%). The Bland-Altman method was performed on data obtained from varying anatomic landmarks (confidence interval, CI= 95%). RESULTS: From 16 healthy and 30 TKA participants, reproducibility analyses revealed a high degree of intraobserver (n=38, RMSCV=0.56%), interobserver (n=38, RMSCV=0.72%), test-retest (n=16, RMSCV=0.87%) and experience-inexperience (n=38, RMSCV=0.73%) reproducibility with variances below 1%. Varying the orientation of tibial and femoral rules according to anatomic landmarks produced a difference that exceeded an a priori limit of agreement of -1.11 degrees to +1.67 degrees. CONCLUSION: Our custom-designed software provides a robust method for measuring KAs within digitized knee radiographs. Although test-retest analyses were only performed in a healthy cohort, we anticipate a similar degree of reproducibility in an OA sample. A standardized set of anatomic landmarks employed for KA measurement is recommended since arbitrary selection of landmarks resulted in imprecise KA measurement even with a computer-assisted technique.

Quantitative, small bore, 1 Tesla, magnetic resonance imaging of the hands of patients with rheumatoid arthritis.

OBJECTIVE: To determine if quantitative hand images obtained from an office-based MRI extremity scanner reliably distinguish patients with rheumatoid arthritis from controls. METHODS: The hands of 39 patients suffering from rheumatoid arthritis were imaged using a small bore, 1.0 Tesla Magnetic Resonance Imager. Non-contrast images of the metacarpophalangeal joints and wrist joints were evaluated using a method based on the validated rheumatoid arthritis magnetic resonance imaging system (RAMRIS). The extent and degree of synovitis, bone edema and bone erosions was assessed. Derived scores were compared with the corresponding scores for groups of younger (n=14) and older (n=27) controls with no signs or symptoms of joint disease. RESULTS: The mean (+/-standard error) total joint scores were 0.3+/-0.2 for young controls, 11.5+/-2.4 for older controls and 34.1+/-6.0 for the patients with rheumatoid arthritis. The greatest difference between rheumatoid patients and older controls was observed for synovitis with scores that were greater by a factor of almost 6.5. Scores for erosions and edema were factors of 2.9 and 2.3 greater in rheumatoid arthritis than in controls. The relationship between scores for the same joints on the dominant and non-dominant sides was generally stronger than the relationship between the metacarpophalangeal and wrist joints of the same hand. CONCLUSION: These observations indicate that scoring of hand images obtained from a small bore, office based, 1.0 Tesla MR imager have clinical validity and may be used to distinguish patients with rheumatoid arthritis from aged matched controls.

Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using stable isotope labelled internal standard assay.

A solid phase microextraction (HS-SPME)-GC-MS methodology was established for the analysis of 3-alkyl-2-methoxypyrazines (MPs) in wine using a stable isotope dilution assay. The compounds analysed were 3-isobutyl-2-methoxypyrazine (IBMP), 3-sec-butyl-2-methoxypyrazine (SBMP), and 3-isopropyl-2-methoxypyrazine (IPMP) using their respective deuterated analogues ([2H3]-IBMP, [2H3]-SBMP, [2H3]-IPMP) as internal standards, synthesised during this work. A divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) fibre was selected for isolation of MPs and the effects of matrix parameters such as pH and ethanol concentration were examined in the development of the method. Best results were obtained at a pH of approximately 6 and with a wine dilution factor of 1:2.5, resulting in an ethanol concentration of approximately 5% (v/v). Relative standard deviations (RSDs) of replicate samples were 5.6-7% for all MPs at 5 ng L(-1) and <5% for 15 and 30 ng L(-1) samples. The limit of detection was <0.5 ng L(-1) in juice and 1-2 ng L(-1) in wine. The recovery efficiencies for spiked wine samples were between 99 and 102% for all three MPs. Using this method, we investigated the impact of the Multicoloured Asian Lady Beetle (MALB) on MPs in wine. In red wine fermented with live MALB, IPMP is the most prevalent MP detected, although SBMP concentrations are also increased and IBMP is unchanged from background levels. MALB that have been dead for 1 day before addition to juice can still contribute to elevated SBMP concentrations in wine, but not if they have been dead for 3 days or longer. Clarifying juice prior to fermentation leads to substantially lower IPMP concentration in the subsequent wine when compared with unclarified juice.

Sex-specific developmental changes in muscle size and bone geometry at the femoral shaft.

INTRODUCTION: When expressed as a percentage of the average result in young adults, bone mineral content lags behind bone length before puberty. Even though this observation has led to speculation about bone fragility in children, such relationships could simply be due to scaling effects when measures with different geometrical dimensions are compared. METHODS: The study population comprised 145 healthy subjects (6-25 years, 94 females). Magnetic resonance imaging and dual-energy X-ray absorptiometry were used to determine femur length, bone mineral content, cortical bone mineral density, cross-sectional bone geometry (bone diameter; cortical thickness; total, cortical and medullary areas; cross-sectional and polar moments of area; bone strength index) and muscle area at the proximal one-third site of the femur. Results were dimensionally scaled by raising two-, three- and four-dimensional variables to the power of 1/2, 1/3 and 1/4, respectively. Sex-differences were also assessed before and after functionally adjusting variables for femur length and weight or muscle size. RESULTS: In prepubertal children, unscaled results expressed as percentages of adult values were lowest for variables with the highest dimensions (e.g., moments of area

Detection thresholds for 2-isopropyl-3-methoxypyrazine in Concord and Niagara grape juice.

2-isopropyl-3-methoxypyrazine (IPMP) is the compound responsible for the off-flavor known as ladybug taint, which occurs when Harmnonia axyridis beetles become incorporated with the grapes during juice processing. It is also an important grape-derived component of juice flavor in some varieties. The main objective of this study was to determine the orthonasal (ON) and retronasal (RN) detection thresholds for IPMP in juice. The ASTM E679 ascending forced choice method of limits was used to determine detection thresholds for 26 individuals in Concord and Niagara juices. Group best estimate thresholds (BETs) averaged 0.93 ng/L and were 50% and 21% higher in Concord than in Niagara juices for ON and RN evaluation, respectively. Group BETs for IPMP (ng/L) for Concord were ON: 1.11; RN: 1.02 and for Niagara were ON: 0.74; RN: 0.84. Variation in individual detection thresholds was observed, although familiarity with ladybug taint was not associated with individual threshold values. We conclude that humans are very sensitive to IPMP in juice, and that detection thresholds are more strongly influenced by grape variety than evaluation mode. These results may assist juice producers in establishing tolerance levels for IPMP in juice affected by ladybug taint or derived from grapes of suboptimal ripeness.

Concentration effect of Riesling Icewine juice on yeast performance and wine acidity.

AIMS: The objective of this study was to determine the effect of increasing juice soluble solids above 40 degrees Brix on wine yeast's ability to grow and ferment the juice, with particular focus on acetic acid production, titratable acidity (TA) changes and the maximum amount of sugar consumed by the yeast. METHODS AND RESULTS: Riesling Icewine juices at 40, 42, 44 and 46 degrees Brix were inoculated with K1- V116 at 0.5 g 1(-1) and fermented at 17 degrees C until sugar consumption ceased. Increasing soluble solids showed strong negative linear correlations with yeast growth, sugar consumption and ethanol production (r = -0.999, -0.997 and 0.984, P < 0.001, respectively). Acetic acid, glycerol and TA production normalized to sugar consumed showed strong positive correlations to the initial juice concentration (r = 0.992, 0.963, and 0.937, P < 0.001 respectively) but no correlation was found for ethanol production. The acetic acid produced as a function of sugar consumed was positively correlated to the glycerol produced (r = 0.970, P < 0.001). The final TA of the wines ranged between 11.8 and 13.7 g 1(-1) tartaric acid, increasing by 2.3-3 g 1(-1) over the starting juice. The increase in TA was positively correlated to the increase in acetic acid produced after normalizing the data to the amount of sugar consumed (r =0.975, P < 0.001). The acid equivalents resulting from the increase in acetic acid accounted for 80-100% of the TA increase when converted to units of tartaric acid. In the final Icewines, acetic acid represented 19-20% of wine TA. CONCLUSIONS: Increasing Icewine juice concentration from 40 to 46 degrees Brix increases the proportion of yeast sugar metabolism towards glycerol and acetic acid production to cope with the increased osmotic stress by decreasing yeast growth, sugar consumption rate, the total amount of sugar consumed and the total amount of ethanol produced. The high proportional contribution of acetic acid to titratable acidity in Riesling Icewine may affect acidity perception. SIGNIFICANCE AND IMPACT OF THE STUDY: We have determined that 10% v/v ethanol would not be achievable with initial juice concentrations above 42 degrees Brix and that Riesling Icewine juice above 52.5 degrees Brix would be theoretically unfermentable. The high proportional contribution of acetic acid to TA may be an important factor in the organoleptic balance of these Icewines.

Determination of ortho- and retronasal detection thresholds for 2-isopropyl-3-methoxypyrazine in wine.

2-Isopropyl-3-methoxypyrazine (IPMP) is a grape-derived component of wine flavor in some wine varieties as well as the causal compound of the off-flavor known as ladybug taint (LBT), which occurs when Harmonia axyridis beetles are incorporated with the grapes during juice and wine processing. The main objective of this study was to obtain robust estimates of the orthonasal (ON) and retronasal (RN) detection thresholds (DTs) for IPMP in wines of differing styles. The ASTM E679 ascending forced choice method of limits was used to determine DTs for 47 individuals in 3 different wines--Chardonnay, Gewürztraminer, and a red wine blend of Baco Noir and Marechel Foch. The group best estimate thresholds (BETs) obtained for IPMP (ng/L) were Chardonnay, ON: 0.32; Gewürztraminer, ON: 1.56, RN: 1.15, and red wine blend, ON: 1.03, RN: 2.29. A large variation in individual DTs was observed. Familiarity with LBT was inversely correlated with DTs for Gewürztraminer, and no difference in thresholds was observed between winemakers and nonwinemakers. We conclude that the human DT for IPMP is extremely low and influenced significantly by wine style and evaluation mode. We recommend against the reporting of single-threshold values for wine flavor compounds, and encourage the determination of consumer rejection thresholds for IPMP in wine.

Response of wine yeast (Saccharomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde stress during Icewine fermentation.

AIMS: We previously reported that the aldehyde dehydrogenase encoded by ALD3 but not ALD6 was responsible, in part, for the increased acetic acid found in Icewines based on the expression profile of these genes during fermentation. We have now completed the expression profile of the remaining yeast aldehyde dehydrogenase genes ALD2, ALD4 and ALD5 during these fermentations to determine their contribution to acetic acid production. The contribution of acetaldehyde stress as a signal to stimulate ALD expression during these fermentations was investigated for all ALD genes. The expression of glycerol-3-phosphate encoded by GPD2 was also followed during these fermentations to determine its role in addition to the role we already identified for GPD1 in the elevated glycerol produced during Icewine fermentation. METHODS AND RESULTS: Icewine juice (38.5 degrees Brix, 398 +/- 5 g l(-1) sugar), diluted Icewine juice (20.8 degrees Brix, 196 +/- 4 g l(-1) sugar) and the diluted juice with sugar levels equal to the original Icewine juice (36.6 degrees Brix, 395 +/- 6 g l(-1) sugar) were fermented in duplicate using the commercial wine yeast K1-V1116. Acetic acid and glycerol production increased 8.4- and 2.7-fold in the Icewine vs the diluted juice fermentation, respectively, accompanied by a fourfold transient increase in acetaldehyde in the Icewine condition during the first week. Both mitochondrial aldehyde dehydrogenases encoded by ALD4 and ALD5 were expressed, with ALD5 expression highest at the start of all fermentations and ALD4 expression increasing during the first week of each condition. ALD2, ALD4, ALD5 and GPD2 showed no differential expression between the three fermentation conditions indicating their lack of involvement in elevating acetic acid and glycerol in Icewine. When yeast fermenting the diluted fermentation was exposed to exogenous acetaldehyde, the transient spike in acetaldehyde increased the expression of ALD3 but this response alone was not sufficient to cause an increase in acetic acid. Expression of the other aldehyde dehydrogenases was unaffected by the acetaldehyde addition. CONCLUSIONS: The aldehyde dehydrogenases encoded by ALD2, ALD4 and ALD5 do not contribute to the elevated acetic acid production during Icewine fermentation. Expression of GPD2 was not upregulated in high sugar fermentations and does not reflect the elevated levels of glycerol found in these wines. Acetaldehyde at a concentration produced during Icewine fermentation stimulates the expression of ALD3, but has no impact on the expression of ALD2, -4, -5 and -6. Upregulation of ALD3 alone in the dilute fermentation is not sufficient to increase acetic acid in wine and requires additional responses found in cells under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms that increased acetic acid and glycerol production during Icewine fermentation follows upregulation of ALD3 and GPD1 respectively, but upregulation of ALD3 alone is not sufficient to increase acetic acid production. Additional responses of cells under osmotic stress are required to increase acetic acid in Icewine.

Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Accuracy and test-retest precision of quantitative cartilage morphology on a 1.0 T peripheral magnetic resonance imaging system.

OBJECTIVE: Quantitative magnetic resonance imaging (qMRI) of knee cartilage morphology is a powerful research tool but relies on expensive and often inaccessible 1.5 T whole-body equipment. Here we examine the reproducibility and accuracy of qMRI at 1.0 T by direct comparison with previously validated technology. METHODS: Coronal images of the knee were obtained in six healthy and six osteoarthritic participants. Two data sets were acquired with a 1.5T whole-body magnetic resonance imaging (MRI) system and two with a 1.0 T peripheral MRI system, with repositioning between scans. Proprietary software was used to analyze surface area, volume, and thickness of femoral and tibial cartilage. RESULTS: At 1.0 T, precision errors for surface areas (root-mean-square (RMS) coefficient of variation (CV%)=1.7-2.6%) were higher than those at 1.5 T (1.0-2.1%). For volume and thickness, precision errors were 2.9-5.5% at 1.0 T compared to 1.6-3.4% at 1.5 T. High levels of agreement were found between the two scanners over all plates. With the exception of lateral femoral cartilage (volume and thickness), no statistically significant systematic bias was found between 1.0 T and 1.5 T. CONCLUSIONS: This is the first reported study to show that knee cartilage morphology can be determined with a reasonable degree of accuracy and precision using a 1.0 T peripheral scanner. Peripheral MRI is less costly, can be performed in clinical offices, and is associated with higher patient comfort and tolerance than 1.5 T whole-body MRI. Implementation of qMRI with peripheral systems may thus permit its more widespread use in clinical research and patient care.