We manipulated the enzyme activity levels of the alcohol fermentation pathway, pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH) in Arabidopsis using sense and antisense overexpression of the corresponding genes (PDC1, PDC2, and ADH1). Transgenic plants were analyzed for levels of fermentation and evaluated for changes in hypoxic survival. Overexpression of either Arabidopsis PDC1 or PDC2 resulted in improved plant survival. In contrast, overexpression of Arabidopsis ADH1 had no effect on flooding survival. These results support the role of PDC as the control step in ethanol fermentation. Although ADH1 null mutants had decreased hypoxic survival, attempts to reduce the level of PDC activity enough to see an effect on plant survival met with limited success. The combination of flooding survival data and metabolite analysis allows identification of critical metabolic flux points. This information can be used to design transgenic strategies to improve hypoxic tolerance in plants.Plants are constantly challenged by environmental stresses that reduce crop yield (e.g. salinity, low temperature, drought, and flooding). Soils with excess water account for 15.7% of the arable land in the United States, and flooding accounted for 16.4% of the crop insurance claims, making it the second highest cause of crop loss in the United States (Boyer, 1982). Traditionally, plant breeders have selected directly for increased stress tolerance, however, selection for flooding tolerance has been performed only in rice (Oryza sativa), where a genetic mapping approach was used to identify major and minor genes involved in submergence tolerance (Sripongpangkul et al., 2000; Xu et al., 2000). Transgenic plants with increased tolerance to drought, salinity, low temperatures, or a combination of these stresses (Nelson et al., 1998; Huang et al., 2000) were obtained by introducing genes for the overaccumulation of benign compounds (e.g. Gly betaine, mannitol, and Pro) that are believed to act as osmolytes. Other groups have increased stress tolerance in Arabidopsis by overexpressing transcription factors (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Yamaguchi-Shinozaki and Shinozaki, 2001) or signal transduction components (Piao et al., 2001; Tamminen et al., 2001).There is substantial variation among terrestrial crop species in their ability to tolerate waterlogging conditions. Even rice, which is adapted to life in a flooded environment, incurs damage if the shoots are completely submerged for periods of time (Drew, 1997; Quimio et al., 2000). Genetic variation in flooding tolerance has been found in rice, maize (Zea mays), barley (Hordeum vulgare), and Arabidopsis. In some cases, this variation results from specific mutants (Harberd and Edwards, 1982; Dolferus et al., 1985; Lemke-Keyes and Sachs, 1989; Ricard et al., 1998). Several studies have focused on the effect of overexpression of genes on flooding tolerance. Zhang et al. (2000) overexpressed a gene involved in cytokinin biosynthesis and demonstrated improve...

SummaryAlanine aminotransferase (AlaAT) catalyses the reversible transfer of an amino group from glutamate to pyruvate to form 2-oxoglutarate and alanine. The regulation of AlaAT in several plant species has been studied in response to low-oxygen stress, light and nitrogen application. In this study, induction of Arabidopsis AlaAT1 and AlaAT2 during hypoxia was observed at the transcriptional level, and an increase in enzyme activity was detected in hypoxically treated roots. In addition, the tissue-specific expression of AlaAT1 and AlaAT2 was analysed using promoter:GUS fusions. The GUS staining patterns indicated that both AlaAT genes are expressed predominantly in vascular tissues. We manipulated AlaAT expression to determine the relative importance of this enzyme in low-oxygen stress tolerance and nitrogen metabolism. This was done by analysing T-DNA mutants and over-expressing barley AlaAT in Arabidopsis. The AlaAT1 knockout mutant (alaat1-1) showed a dramatic reduction in AlaAT activity, suggesting that AlaAT1 is the major AlaAT isozyme in Arabidopsis. Over-expression of barley AlaAT significantly increased the AlaAT activity in the transgenic plants. These plants were analysed for metabolic changes over a period of hypoxic stress and during their subsequent recovery. The results showed that alaat1-1 plants accumulate more alanine than wild-type plants during the early phase of hypoxia, and the decline in accumulated alanine was delayed in the alaat1-1 line during the post-hypoxia recovery period. When alanine was supplied as the nitrogen source, alaat1-1 plants utilized alanine less efficiently than wild-type plants did. These results indicate that the primary role of AlaAT1 is to break down alanine when it is in excess. Therefore, AlaAT appears to be crucial for the rapid conversion of alanine to pyruvate during recovery from low-oxygen stress.

Plants, like animals, are obligate aerobes, but due to their inability to move, have evolved adaptation mechanisms that enable them to survive short periods of low oxygen supply, such as those occurring after heavy rain or flooding. Crop plants are often grown on soils subject to waterlogging and many are sensitive to waterlogging of the root zone. The combination of unfavourable weather conditions and suboptimal soil and irrigation techniques can result in severe yield losses. The molecular basis of the adaptation to transient low oxygen conditions has not been completely characterized, but progress has been made towards identifying genes and gene products induced during low oxygen conditions. Promoter elements and transcription factors involved in the regulation of anaerobically induced genes have been characterized. In this paper an account is presented of the molecular strategies that have been used in an attempt to increase flooding tolerance of crop plants.

During waterlogging conditions plants switch from aerobic respiration to anaerobic fermentation to cope with the lack of available oxygen. Plants have two main fermentation pathways: ethanol and lactic acid fermentation. In this paper we carry out a functional analysis of the Arabidopsis lactate dehydrogenase gene, LDH1. Our results indicate that LDH1, like some other anaerobic genes, is expressed in a root-specific manner and is affected by a variety of abiotic stresses (hypoxia, drought, cold) and mechanical wounding. Functional analysis of LDH1 was carried out using transgenic Arabidopsis overexpressing the gene (35S promoter) and a T-DNA knockout line. Overexpression of LDH1 resulted in improved survival of low oxygen stress conditions in roots but not in shoots. Increased lactic acid fermentation also resulted in significantly higher activities of pyruvate decarboxylase (PDC). Knockout mutants of LDH1 showed reduced survival under low oxygen conditions and PDC activity levels were not changed compared with the wild type. Our data suggest that there is an interdependency between the lactic and ethanol fermentation pathways and that lactic acid fermentation may play a role in stimulating ethanol fermentation and improving plant survival. We show also that Arabidopsis plants are able to exude lactate efficiently into the medium, preventing it accumulating to toxic levels in the cells.

Plants, like animals, are obligate aerobes, but due to their inability to move, have evolved adaptation mechanisms that enable them to survive short periods of low oxygen supply, such as those occurring after heavy rain or flooding. Crop plants are often grown on soils subject to waterlogging and many are sensitive to waterlogging of the root zone. The combination of unfavourable weather conditions and suboptimal soil and irrigation techniques can result in severe yield losses. The molecular basis of the adaptation to transient low oxygen conditions has not been completely characterized, but progress has been made towards identifying genes and gene products induced during low oxygen conditions. Promoter elements and transcription factors involved in the regulation of anaerobically induced genes have been characterized. In this paper an account is presented of the molecular strategies that have been used in an attempt to increase flooding tolerance of crop plants.

BACKGROUND Malnutrition is prevalent in inflammatory bowel disease (IBD). Multiple nutrition screening (NST) and assessment tools (NAT) have been developed for general populations, but the evidence in patients with IBD remains unclear. AIM To systematically review the prevalence of abnormalities on NSTs and NATs, whether NSTs are associated with NATs, and whether they predict clinical outcomes in patients with IBD. METHODS Comprehensive searches performed in Medline, CINAHL Plus and PubMed. Included: English language studies correlating NSTs with NATs or NSTs/NATs with clinical outcomes in IBD. Excluded: Review articles/case studies; use of body mass index/laboratory values as sole NST/NAT; age < 16. RESULTS Of 16 studies and 1618 patients were included, 72% Crohn’s disease and 28% ulcerative colitis. Four NSTs (the Malnutrition Universal Screening Tool, Malnutrition Inflammation Risk Tool (MIRT), Saskatchewan Inflammatory Bowel Disease Nutrition Risk Tool (SaskIBD-NRT) and Nutrition Risk Screening 2002 (NRS-2002) were significantly associated with nutritional assessment measures of sarcopenia and the Subjective Global Assessment (SGA). Three NSTs (MIRT, NRS-2002 and Nutritional Risk Index) were associated with clinical outcomes including hospitalizations, need for surgery, disease flares, and length of stay (LOS). Sarcopenia was the most commonly evaluated NAT associated with outcomes including the need for surgery and post-operative complications. The SGA was not associated with clinical outcomes aside from LOS. CONCLUSION There is limited evidence correlating NSTs, NATs and clinical outcomes in IBD. Although studies support the association of NSTs/NATs with relevant outcomes, the heterogeneity calls for further studies before an optimal tool can be recommended. The NRS-2002, measures of sarcopenia and developments of novel NSTs/NATs, such as the MIRT, represent key, clinically-relevant areas for future exploration.

Aim To investigate patient experiences and perceptions of advance care planning (ACP) process in cirrhosis. Methods Purposive sampling was used to identify and recruit participants (N = 17) from discrete patient groups: compensated with no prior decompensation, decompensated and not yet listed for transplant, transplant wait listed, medical contraindications for transplant, and low socioeconomic status. Review and discussion of local ACP videos, documents, and experiences with ACP occurred in two individual interviews and four focus groups. Data were analyzed using inductive content analysis including iterative processes of open coding, categorization, and abstraction. Results Three overarching categories emerged: (1) lack of understanding about disease trajectories and ACP processes, (2) roles of alternate decision makers, and (3) preferences for receiving ACP information. Most patients desired advanced care-planning conversations before the onset of decompensation (specifically hepatic encephalopathy) with a care provider with whom they had a trusting, preexisting relationship. Involvement of the alternate decision makers was of critical importance to participants, as was the use of direct, easy to understand patient education tools that address practical issues. Conclusion Our findings support the need for early advance care planning in the outpatient setting. Outpatient clinicians may play a key role in facilitating these discussions.

Background: Population-based screening programs are credited with earlier colorectal cancer diagnoses and treatment initiation, which reduce mortality rates and improve patient health outcomes. However, recommended screening methods are unsatisfactory as they are invasive, are resource intensive, suffer from low uptake, or have poor diagnostic performance. Our goal was to identify a urine metabolomic-based biomarker panel for the detection of colorectal cancer that has the potential for global population-based screening.Methods: Prospective urine samples were collected from study participants. Based upon colonoscopy and histopathology results, 342 participants (colorectal cancer, 171; healthy controls, 171) from two study sites (Canada, United States) were included in the analyses. Targeted liquid chromatography-mass spectrometry (LC-MS) was performed to quantify 140 highly valuable metabolites in each urine sample. Potential biomarkers for colorectal cancer were identified by com-paring the metabolomic profiles from colorectal cancer versus controls. Multiple models were constructed leading to a good separation of colorectal cancer from controls.Results: A panel of 17 metabolites was identified as possible biomarkers for colorectal cancer. Using only two of the selected metabolites, namely diacetylspermine and kynurenine, a predictor for detecting colorectal cancer was developed with an AUC of 0.864, a specificity of 80.0%, and a sensitivity of 80.0%.Conclusions: We present a potentially "universal" metabolomic biomarker panel for colorectal cancer independent of cohort clinical features based on a North American population. Further research is needed to confirm the utility of the profile in a prospective, population-based colorectal cancer screening trial.Impact: A urinary metabolomic biomarker panel was identified for colorectal cancer with the potential of clinical application.