ABSTRACT
BACKGROUND: Patients with cardiogenic shock or end-stage heart failure can be maintained on mechanical circulatory support (MCS) devices. Once a patient undergoes placement of a device, obtaining and maintaining therapeutic anticoagulation is vital. Guidelines recommend the use of institutional protocols to assist in dosing and titration of anticoagulants. OBJECTIVE: The purpose of this study was to characterize the use of bivalirudin before and after the implementation of a standardized titration protocol in patients with MCS. METHODS: A retrospective review of patients who received bivalirudin for MCS (VA ECMO [veno-arterial extracorporeal membrane oxygenation], Impella, or LVAD [left ventricular assist device]) before and after the implementation of the titration protocol into the electronic health record (EHR) was conducted. The primary outcome was to compare the proportion of therapeutic activated partial thromboplastin time (aPTT). Secondary outcomes included number of subtherapeutic and supratherapeutic aPTTs, incidence of bleeding and clotting events, bivalirudin titrations per day, and percentage of patients with therapeutic aPTT level. RESULTS: A total of 100 patients were included (precohort = 67; postcohort = 33). The proportion of therapeutic aPTTs was significantly higher in the postcohort than that in the precohort (62% vs 48%; P < 0.001). The postcohort had 0% of patients failing to achieve therapeutic aPTT levels. The number of titrations per day was significantly lower in the postcohort, with 1.20 titrations per day versus 1.93 in the precohort (P < 0.001). CONCLUSIONS: Implementation of the bivalirudin titration nomograms within the EHR significantly increased the number of therapeutic aPTTs, reduced the number of patients who never achieved a therapeutic aPTT, and reduced the required number of titrations per day.
ABSTRACT
Pharmacist contraception services are growing across the United States. Several states have authorized pharmacists to prescribe contraception, and the interest in other states continues to grow. Opposition to these practices exists and centers on discussions related to safety, training, cost, and fragmentation of care. We review these arguments and provide evidence refuting these concerns. Pharmacist-prescribed contraception increases access to care, and patients express interest in utilizing this service at the pharmacy. Pharmacists follow evidence-based recommendations. Counseling on preventative services and referral to other providers is part of contraception care by pharmacists. Training programs have been developed to equip both pharmacy students and pharmacists with the knowledge, skills, and tools needed to successfully provide these services. This article can serve as a guide for pharmacists and advocates when discussing pharmacist-prescribed contraception with policymakers, patients, and other healthcare professionals.
ABSTRACT
Engineering high biomass plants that produce oil (triacylglycerol or TAG) in vegetative rather than seed-related tissues could help meet our growing demand for plant oil. Several studies have already demonstrated the potential of this approach by creating transgenic crop and model plants that accumulate TAG in their leaves and stems. However, TAG synthesis may compete with other important carbon and energy reserves, including carbohydrate production, and thereby limit plant growth. The aims of this study were thus: first, to investigate the effect of TAG accumulation on growth and development of previously generated high leaf oil tobacco plants; and second, to increase plant growth and/or oil yields by further altering carbon fixation and partitioning. This study showed that TAG accumulation varied with leaf and plant developmental stage, affected leaf carbon and nitrogen partitioning and reduced the relative growth rate and final biomass of high leaf oil plants. To overcome these growth limitations, four genes related to carbon fixation (encoding CBB cycle enzymes SBPase and chloroplast-targeted FBPase) or carbon partitioning (encoding sucrose biosynthetic enzyme cytosolic FBPase and lipid-related transcription factor DOF4) were overexpressed in high leaf oil plants. In glasshouse conditions, all four constructs increased early growth without affecting TAG accumulation while chloroplast-targeted FBPase and DOF4 also increased final biomass and oil yields. These results highlight the reliance of plant growth on carbon partitioning, in addition to carbon supply, and will guide future attempts to improve biomass and TAG accumulation in transgenic leaf oil crops.
ABSTRACT
This article examines contemporary representations of female and male scientists in The New York Times with a particular emphasis on stereotypes related to gender and science as a profession. The selected series of profiles is approximately proportional in its representation of women in science and generally gives a rounded and diverse picture of their subjects. Traditionally 'masculine' characteristics (e.g. individual drive and brilliance) as well as 'feminine' communal skills (e.g. collaboration, communication and teamwork) are attributed to both male and female scientists. Nevertheless, textual and image analyses reveal that some differences remain in the treatment of male and female subjects, particularly in the unequal focus on combining family and career. This research identifies progress in media representations of scientists in comparison to previous studies. However, there is still room for improvement, especially in the representation of scientists from culturally and linguistically diverse backgrounds.
ABSTRACT
Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.
Subject(s)
Lipids/biosynthesis , Plant Leaves/metabolism , Plant Oils/analysis , Sorghum/metabolism , Amino Acids/analysis , Amino Acids/metabolism , Lipid Metabolism , Lipids/analysis , Plant Leaves/chemistry , Plant Oils/metabolism , Plants, Genetically Modified/metabolism , Sorghum/chemistry , Starch/analysis , Starch/metabolism , Triglycerides/metabolism , Up-RegulationABSTRACT
OBJECTIVE: To measure the association between health literacy and both patient-reported and clinical outcomes in patients with non-insulin-treated type 2 diabetes. RESEARCH DESIGN AND METHODS: We surveyed patients with non-insulin-treated type 2 diabetes (n = 448) from 15 primary care practices. The association between health literacy and patient-reported and clinical outcomes, including numeracy of self-monitoring of blood glucose (SMBG) use, how often physicians advised patients to conduct SMBG testing, and glycemic control (as measured by A1C), was investigated. RESULTS: Study participants included 448 patients with non-insulin-treated type 2 diabetes located within central North Carolina. Participants with limited health literacy had poorer glycemic control (A1C 7.7 ± 1.1% vs. 7.5 ± 1.0%, P = 0.016) despite using SMBG testing more frequently (daily SMBG testing 49.3 vs. 30.7%, P = 0.001) compared to individuals with adequate health literacy. The difference in how often physicians advised patients to conduct SMBG testing between limited and adequate health literacy groups was not significant (P = 0.68). CONCLUSION: Limited health literacy was associated with poorer glycemic control and an increased frequency of SMBG testing in patients with non-insulin-treated type 2 diabetes. There was no significant difference in how often physicians advised patients to conduct SMBG testing between patients with limited and adequate health literacy.
ABSTRACT
BACKGROUND: Diabetes management is influenced by a number of factors beyond the individual-level. This study examined how neighborhood social disorganization (i.e., neighborhoods characterized by high economic disadvantage, residential instability, and ethnic heterogeneity), is associated with diabetes-related outcomes. METHODS: We used a multilevel modeling approach to investigate the associations between census-tract neighborhood social disorganization, A1c, and self-reported use of acute or emergency health care services for a sample of 424 adults with type 2 diabetes. RESULTS: Individuals living in neighborhoods with high social disorganization had higher A1c values than individuals living in neighborhoods with medium social disorganization (B = 0.39, p = 0.01). Individuals living in neighborhoods with high economic disadvantage had higher self-reported use of acute or emergency health care services than individuals living in neighborhoods with medium economic disadvantage (B = 0.60, p = 0.02). CONCLUSIONS: High neighborhood social disorganization was associated with higher A1c values and high neighborhood economic disadvantage was associated with greater self-reported use of acute or emergency health care services. Controlling for individual level variables diminished this effect for A1c, but not acute or emergency health care use. Comprehensive approaches to diabetes management should include attention to neighborhood context. Failure to do so may help explain the continuing disproportionate diabetes burden in many neighborhoods despite decades of attention to individual-level clinical care and education. TRIAL REGISTRATION: For this study, we used baseline data from a larger study investigating the impacts on patient-centered outcomes of three different approaches to self-monitoring of blood glucose among 450 adults with non-insulin dependent type 2 diabetes living in North Carolina. This study was registered as a clinical trial on 1/7/2014 (https://clinicaltrials.gov/ct2/show/NCT02033499).
ABSTRACT
Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting environments, and in eukaryotic microalgae localisation of Rubisco to a microcompartment called the pyrenoid is key. In the model green alga Chlamydomonas reinhardtii, Rubisco preferentially relocalises to the pyrenoid during CCM induction and pyrenoid-less mutants lack a functioning CCM and grow very poorly at low CO2. The aim of this study was to investigate the CO2 response of pyrenoid-positive (pyr+) and pyrenoid-negative (pyr-) mutant strains to determine the effect of pyrenoid absence on CCM induction and gene expression. Shotgun proteomic analysis of low-CO2-adapted strains showed reduced accumulation of some CCM-related proteins, suggesting that pyr- has limited capacity to respond to low-CO2 conditions. Comparisons between gene transcription and protein expression revealed potential regulatory interactions, since Rubisco protein linker (EPYC1) protein did not accumulate in pyr- despite increased transcription, while elements of the LCIB/LCIC complex were also differentially expressed. Furthermore, pyr- showed altered abundance of a number of proteins involved in primary metabolism, perhaps due to the failure to adapt to low CO2. This work highlights two-way regulation between CCM induction and pyrenoid formation, and provides novel candidates for future studies of pyrenoid assembly and CCM function.
Subject(s)
Algal Proteins/genetics , Carbon/metabolism , Chlamydomonas reinhardtii/genetics , Chloroplasts/metabolism , Gene Expression , Photosynthesis , Algal Proteins/metabolism , Chlamydomonas reinhardtii/metabolism , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNAABSTRACT
Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers, and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2-1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.
ABSTRACT
Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.
Subject(s)
Gene Expression Regulation, Plant/genetics , Genetic Enhancement/methods , Metabolic Engineering/methods , Plant Oils/metabolism , Plant Tubers/genetics , Plant Tubers/metabolism , Solanum tuberosum/genetics , Carbohydrates/analysis , Fatty Acids/analysis , Fatty Acids/chemistry , Fatty Acids/metabolism , Galactolipids/metabolism , Genes, Plant , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Phospholipids/metabolism , Plant Oils/analysis , Plant Oils/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Tubers/cytology , Plants, Genetically Modified , Solanum tuberosum/cytology , Starch/analysis , Starch/metabolism , Transformation, Genetic , Triglycerides/metabolismABSTRACT
Residents of assisted living (AL) communities are at high risk for falls, which result in negative outcomes and high health care costs. Adapting effective falls prevention programs for AL quality improvement (QI) has the potential to reduce falls, improve resident quality of life, and reduce costs. This project tested the feasibility and outcomes of an evidence-based multi-component QI program, the Assisted Living Falls Prevention and Monitoring Program (AL-FPMP). Resident posture and gait improved, likely due to exercise and/or physical therapy. Effective falls prevention QI programs can be implemented in AL, and are advised to (1) establish and maintain a falls team to create a culture focused on the reduction of falls risk; (2) teach staff to assess residents using the Morse Falls Scale to increase their awareness of residents' falls risk and improvement; and (3) modify existing exercise programs to address balance and lower body strength.
Subject(s)
Accidental Falls/prevention & control , Assisted Living Facilities , Exercise/physiology , Quality Improvement , Accidental Falls/economics , Aged , Female , Gait , Humans , Male , Pilot Projects , Postural Balance/physiology , Risk FactorsABSTRACT
Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.
Subject(s)
Genetic Enhancement/methods , Metabolic Engineering/methods , Metabolic Networks and Pathways/physiology , Nicotiana/physiology , Plant Leaves/physiology , Plant Oils/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Plant Oils/isolation & purification , Transcription Factors/geneticsABSTRACT
Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.
Subject(s)
Carbon Dioxide/metabolism , Chlamydomonas reinhardtii/enzymology , Organelles/enzymology , Ribulose-Bisphosphate Carboxylase/metabolism , Chlamydomonas reinhardtii/genetics , Organelles/genetics , Ribulose-Bisphosphate Carboxylase/geneticsABSTRACT
In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction.
Subject(s)
Carbon/metabolism , Chlamydomonas reinhardtii/physiology , Darkness , Light , Plant Proteins/metabolism , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Gene Expression , Genes, Plant , Plant Proteins/biosynthesisABSTRACT
OBJECTIVES: To develop and test a person-centered, evidence-based mouth care program in nursing homes. DESIGN: Pre-post assessment, with an 8-week intervention period and a pilot 6-month extension at one site. SETTING: Three North Carolina nursing homes. PARTICIPANTS: Ninety-seven residents and six certified nursing assistants (CNAs). INTERVENTION: CNAs already working in the facilities were trained as dedicated mouth care aides. A psychologist and dental hygienist provided didactic and hands-on training in evidence-based mouth care products and techniques and in person-centered behavioral care. MEASUREMENTS: Primary outcome measures for natural teeth were the Plaque Index for Long-Term Care (PI-LTC) and Gingival Index for Long-Term Care(GI-LTC) and for dentures the Denture Plaque Index (DPI); a dentist unmasked to study design obtained measures. Secondary outcomes included quantity and quality of care provided. RESULTS: Outcome scores significantly improved (P < .001 for PI-LTC and GI-LTC; P = .04 for DPI). Coding of videotaped care episodes indicated that care was more thorough (P < .001-P = .03) but took more time (P < .001) after training. Consistency of care appeared to be more important for natural teeth than dentures. CONCLUSION: As little as 8 weeks of mouth care can significantly improve oral hygiene outcomes. Given the consequences of poor oral hygiene, greater attention to mouth care education and provision are merited. The dedicated worker model is controversial, and future work should assess whether other models of care are equally beneficial.
Subject(s)
Nursing Homes , Oral Hygiene/methods , Oral Hygiene/nursing , Outcome and Process Assessment, Health Care , Patient-Centered Care/methods , Aged , Aged, 80 and over , Evidence-Based Medicine , Female , Humans , Linear Models , Male , North Carolina , Nursing Assistants , Quality of Health CareABSTRACT
PURPOSE: This study examined how characteristics of practice leadership affect the change process in a statewide initiative to improve the quality of diabetes and asthma care. METHODS: We used a mixed methods approach, involving analyses of existing quality improvement data on 76 practices with at least 1 year of participation and focus groups with clinicians and staff in a 12-practice subsample. Existing data included monthly diabetes or asthma measures (clinical measures) and monthly practice implementation, leadership, and practice engagement scores rated by an external practice coach. RESULTS: Of the 76 practices, 51 focused on diabetes and 25 on asthma. In aggregate, 50% to 78% made improvements within in each clinical measure in the first year. The odds of making practice changes were greater for practices with higher leadership scores (odds ratios = 2.41-4.20). Among practices focused on diabetes, those with higher leadership scores had higher odds of performing nephropathy screening (odds ratio = 1.37, 95% CI, 1.08-1.74); no significant associations were seen for the intermediate outcome measures of hemoglobin A1c, blood pressure, and cholesterol. Focus groups revealed the importance of a leader, typically a physician, who believed in the transformation work (ie, a visionary leader) and promoted practice engagement through education and cross-training. Practices with greater change implementation also mentioned the importance of a midlevel operational leader who helped to create and sustain practice changes. This person communicated and interacted well with, and was respected by both clinicians and staff. CONCLUSIONS: In the presence of a vision for transformation, operational leaders within practices can facilitate practice changes that are associated with clinical improvement.
Subject(s)
Delivery of Health Care/organization & administration , Leadership , Primary Health Care/organization & administration , Quality Improvement , Asthma/therapy , Diabetes Mellitus/therapy , Humans , Quality Assurance, Health CareABSTRACT
The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO(2)-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C(4) pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future.
Subject(s)
Chlamydomonas/enzymology , Organelles/metabolism , Ribulose-Bisphosphate Carboxylase/chemistry , Ribulose-Bisphosphate Carboxylase/metabolism , Amino Acid Sequence , Autotrophic Processes/drug effects , Carbon/metabolism , Carbon Dioxide/pharmacology , Chlamydomonas/drug effects , Chlamydomonas/growth & development , Chlamydomonas/ultrastructure , Gene Deletion , Kinetics , Molecular Sequence Data , Organelles/ultrastructure , Oxygen/metabolism , Phenotype , Photosynthesis/drug effects , Protein Structure, Secondary , Spinacia oleracea/drug effects , Spinacia oleracea/enzymology , Structure-Activity RelationshipABSTRACT
BACKGROUND: The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. RESULTS: Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. CONCLUSIONS: The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.