Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Simultaneous stimulation of sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphophate aldolase and the photorespiratory glycine decarboxylase-H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis.

In this article, we have altered the levels of three different enzymes involved in the Calvin-Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7-bisphosphatase (SBPase), fructose 1,6-bisphophate aldolase (FBPA) and the glycine decarboxylase-H protein (GDC-H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO2 fixation in lines over-expressing SBPase and FBPA. Moreover, the co-expression of GDC-H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene stacking for improving the productivity of food and energy crops.

Effects of kinetics of light-induced stomatal responses on photosynthesis and water-use efficiency.

Both photosynthesis (A) and stomatal conductance (gs ) respond to changing irradiance, yet stomatal responses are an order of magnitude slower than photosynthesis, resulting in noncoordination between A and gs in dynamic light environments. Infrared gas exchange analysis was used to examine the temporal responses and coordination of A and gs to a step increase and decrease in light in a range of different species, and the impact on intrinsic water use efficiency was evaluated. The temporal responses revealed a large range of strategies to save water or maximize photosynthesis in the different species used in this study but also displayed an uncoupling of A and gs in most of the species. The shape of the guard cells influenced the rapidity of response and the overall gs values achieved, with different impacts on A and Wi . The rapidity of gs in dumbbell-shaped guard cells could be attributed to size, whilst in elliptical-shaped guard cells features other than anatomy were more important for kinetics. Our findings suggest significant variation in the rapidity of stomatal responses amongst species, providing a novel target for improving photosynthesis and water use.

A meta-analysis to compare Dacron versus polytetrafluroethylene grafts for above-knee femoropopliteal artery bypass.

BACKGROUND: Surgical revascularization for lower limb ischemia remains an important component for optimization of quality of life and symptoms in patients with peripheral arterial disease. In the absence of a vein graft, prosthetic alternatives are considered. The objective of this meta-analysis was to establish which prosthetic graft, Dacron or polytetrafluroethylene (PTFE), has the better long-term patency in patients undergoing an above-knee femoropopliteal arterial bypass. METHODS: This meta-analysis was performed by use of Cochrane and Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. An electronic search of all relevant databases was performed from 1990 to 2013 with the Medical Subject Headings "Dacron," "polytetrafluroethylene," "PTFE," "above knee," "femoropopliteal," and "bypass" combined with the Boolean operator "AND." The inclusion criteria were randomized controlled trials, use of Dacron vs PTFE prosthetic conduits, and completion of an above-knee femoropopliteal arterial bypass involving adult patients older than 18 years presenting with disabling claudication, rest pain or tissue loss, occlusion of the superficial femoral artery, and reconstitution of the above-knee popliteal artery. Whenever studies included above- and below-knee data, only the above-knee arterial bypass data were extracted and analyzed. Graft patency rates were calculated with RevMan 5.1 software provided by the Cochrane Collaboration. RESULTS: Ninety-one publications were reviewed. After exclusion of duplicate, nonrandomized, and alternative bypass surgery studies, eight randomized controlled trials were identified and included in the meta-analysis. Two of the included trials represented follow-up evaluation of two previous studies, and for the purpose of this analysis, the initial and follow-up studies were subsequently evaluated as one trial. In this meta-analysis, 1192 patients were assessed, including 601 Dacron and 591 PTFE above-knee lower limb arterial bypasses. Primary patency was calculated from all included studies. However, only four studies provided data to evaluate secondary patency. Mean age reported was 66 years. Although all studies described cardiovascular comorbidities and risk factors including myocardial ischemia, diabetes, hypertension, and smoking, exact patient numbers were not consistently provided. Included studies evaluated grafts from 5 to 8 mm. Although primary and secondary patency rates at 12 months were not significantly different (relative risk [RR], 0.78; P = .08, and RR, 0.84; P = .52), 24-, 36-, and 60-month primary patency rates were significantly better with Dacron compared with PTFE grafts (RR, 0.79; P = .003; RR, 0.80; P = .03; RR, 0.85; P = .02). Statistical analysis also supported higher secondary patency rates for Dacron at 24 months (RR, 0.75; P = .02) and 60 months (RR, 0.76-0.77; P = .03-.27). Although primary patency was similar between grafts (28% vs 28%; P = .12), secondary patencies were better with Dacron at 10 years (49% vs 35%; P = .01). Antiplatelet and anticoagulation protocols varied between the trials. There was no difference in amputation, overall morbidity, or mortality rates between the two surgical graft populations. CONCLUSIONS: Current evidence suggests that Dacron prosthetic grafts are superior to PTFE grafts in above-knee femoropopliteal arterial bypass procedures. Further randomized trials targeting standardization of confounding variables, particularly graft size and best medical therapy, are warranted.

Engineering α-carboxysomes into plant chloroplasts to support autotrophic photosynthesis.

The growth in world population, climate change, and resource scarcity necessitate a sustainable increase in crop productivity. Photosynthesis in major crops is limited by the inefficiency of the key CO2-fixing enzyme Rubisco, owing to its low carboxylation rate and poor ability to discriminate between CO2 and O2. In cyanobacteria and proteobacteria, carboxysomes function as the central CO2-fixing organelles that elevate CO2 levels around encapsulated Rubisco to enhance carboxylation. There is growing interest in engineering carboxysomes into crop chloroplasts as a potential route for improving photosynthesis and crop yields. Here, we generate morphologically correct carboxysomes in tobacco chloroplasts by transforming nine carboxysome genetic components derived from a proteobacterium. The chloroplast-expressed carboxysomes display a structural and functional integrity comparable to native carboxysomes and support autotrophic growth and photosynthesis of the transplastomic plants at elevated CO2. Our study provides proof-of-concept for a route to engineering fully functional CO2-fixing modules and entire CO2-concentrating mechanisms into chloroplasts to improve crop photosynthesis and productivity.

Three-dimensional (3D) printed endovascular simulation models: a feasibility study.

BACKGROUND: Three-dimensional (3D) printing is a manufacturing process in which an object is created by specialist printers designed to print in additive layers to create a 3D object. Whilst there are initial promising medical applications of 3D printing, a lack of evidence to support its use remains a barrier for larger scale adoption into clinical practice. Endovascular virtual reality (VR) simulation plays an important role in the safe training of future endovascular practitioners, but existing VR models have disadvantages including cost and accessibility which could be addressed with 3D printing. METHODS: This study sought to evaluate the feasibility of 3D printing an anatomically accurate human aorta for the purposes of endovascular training. RESULTS: A 3D printed model was successfully designed and printed and used for endovascular simulation. The stages of development and practical applications are described. Feedback from 96 physicians who answered a series of questions using a 5 point Likert scale is presented. CONCLUSIONS: Initial data supports the value of 3D printed endovascular models although further educational validation is required.

Developing cross-specialty endovascular simulation training.

BACKGROUND: Simulation is increasingly recognised as a valuable tool in training tomorrow's doctors. This technology has the potential to improve patient safety and address some of the challenges posed by recent changes in doctors' training, yet the uptake has been slow in the majority of institutions. In our own centre, we noted existing equipment was used infrequently. We sought to address this problem through the development of a 1-day training course in simulation (SIM) and basic interventional skills aimed at trainees from across different endovascular specialties. METHODS: A 1-day course for trainees in cardiology, interventional radiology and vascular surgery was piloted. A variety of endovascular simulators were used to teach core skills common to all three specialties, under the umbrella theme of safe access, safe navigation and safe closure. Independent continuing use of SIM-based training was encouraged. Trainee and faculty member experiences of SIM training in a cross-specialty environment were explored by interview and online questionnaire. RESULTS: Thirty-six trainees completed the pilot training course. Feedback was almost universally positive, with all trainees agreeing that SIM was useful in achieving the course's objectives, and that they would recommend the course to a colleague. Cross-specialty training was viewed positively by trainees and trainers alike, with benefits identified in knowledge and skills sharing, as well as fostering better clinical collaboration. SIM-based training was perceived as useful in promoting patient safety, and is considered to be a desirable component of future training. DISCUSSION: We present a SIM-based model that achieves economies of scale by delivering common skill-set training for doctors from different specialties. Through our experiences piloting this course we discuss how the recognised barriers to adopting this new technology may be addressed and offer insights into how SIM may be integrated into existing training programmes.

Glomus tumor of the mediastinum.

Glomus tumors are rare benign myoepithelial neoplasms that can present with intractable pain. We report the case of a 59-year-old gentleman who presented with upper abdominal and chest pain caused by a posterior mediastinal glomus tumor arising from the spinal column, which required surgical resection. As glomus tumors usually develop in the limbs, this case highlights the complexity of diagnosis and treatment of glomus tumors when they present in rare locations.

Colorectal cancer screening: the northern trust experience.

OBJECTIVE: Colorectal cancer (CRC) is the third most common type of cancer with resulting major mortality. In a bid to reduce the mortality, bowel cancer screening has been established in the United Kingdom. The screening programme was commenced in Northern Ireland in 2010 within the Northern Health and Social Care Trust, following its implementation in England and Scotland. This study aimed to look at early outcome data for bowel cancer screening in Northern Ireland and compare data with other regions in the UK. DESIGN: A retrospective analysis was conducted of patients who tested faecal occult blood (FOB) positive and attended for pre-assessment between May 2010 and May 2011. Data was also collected from the computerised endoscopy database (Endoscribe®). Patient demographics, colonoscopic depth of insertion, findings and complications were documented. Subsequent surgical management, pathological staging and final outcome were also noted. RESULTS: 182 patients attended for pre-assessment in the time frame and 178 patients proceeded to colonoscopy. The commonest pathology encountered was polyps, identified in 95 (52.7%) patients. Macroscopically 13 cancers were seen on endoscopy and a further two were found on post-operative histology of polyps that were not amenable to endoscopic resection. In addition, 5 malignant polyps were found on histological analysis of the excised polyps. The staging of cancers was favourable with 35% being Dukes' A stage. CONCLUSION: Outcomes from the first year of colorectal cancer screening in the Northern Trust are in keeping with early results from previous studies in terms of cancer detection rates per colonoscopy and proportion of early stage cancers. However, the adenoma detection rate was higher than anticipated.

Defunctioning loop ileostomy: not always a safe option.

A 74-year-old woman underwent a low anterior resection and defunctioning loop ileostomy for a T1 N1 M0 rectal adenocarcinoma. Three months following surgery she attended complaining of pain inferior to the loop ileostomy. A clinical examination demonstrated an extensive area of spreading cellulitis on the lower abdominal wall inferior to the loop ileostomy with associated crepitus and skin necrosis. The clinical diagnosis of necrotising fasciitis was confirmed radiologically on emergency computed tomography. The patient underwent an emergency debridement of the anterior abdominal wall.

The cyclooxygenase-2 inhibitor GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] is effective in animal models of neuropathic pain and central sensitization.

The pathogenic form of the cyclooxygenase (COX) enzyme, COX-2, is also constitutively present in the spinal cord and has been implicated in chronic pain states in rat and man. A number of COX-2 inhibitors, including celecoxib and rofecoxib, are already used in man for the treatment of inflammatory pain. Preclinically, the dual-acting COX-2 inhibitor, GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine, where X denotes the free base], is as effective as rofecoxib and celecoxib in the rat established Freund's Complete Adjuvant model with an ED(50) of 1.5 mg/kg p.o. compared with 1.0 mg/kg p.o. for rofecoxib and 6.6 mg/kg p.o. for celecoxib. However, in contrast to celecoxib (5 mg/kg p.o. b.i.d.) and rofecoxib (5 mg/kg p.o. b.i.d.), which were without significant effect, GW406381X (5 mg/kg p.o. b.i.d.) fully reversed mechanical allodynia in the chronic constriction injury model and reversed thermal hyperalgesia in the mouse partial ligation model, both models of neuropathic pain. GW406381X, was also effective in a rat model of capsaicin-induced central sensitization, when given intrathecally (ED(50) = 0.07 mug) and after chronic but not acute oral dosing. Celecoxib and rofecoxib had no effect in this model. Several hypotheses have been proposed to try to explain these differences in efficacy, including central nervous system penetration, enzyme kinetics, and potency. The novel finding of effectiveness of GW406381X in these models of neuropathic pain/central sensitization, in addition to activity in inflammatory pain models and together with its central efficacy, suggests dual activity of GW406381X compared with celecoxib and rofecoxib, which may translate into greater efficacy in a broader spectrum of pain states in the clinic.