A systematic review of frailty assessment tools used in vascular surgery research.

OBJECTIVE: Frailty is common in vascular patients and is recognized for its prognostic value. In the absence of consensus, a multitude of frailty assessment tools exist. This systematic review aimed to quantify the variety in these tools and describe their content and application to inform future research and clinical practice. METHODS: Multiple cross-disciplinary electronic literature databases were searched from inception to August 2022. Studies describing frailty assessment in a vascular surgical population were eligible. Data extraction to a validated template included patient demographics, tool content, and analysis methods. A secondary systematic search for papers describing the psychometric properties of commonly used frailty tools was then performed. RESULTS: Screening 5358 records identified 111 eligible studies, with an aggregate population of 5,418,236 patients. Forty-three differing frailty assessment tools were identified. One-third of these failed to assess frailty as a multidomain deficit and there was a reliance on assessing function and presence of comorbidity. Substantial methodological variability in data analysis and lack of methodological description was also identified. Published psychometric assessment was available for only 4 of the 10 most commonly used frailty tools. The Clinical Frailty Scale was the most studied and demonstrates good psychometric properties within a surgical population. CONCLUSIONS: Substantial heterogeneity in frailty assessment is demonstrated, precluding meaningful comparisons of services and data pooling. A uniform approach to assessment is required to guide future frailty research. Based on the literature, we make the following recommendations: frailty should be considered a continuous construct and the reporting of frailty tools' application needs standardized. In the absence of consensus, the Clinical Frailty Scale is a validated tool with good psychometric properties that demonstrates usefulness in vascular surgery.

Dynamics of Rubisco regulation by sugar phosphate derivatives and their phosphatases.

Regulating the central CO2-fixing enzyme Rubisco is as complex as its ancient reaction mechanism and involves interaction with a series of cofactors and auxiliary proteins that activate catalytic sites and maintain activity. A key component among the regulatory mechanisms is the binding of sugar phosphate derivatives that inhibit activity. Removal of inhibitors via the action of Rubisco activase is required to restore catalytic competency. In addition, specific phosphatases dephosphorylate newly released inhibitors, rendering them incapable of binding to Rubisco catalytic sites. The best studied inhibitor is 2-carboxy-d-arabinitol 1-phosphate (CA1P), a naturally occurring nocturnal inhibitor that accumulates in most species during darkness and low light, progressively binding to Rubisco. As light increases, Rubisco activase removes CA1P from Rubisco, and the specific phosphatase CA1Pase dephosphorylates CA1P to CA, which cannot bind Rubisco. Misfire products of Rubisco's complex reaction chemistry can also act as inhibitors. One example is xylulose-1,5-bisphosphate (XuBP), which is dephosphorylated by XuBPase. Here we revisit key findings related to sugar phosphate derivatives and their specific phosphatases, highlighting outstanding questions and how further consideration of these inhibitors and their role is important for better understanding the regulation of carbon assimilation.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Rubiscosome gene expression is balanced across the hexaploid wheat genome.

Functional and active Rubisco is essential for CO2 fixation and is a primary target for engineering approaches to increasing crop yields. However, the assembly and maintenance of active Rubisco are dependent on the coordinated biosynthesis of at least 11 nuclear-encoded proteins, termed the 'Rubiscosome'. Using publicly available gene expression data for wheat (Triticum aestivum L.), we show that the expression of Rubiscosome genes is balanced across the three closely related subgenomes that form the allohexaploid genome. Each subgenome contains a near complete set of homoeologous genes and contributes equally to overall expression, both under optimal and under heat stress conditions. The expression of the wheat thermo-tolerant Rubisco activase isoform 1ß increases under heat stress and remains balanced across the subgenomes, albeit with a slight shift towards greater contribution from the D subgenome. The findings show that the gene copies in all three subgenomes need to be accounted for when designing strategies for crop improvement.

Heat-induced changes in the abundance of wheat Rubisco activase isoforms.

The Triticum aestivum (wheat) genome encodes three isoforms of Rubisco activase (Rca) differing in thermostability, which could be exploited to improve the resilience of this crop to global warming. We hypothesized that elevated temperatures would cause an increase in the relative abundance of heat-stable Rca1ß. Wheat plants were grown at 25° C : 18°C (day : night) and exposed to heat stress (38° C : 22°C) for up to 5 d at pre-anthesis. Carbon (C) assimilation, Rubisco activity, CA1Pase activity, transcripts of Rca1ß, Rca2ß, and Rca2α, and the quantities of the corresponding protein products were measured during and after heat stress. The transcript of Rca1ß increased 40-fold in 4 h at elevated temperatures and returned to the original level after 4 h upon return of plants to control temperatures. Rca1ß comprised up to 2% of the total Rca protein in unstressed leaves but increased three-fold in leaves exposed to elevated temperatures for 5 d and remained high at 4 h after heat stress. These results show that elevated temperatures cause rapid changes in Rca gene expression and adaptive changes in Rca isoform abundance. The improved understanding of the regulation of C assimilation under heat stress will inform efforts to improve wheat productivity and climate resilience.

Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation.

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO2-fixing enzyme Rubisco. Cyanobacteria utilize a CO2-concentrating mechanism (CCM), which encapsulates Rubisco with poor specificity but a relatively fast catalytic rate within a carboxysome microcompartment. Alongside the active transport of bicarbonate into the cell and localization of carbonic anhydrase within the carboxysome shell with Rubisco, cyanobacteria are able to overcome the limitations of Rubisco via localization within a high-CO2 environment. As part of ongoing efforts to engineer a ß-cyanobacterial CCM into land plants, we investigated the potential for Rubisco large subunits (LSU) from the ß-cyanobacterium Synechococcus elongatus (Se) to form aggregated Rubisco complexes with the carboxysome linker protein CcmM35 within tobacco (Nicotiana tabacum) chloroplasts. Transplastomic plants were produced that lacked cognate Se Rubisco small subunits (SSU) and expressed the Se LSU in place of tobacco LSU, with and without CcmM35. Plants were able to form a hybrid enzyme utilizing tobacco SSU and the Se LSU, allowing slow autotrophic growth in high CO2 CcmM35 was able to form large Rubisco aggregates with the Se LSU, and these incorporated small amounts of native tobacco SSU. Plants lacking the Se SSU showed delayed growth, poor photosynthetic capacity, and significantly reduced Rubisco activity compared with both wild-type tobacco and lines expressing the Se SSU. These results demonstrate the ability of the Se LSU and CcmM35 to form large aggregates without the cognate Se SSU in planta, harboring active Rubisco that enables plant growth, albeit at a much slower pace than plants expressing the cognate Se SSU.

Photosynthesis across African cassava germplasm is limited by Rubisco and mesophyll conductance at steady state, but by stomatal conductance in fluctuating light.

Sub-Saharan Africa is projected to see a 55% increase in food demand by 2035, where cassava (Manihot esculenta) is the most widely planted crop and a major calorie source. Yet, cassava yield in this region has not increased significantly for 13 yr. Improvement of genetic yield potential, the basis of the first Green Revolution, could be realized by improving photosynthetic efficiency. First, the factors limiting photosynthesis and their genetic variability within extant germplasm must be understood. Biochemical and diffusive limitations to leaf photosynthetic CO2 uptake under steady state and fluctuating light in 13 farm-preferred and high-yielding African cultivars were analyzed. A cassava leaf metabolic model was developed to quantify the value of overcoming limitations to leaf photosynthesis. At steady state, in vivo Rubisco activity and mesophyll conductance accounted for 84% of the limitation. Under nonsteady-state conditions of shade to sun transition, stomatal conductance was the major limitation, resulting in an estimated 13% and 5% losses in CO2 uptake and water use efficiency, across a diurnal period. Triose phosphate utilization, although sufficient to support observed rates, would limit improvement in leaf photosynthesis to 33%, unless improved itself. The variation of carbon assimilation among cultivars was three times greater under nonsteady state compared to steady state, pinpointing important overlooked breeding targets for improved photosynthetic efficiency in cassava.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.

Overexpression of ca1pase Decreases Rubisco Abundance and Grain Yield in Wheat.

Rubisco catalyzes the fixation of CO2 into organic compounds that are used for plant growth and the production of agricultural products, and specific sugar-phosphate derivatives bind tightly to the active sites of Rubisco, locking the enzyme in a catalytically inactive conformation. 2-carboxy-d-arabinitol-1-phosphate phosphatase (CA1Pase) dephosphorylates such tight-binding inhibitors, contributing to the maintenance of Rubisco activity. Here, we investigated the hypothesis that overexpressing ca1pase would decrease the abundance of Rubisco inhibitors, thereby increasing the activity of Rubisco and enhancing photosynthetic performance and productivity in wheat (Triticum aestivum). Plants of four independent wheat transgenic lines overexpressing ca1pase showed up to 30-fold increases in ca1pase expression compared to the wild type. Plants overexpressing ca1pase had lower numbers of Rubisco tight-binding inhibitors and higher Rubisco activation state than the wild type; however, there were 17% to 60% fewer Rubisco active sites in the four transgenic lines than in the wild type. The lower Rubisco content in plants overexpressing ca1pase resulted in lower initial and total carboxylating activities measured in flag leaves at the end of the vegetative stage and lower aboveground biomass and grain yield measured in fully mature plants. Hence, contrary to what would be expected, ca1pase overexpression decreased Rubisco content and compromised wheat grain yields. These results support a possible role for Rubisco inhibitors in protecting the enzyme and maintaining an adequate number of Rubisco active sites to support carboxylation rates in planta.

During photosynthetic induction, biochemical and stomatal limitations differ between Brassica crops.

Interventions to increase crop radiation use efficiency rely on understanding of how biochemical and stomatal limitations affect photosynthesis. When leaves transition from shade to high light, slow increases in maximum Rubisco carboxylation rate and stomatal conductance limit net CO2 assimilation for several minutes. However, as stomata open intercellular [CO2 ] increases, so electron transport rate could also become limiting. Photosynthetic limitations were evaluated in three important Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. Measurements of induction after a period of shade showed that net CO2 assimilation by B. rapa and B. napus saturated by 10 min. A new method of analyzing limitations to induction by varying intercellular [CO2 ] showed this was due to co-limitation by Rubisco and electron transport. By contrast, in B. oleracea persistent Rubisco limitation meant that CO2 assimilation was still recovering 15 min after induction. Correspondingly, B. oleracea had the lowest Rubisco total activity. The methodology developed, and its application here, shows a means to identify the basis of variation in photosynthetic efficiency in fluctuating light, which could be exploited in breeding and bioengineering to improve crop productivity.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

Surveying Rubisco Diversity and Temperature Response to Improve Crop Photosynthetic Efficiency.

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally "better" compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.

Uncertainty in measurements of the photorespiratory CO2 compensation point and its impact on models of leaf photosynthesis.

Rates of carbon dioxide assimilation through photosynthesis are readily modeled using the Farquhar, von Caemmerer, and Berry (FvCB) model based on the biochemistry of the initial Rubisco-catalyzed reaction of net C3 photosynthesis. As models of CO2 assimilation rate are used more broadly for simulating photosynthesis among species and across scales, it is increasingly important that their temperature dependencies are accurately parameterized. A vital component of the FvCB model, the photorespiratory CO2 compensation point (Γ *), combines the biochemistry of Rubisco with the stoichiometry of photorespiratory release of CO2. This report details a comparison of the temperature response of Γ * measured using different techniques in three important model and crop species (Nicotiana tabacum, Triticum aestivum, and Glycine max). We determined that the different Γ * determination methods produce different temperature responses in the same species that are large enough to impact higher-scale leaf models of CO2 assimilation rate. These differences are largest in N. tabacum and could be the result of temperature-dependent increases in the amount of CO2 lost from photorespiration per Rubisco oxygenation reaction.

Rubisco catalytic properties of wild and domesticated relatives provide scope for improving wheat photosynthesis.

Rubisco is a major target for improving crop photosynthesis and yield, yet natural diversity in catalytic properties of this enzyme is poorly understood. Rubisco from 25 genotypes of the Triticeae tribe, including wild relatives of bread wheat (Triticum aestivum), were surveyed to identify superior enzymes for improving photosynthesis in this crop. In vitro Rubisco carboxylation velocity (V c), Michaelis-Menten constants for CO2 (K c) and O2 (K o) and specificity factor (S c/o) were measured at 25 and 35 °C. V c and K c correlated positively, while V c and S c/o were inversely related. Rubisco large subunit genes (rbcL) were sequenced, and predicted corresponding amino acid differences analysed in relation to the corresponding catalytic properties. The effect of replacing native wheat Rubisco with counterparts from closely related species was analysed by modelling the response of photosynthesis to varying CO2 concentrations. The model predicted that two Rubisco enzymes would increase photosynthetic performance at 25 °C while only one of these also increased photosynthesis at 35 °C. Thus, under otherwise identical conditions, catalytic variation in the Rubiscos analysed is predicted to improve photosynthetic rates at physiological CO2 concentrations. Naturally occurring Rubiscos with superior properties amongst the Triticeae tribe can be exploited to improve wheat photosynthesis and crop productivity.

Purification of Rubisco from Leaves.

Rubisco fixes CO2 through the carboxylation of ribulose 1,5-bisphosphate (RuBP) during photosynthesis, enabling the synthesis of organic compounds. The natural diversity of Rubisco properties represents an opportunity to improve its performance and there is considerable research effort focusing on better understanding the properties and regulation of the enzyme. This chapter describes a method for large-scale purification of Rubisco from leaves. After the extraction of Rubisco from plant leaves, the enzyme is separated from other proteins by fractional precipitation with polyethylene glycol followed by ion-exchange chromatography. This method enables the isolation of Rubisco in large quantities for a wide range of biochemical applications.

Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Frailty Assessment in Vascular OUtpatients Review (FAVOUR) protocol: single-centre prospective cohort study comparing feasibility and prognostic value of commonly used frailty assessment tools.

INTRODUCTION: Frailty has consistently demonstrated associations with poorer healthcare outcomes. Vascular guidelines have recognised the importance of frailty assessment. However, an abundance of frailty tools and a lack of prospective studies confirming suitability of routine frailty assessment in clinical practice has delayed the uptake of these guidelines. The Frailty Assessment in Vascular OUtpatients Review study speaks to this evidence gap. The primary aim is to assess feasibility of implementing routine frailty assessment in a reproducible outpatient setting. Secondary objectives include comparing prognostic values and interuser agreement across five frailty assessment tools. METHODS AND ANALYSIS: This single-centre prospective cohort study of feasibility is conducted in a rapid-referral vascular surgery clinic, serving a population of 2 million. Adults with capacity (>18 years), attending a clinic for any reason, are eligible for inclusion. Five assessments are completed by patient (Rockwood Clinical Frailty Scale (CFS) and Frail NonDisabled Questionnaire), clinician (CFS, Healthcare Improvement Scotland FRAIL tool and 'Initial Clinical Evaluation') and researcher (11-item modified Frailty Index). Consistent with feasibility objectives, outcome measures include recruitment rates, frailty assessment completion rates, time-to-complete assessments and interuser variability. Electronic follow-up at 30 days and 1 year will assess home-time and mortality as prognostic indicators. Patients treated surgically/endovascularly will undergo additional 30-day and 1-year postoperative follow-up, outcome measures include: surgical procedure, mortality, complications (according to Clavien-Dindo Classification), length of stay, readmission rates, non-home discharge, home-time, higher social care requirements on discharge and amputation-free survival. Prognostic value will be compared by area under receiver operating characteristic curves. Continuous outcome variables will be analysed using Spearman's rank correlation coefficient. Interuser agreement will be compared by percentage agreement in Cohen's kappa coefficient.  ETHICS AND DISSEMINATION: The study is sponsored by National Health Service Greater Glasgow and Clyde (R&IUGN23CE014). London-Riverside REC (23/PR/0062) granted ethical approval. Results will be disseminated through publication in peer-reviewed vascular surgery and geriatric medicine themed journals and presentation at similar scientific conferences. TRIALS REGISTRATION NUMBER: NCT06040658. Stage of study: pre-results.

Structural plasticity enables evolution and innovation of RuBisCO assemblies.

Oligomerization is a core structural feature that defines the form and function of many proteins. Most proteins form molecular complexes; however, there remains a dearth of diversity-driven structural studies investigating the evolutionary trajectory of these assemblies. Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is one such enzyme that adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Here, we retrace the evolution of ancestral and extant form II RuBisCOs, revealing a complex and diverse history of oligomerization. We structurally characterize a newly discovered tetrameric RuBisCO, elucidating how solvent-exposed surfaces can readily adopt new interactions to interconvert or give rise to new oligomeric states. We further use these principles to engineer and demonstrate how changes in oligomerization can be mediated by relatively few mutations. Our findings yield insight into how structural plasticity may give rise to new oligomeric states.

Pharmacotherapy to improve outcomes in infrainguinal bypass graft surgery: a review of current treatment strategies.

A total of 12,000 infrainguinal bypass grafts are performed annually in the United Kingdom, with outcomes suboptimal: 20% of above-knee vein grafts require intervention by 3 years. Transatlantic Inter-Society Consensus (TASC) guidelines exist on pharmacological management of peripheral vascular disease patients, however, little is recommended regarding optimum pharmacological management following revascularization to improve graft patency. The current recommendation is that all patients are on an antiplatelet agent following bypass grafting, the only intervention with significant evidence supporting use. This article will review pharmacological strategies aimed at improving the survival of infrainguinal vein grafts and the current evidence base for their use.