Effects of RBT-1 on preconditioning response biomarkers in patients undergoing coronary artery bypass graft or heart valve surgery: a multicentre, double-blind, randomised, placebo-controlled phase 2 trial.

Background: RBT-1 is a combination drug of stannic protoporfin (SnPP) and iron sucrose (FeS) that elicits a preconditioning response through activation of antioxidant, anti-inflammatory, and iron-scavenging pathways, as measured by heme oxygenase-1 (HO-1), interleukin-10 (IL-10), and ferritin, respectively. Our primary aim was to determine whether RBT-1 administered before surgery would safely and effectively elicit a preconditioning response in patients undergoing cardiac surgery. Methods: This phase 2, double-blind, randomised, placebo-controlled, parallel-group, adaptive trial, conducted in 19 centres across the USA, Canada, and Australia, enrolled patients scheduled to undergo non-emergent coronary artery bypass graft (CABG) and/or heart valve surgery with cardiopulmonary bypass. Patients were randomised (1:1:1) to receive either a single intravenous infusion of high-dose RBT-1 (90 mg SnPP/240 mg FeS), low-dose RBT-1 (45 mg SnPP/240 mg FeS), or placebo within 24-48 h before surgery. The primary outcome was a preoperative preconditioning response, measured by a composite of plasma HO-1, IL-10, and ferritin. Safety was assessed by adverse events and laboratory parameters. Prespecified adaptive criteria permitted early stopping and enrichment. This trial is registered with ClinicalTrials.gov, NCT04564833. Findings: Between Aug 4, 2021, and Nov 9, 2022, of 135 patients who were enrolled and randomly allocated to a study group (46 high-dose, 45 low-dose, 44 placebo), 132 (98%) were included in the primary analysis (46 high-dose, 42 low-dose, 44 placebo). At interim, the trial proceeded to full enrollment without enrichment. RBT-1 led to a greater preconditioning response than did placebo at high-dose (geometric least squares mean [GLSM] ratio, 3.58; 95% CI, 2.91-4.41; p < 0.0001) and low-dose (GLSM ratio, 2.62; 95% CI, 2.11-3.24; p < 0.0001). RBT-1 was generally well tolerated by patients. The primary drug-related adverse event was dose-dependent photosensitivity, observed in 12 (26%) of 46 patients treated with high-dose RBT-1 and in six (13%) of 45 patients treated with low-dose RBT-1 (safety population). Interpretation: RBT-1 demonstrated a statistically significant cytoprotective preconditioning response and a manageable safety profile. Further research is needed. A phase 3 trial is planned. Funding: Renibus Therapeutics, Inc.

A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

Age- and sex-specific associations of frailty with mortality and healthcare utilization in community-dwelling adults from ontario, Canada.

BACKGROUND: Understanding how health trajectories are related to the likelihood of adverse outcomes and healthcare utilization is key to planning effective strategies for improving health span and the delivery of care to older adults. Frailty measures are useful tools for risk stratification in community-based and primary care settings, although their effectiveness in adults younger than 60 is not well described. METHODS: We performed a 10-year retrospective analysis of secondary data from the Ontario Health Study, which included 161,149 adults aged ≥ 18. Outcomes including all-cause mortality and hospital admissions were obtained through linkage to ICES administrative databases with a median follow-up of 7.1-years. Frailty was characterized using a 30-item frailty index. RESULTS: Frailty increased linearly with age and was higher for women at all ages. A 0.1-increase in frailty was significantly associated with mortality (HR = 1.47), the total number of outpatient (IRR = 1.35) and inpatient (IRR = 1.60) admissions over time, and length of stay (IRR = 1.12). However, with exception to length of stay, these estimates differed depending on age and sex. The hazard of death associated with frailty was greater at younger ages, particularly in women. Associations with admissions also decreased with age, similarly between sexes for outpatient visits and more so in men for inpatient. CONCLUSIONS: These findings suggest that frailty is an important health construct for both younger and older adults. Hence targeted interventions to reduce the impact of frailty before the age of 60 would likely have important economic and social implications in both the short- and long-term.

Association of insulin resistance with the accumulation of saturated intramyocellular lipid: A comparison with other fat stores.

It has been shown using proton magnetic resonance spectroscopy (1 H MRS) that, in a group of females, whole-body insulin resistance was more closely related to accumulation of saturated intramyocellular lipid (IMCL) than to IMCL concentration alone. This has not been investigated in males. We investigated whether age- and body mass index-matched healthy males differ from the previously reported females in IMCL composition (measured as CH2 :CH3 ) and IMCL concentration (measured as CH3 ), and in their associations with insulin resistance. We ask whether saturated IMCL accumulation is more strongly associated with insulin resistance than other ectopic and adipose tissue lipid pools and remains a significant predictor when these other pools are taken into account. In this group of males, who had similar overall insulin sensitivity to the females, IMCL was similar between sexes. The males demonstrated similar and even stronger associations of IMCL with insulin resistance, supporting the idea that a marker reflecting the accumulation of saturated IMCL is more strongly associated with whole-body insulin resistance than IMCL concentration alone. However, this marker ceased to be a significant predictor of whole-body insulin resistance after consideration of other lipid pools, which implies that this measure carries no more information in practice than the other predictors we found, such as intrahepatic lipid and visceral adipose tissue. As the marker of saturated IMCL accumulation appears to be related to these two predictors and has a much smaller dynamic range, this finding does not rule out a role for it in the pathogenesis of insulin resistance.

Protein design using structure-based residue preferences.

Recent developments in protein design rely on large neural networks with up to 100s of millions of parameters, yet it is unclear which residue dependencies are critical for determining protein function. Here, we show that amino acid preferences at individual residues-without accounting for mutation interactions-explain much and sometimes virtually all of the combinatorial mutation effects across 8 datasets (R2 ~ 78-98%). Hence, few observations (~100 times the number of mutated residues) enable accurate prediction of held-out variant effects (Pearson r > 0.80). We hypothesized that the local structural contexts around a residue could be sufficient to predict mutation preferences, and develop an unsupervised approach termed CoVES (Combinatorial Variant Effects from Structure). Our results suggest that CoVES outperforms not just model-free methods but also similarly to complex models for creating functional and diverse protein variants. CoVES offers an effective alternative to complicated models for identifying functional protein mutations.

Factors associated with frequent buprenorphine / naloxone initiation in a national survey of Canadian emergency physicians.

OBJECTIVE: To identify individual and site-related factors associated with frequent emergency department (ED) buprenorphine/naloxone (BUP) initiation. BUP initiation, an effective opioid use disorder (OUD) intervention, varies widely across Canadian EDs. METHODS: We surveyed emergency physicians in 6 Canadian provinces from 2018 to 2019 using bilingual paper and web-based questionnaires. Survey domains included BUP-related practice, demographics, attitudes toward BUP, and site characteristics. We defined frequent BUP initiation (the primary outcome) as at least once per month, high OUD prevalence as at least one OUD patient per shift, and high OUD resources as at least 3 out of the following 5 resources: BUP initiation pathways, BUP in ED, peer navigators, accessible addiction specialists, and accessible follow-up clinics. We excluded responses from sites with <50% participation (to minimize non-responder bias) and those missing the primary outcome. We used univariate analysis to identify associations between frequent BUP initiation and factors of interest, stratifying by OUD prevalence. RESULTS: We excluded 3 responses for missing BUP initiation frequency and 9 for low response rate at one ED. Of the remaining 649 respondents from 34 EDs, 374 (58%) practiced in metropolitan areas, 384 (59%) reported high OUD prevalence, 312 (48%) had high OUD resources, and 161 (25%) initiated BUP frequently. Age, gender, board certification and years in practice were not associated with frequent BUP initiation. Site-specific factors were associated with frequent BUP initiation (high OUD resources [OR 6.91], high OUD prevalence [OR 4.45], and metropolitan location [OR 2.39],) as were individual attitudinal factors (willingness, confidence, and responsibility to initiate BUP.) Similar associations persisted in the high OUD prevalence subgroup. CONCLUSIONS: Individual attitudinal and site-specific factors were associated with frequent BUP initiation. Training to increase physician confidence and increasing OUD resources could increase BUP initiation and benefit ED patients with OUD.

Mortality and risk factors associated with misdiagnosis of acute aortic syndrome in Ontario, Canada: a population-based study.

INTRODUCTION: Acute aortic syndrome (AAS) is a life-threatening aortic emergency. It describes three diagnoses: acute aortic dissection, acute intramural haematoma and penetrating atherosclerotic ulcer. Unfortunately, there are no accurate estimates of the miss rate for AAS, risk factors for missed diagnosis or its effect on outcomes. METHODS: A population-based retrospective cohort study of anonymously linked data for residents of Ontario, Canada, was carried out. Incident cases of AAS were identified between 2003 and 2018 using a validated algorithm based on ICD codes and death. Before multivariate modelling, all categorical variables were analysed for an association with missed AAS diagnosis using χ2 tests. These preliminary analyses were unadjusted for clustering or any covariates. Finally, we performed multilevel logistic regression analysis using a generalised linear mixed model approach to model the probability of a missed case occurring. RESULTS: There were 1299 cases of AAS (age mean (SD) 68.03±14.70, woman 500 (38.5%), rural areas (n=111, 8.55%)) over the study period. Missed cases accounted for 163 (12.5%) of the cohort. Mortality (non-missed AAS 59.7% vs missed AAS 54.6%) and surgical intervention (non-missed AAS 31% vs missed AAS 30.7%) were similar in missed and non-missed cases. However, lower acuity (Canadian triage acuity scale >2 (OR 2.45 95% CI 1.71 to 3.52) (the scale is from 1 to 5, with 1 indicating high acuity) had a higher odds of being a missed case and non-ambulatory presentation (OR 0.47 95% CI 0.33 to 0.67) and presenting to a teaching (OR 0.60 95% CI 0.40 to 0.90)) or cardiac centre (OR 0.41 95% CI 0.27 to 0.62) were associated with a lower odds of being a missed case. CONCLUSIONS: The high rate of misdiagnosis has remained stable for over a decade. Non-teaching and non-cardiac hospitals had a higher incidence of missed cases. Mortality and rates of surgery were not associated with a missed diagnosis of AAS. Educational interventions should be prioritised in non-teaching hospitals and non-cardiac centres.

Residential mobility restrictions and adverse mental health outcomes during the COVID-19 pandemic in the UK.

During the COVID-19 pandemic, several governments tried to contain the spread of SARS-CoV-2, the virus that causes COVID-19, with lockdowns that prohibited leaving one's residence unless carrying out a few essential services. We investigate the relationship between limitations to mobility and mental health in the UK during the first year and a half of the pandemic using a unique combination of high-frequency mobility data from Google and monthly longitudinal data collected through the Understanding Society survey. We find a strong and statistically robust correlation between mobility data and mental health survey data and show that increased residential stationarity is associated with the deterioration of mental wellbeing even when regional COVID-19 prevalence and lockdown stringency are controlled for. The relationship is heterogeneous, as higher levels of distress are seen in young, healthy people living alone; and in women, especially if they have young children.

Autotrophic biofilms sustained by deeply sourced groundwater host diverse bacteria implicated in sulfur and hydrogen metabolism.

BACKGROUND: Biofilms in sulfide-rich springs present intricate microbial communities that play pivotal roles in biogeochemical cycling. We studied chemoautotrophically based biofilms that host diverse CPR bacteria and grow in sulfide-rich springs to investigate microbial controls on biogeochemical cycling. RESULTS: Sulfide springs biofilms were investigated using bulk geochemical analysis, genome-resolved metagenomics, and scanning transmission X-ray microscopy (STXM) at room temperature and 87 K. Chemolithotrophic sulfur-oxidizing bacteria, including Thiothrix and Beggiatoa, dominate the biofilms, which also contain CPR Gracilibacteria, Absconditabacteria, Saccharibacteria, Peregrinibacteria, Berkelbacteria, Microgenomates, and Parcubacteria. STXM imaging revealed ultra-small cells near the surfaces of filamentous bacteria that may be CPR bacterial episymbionts. STXM and NEXAFS spectroscopy at carbon K and sulfur L2,3 edges show that filamentous bacteria contain protein-encapsulated spherical elemental sulfur granules, indicating that they are sulfur oxidizers, likely Thiothrix. Berkelbacteria and Moranbacteria in the same biofilm sample are predicted to have a novel electron bifurcating group 3b [NiFe]-hydrogenase, putatively a sulfhydrogenase, potentially linked to sulfur metabolism via redox cofactors. This complex could potentially contribute to symbioses, for example, with sulfur-oxidizing bacteria such as Thiothrix that is based on cryptic sulfur cycling. One Doudnabacteria genome encodes adjacent sulfur dioxygenase and rhodanese genes that may convert thiosulfate to sulfite. We find similar conserved genomic architecture associated with CPR bacteria from other sulfur-rich subsurface ecosystems. CONCLUSIONS: Our combined metagenomic, geochemical, spectromicroscopic, and structural bioinformatics analyses of biofilms growing in sulfide-rich springs revealed consortia that contain CPR bacteria and sulfur-oxidizing Proteobacteria, including Thiothrix, and bacteria from a new family within Beggiatoales. We infer roles for CPR bacteria in sulfur and hydrogen cycling. Video Abstract.

α-Carboxysome Size Is Controlled by the Disordered Scaffold Protein CsoS2.

Carboxysomes are protein microcompartments that function in the bacterial CO2 concentrating mechanism (CCM) to facilitate CO2 assimilation. To do so, carboxysomes assemble from thousands of constituent proteins into an icosahedral shell, which encapsulates the enzymes Rubisco and carbonic anhydrase to form structures typically > 100 nm and > 300 megadaltons. Although many of the protein interactions driving the assembly process have been determined, it remains unknown how size and composition are precisely controlled. Here, we show that the size of α-carboxysomes is controlled by the disordered scaffolding protein CsoS2. CsoS2 contains two classes of related peptide repeats that bind to the shell in a distinct fashion, and our data indicate that size is controlled by the relative number of these interactions. We propose an energetic and structural model wherein the two repeat classes bind at the junction of shell hexamers but differ in their preferences for the shell contact angles, and thus the local curvature. In total, this model suggests that a set of specific and repeated interactions between CsoS2 and shell proteins collectively achieve the large size and monodispersity of α-carboxysomes.

The role of lipid droplet associated proteins in inherited human disorders.

Proteins which associate with the surface of lipid droplets are intimately involved in the regulation of the droplets. Several human inherited disorders have now been linked to loss- and, in some cases, likely gain-of-function mutations in the genes encoding these proteins. These are summarised in this Graphical Review.

Can you accurately rule out acute aortic syndrome by restricting imaging of the aorta to the area of the patient's pain?

BACKGROUND: Acute aortic syndrome (AAS) is a life-threatening condition necessitating timely and accurate diagnosis for appropriate treatment. Currently, the only way to rule out the diagnosis is advanced imaging. The most accessible is computed tomography of the entire aorta. Most scans are negative, exposing patients to radiation, increased time in the emergency department (ED), and non-significant incidental findings. This study investigated whether restricting imaging to the area of aortic-related pain accurately rules out AAS. METHODS: A health records review was conducted on consecutive cases from three academic EDs between 2015 and 2020. Data were extracted and verified from multiple sources. Participants included adults diagnosed with AAS based on radiological evidence. The diagnostic performance of the restricted imaging strategy was assessed; sensitivity and likelihood ratios with 95% confidence intervals were calculated. RESULTS: Data from 149 cases of AAS were collected, with the majority presenting with chest pain (46%) or abdominal pain (24%). The restricted imaging strategy demonstrated a sensitivity of 96% (95% CI 91.4-98.5%) in ruling out AAS. In a subset of patients with systolic blood pressure > 90 mmHg and without aortic aneurysm/repair (n = 86), the sensitivity was 100% (95% CI 96-100%). CONCLUSION: Restricting imaging to the area of pain in hemodynamically stable patients without known aortic aneurysm provides a highly sensitive approach to ruling out AAS.

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection.

Eukaryotic viruses assemble compartments required for genome replication, but no such organelles are known to be essential for prokaryotic viruses. Bacteriophages of the family Chimalliviridae sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of viral protein ChmA. Using the dRfxCas13d-based knockdown system CRISPRi-ART, we show that ChmA is essential for the E. coli phage Goslar life cycle. Without ChmA, infections are arrested at an early stage in which the injected phage genome is enclosed in a membrane-bound vesicle capable of gene expression but not DNA replication. Not only do we demonstrate that the phage nucleus is essential for genome replication, but we also show that the Chimalliviridae early phage infection (EPI) vesicle is a transcriptionally active, phage-generated organelle.

Identification of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome.

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Evaluation of the Canadian Clinical Practice Guidelines Risk Prediction Tool for Acute Aortic Syndrome: The RIPP Score.

Introduction: Acute aortic syndrome (AAS) is a rare clinical syndrome with a high mortality rate. The Canadian clinical practice guideline for the diagnosis of AAS was developed in order to reduce the frequency of misdiagnoses. As part of the guideline, a clinical decision aid was developed to facilitate clinician decision-making (RIPP score). The aim of this study is to validate the diagnostic accuracy of this tool and assess its performance in comparison to other risk prediction tools that have been developed. Methods: This was a historical case-control study. Consecutive cases and controls were recruited from three academic emergency departments from 2002-2020. Cases were identified through an admission, discharge, or death certificated diagnosis of acute aortic syndrome. Controls were identified through presenting complaint of chest, abdominal, flank, back pain, and/or perfusion deficit. We compared the clinical decision tools' C statistic and used the DeLong method to test for the significance of these differences and report sensitivity and specificity with 95% confidence intervals. Results: We collected data on 379 cases of acute aortic syndrome and 1340 potential eligible controls; 379 patients were randomly selected from the final population. The RIPP score had a sensitivity of 99.7% (98.54-99.99). This higher sensitivity resulted in a lower specificity (53%) compared to the other clinical decision aids (63-86%). The DeLong comparison of the C statistics found that the RIPP score had a higher C statistic than the ADDRS (-0.0423 (95% confidence interval -0.07-0.02); P < 0.0009) and the AORTAs score (-0.05 (-0.07 to -0.02); P = 0.0002), no difference compared to the Lovy decision tool (0.02 (95% CI -0.01-0.05 P < 0.25)) and decreased compared to the Von Kodolitsch decision tool (0.04 (95% CI 0.01-0.07 P < 0.008)). Conclusion: The Canadian clinical practice guideline's AAS clinical decision aid is a highly sensitive tool that uses readily available clinical information. It has the potential to improve diagnosis of AAS in the emergency department.

An assessment of mass casualty triage systems using the Alberta trauma registry.

OBJECTIVE: Triage is the process of identifying patients with both the greatest clinical need and the greatest likelihood of benefit in the setting of limited clinical resources. The primary objective of this study was to assess the ability of formal mass casualty incident triage tools to identify patients requiring urgent lifesaving interventions. METHODS: Data from the Alberta Trauma Registry (ATR) was used to assess seven triage tools: START, JumpSTART, SALT, RAMP, MPTT, BCD and MITT. Clinical data captured in the ATR was used to determine which triage category each of the seven tools would have applied to each patient. These categorizations were compared to a reference standard definition based on the patients' need for specific urgent lifesaving interventions. RESULTS: Of the 9448 records that were captured 8652 were included in our analysis. The most sensitive triage tool was MPTT, which demonstrated a sensitivity of 0.76 (0.75, 0.78). Four of the seven triage tools evaluated had sensitivities below 0.45. JumpSTART had the lowest sensitivity and the highest under-triage rate for pediatric patients. All the triage tools evaluated had a moderate to high positive predictive value (> 0.67) for patients who had experienced penetrating trauma. CONCLUSIONS: There was a wide range in the sensitivity of triage tools to identify patients requiring urgent lifesaving interventions. MPTT, BCD and MITT were the most sensitive triage tools assessed. All of the triage tools assessed should be employed with caution during mass casualty incidents as they may fail to identify a large proportion of patients requiring urgent lifesaving interventions.

ABSTRAIT: OBJECTIFS: Le triage est le processus qui consiste à identifier les patients qui ont à la fois les besoins cliniques les plus importants et les avantages les plus probables dans le contexte de ressources cliniques limitées. Le principal objectif de cette étude était d'évaluer la capacité des outils formels de triage des incidents impliquant des blessés de masse à identifier les patients nécessitant des interventions urgentes de sauvetage. MéTHODES: Les données du Alberta Trauma Registry (ATR) ont été utilisées pour évaluer sept outils de triage : START, JumpSTART, SALT, RAMP, MPTT, BCD et MITT. Les données cliniques saisies dans l'AR ont servi à déterminer la catégorie de triage que chacun des sept outils aurait appliquée à chaque patient. Ces catégories ont été comparées à une définition standard de référence fondée sur le besoin des patients d'interventions de sauvetage urgentes. RéSULTATS: Sur les 9448 enregistrements saisis, 8652 ont été inclus dans notre analyse. L'outil de triage le plus sensible était le TPMD, qui présentait une sensibilité de 0,76 (0,75, 0,78). Quatre des sept outils de triage évalués présentaient une sensibilité inférieure à 0,45. JumpSTART avait la sensibilité la plus faible et le taux de sous-triage le plus élevé chez les patients pédiatriques. Tous les outils de triage évalués avaient une valeur prédictive positive modérée à élevée (>0,67) pour les patients qui avaient subi un traumatisme pénétrant. CONCLUSION: La sensibilité des outils de triage pour identifier les patients nécessitant des interventions de sauvetage urgentes variait grandement. Les outils de triage les plus sensibles ont été le TCPR, le BCD et le MITT. Tous les outils de triage évalués doivent être utilisés avec prudence lors d'incidents impliquant des pertes massives, car ils peuvent ne pas identifier une grande proportion de patients nécessitant des interventions de sauvetage urgentes.

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.