Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.

Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve pentacoordinate transition states. We demonstrate that meta-vanadate ion (VO(3)(-)) and appropriate DNA substrates assemble a transition-state analog-like complex in the Cre active site. Meta-vanadate inhibits recombination of LoxP-derived oligonucleotide substrates that contain a gap at either or both scissile phosphates, but does not inhibit reactions with intact LoxP. The 3(')-hydroxyl group of the gapped substrate is required for inhibition, suggesting that vanadate is ligated by three oxo ligands. Assembly of the inhibited complex is slow (t(1/2)=19min at 4mM NaVO(3)) and requires Cre, substrates, and meta-vanadate. Holliday junction intermediates accumulated at lower meta-vanadate concentrations, suggesting that the second strand exchange is inhibited more readily than the first. The apparent K(D) for meta-vanadate is 1.5-2mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions

Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.

Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve pentacoordinate transition states. We demonstrate that meta-vanadate ion (VO(3)(-)) and appropriate DNA substrates assemble a transition-state analog-like complex in the Cre active site. Meta-vanadate inhibits recombination of LoxP-derived oligonucleotide substrates that contain a gap at either or both scissile phosphates, but does not inhibit reactions with intact LoxP. The 3(')-hydroxyl group of the gapped substrate is required for inhibition, suggesting that vanadate is ligated by three oxo ligands. Assembly of the inhibited complex is slow (t(1/2)=19min at 4mM NaVO(3)) and requires Cre, substrates, and meta-vanadate. Holliday junction intermediates accumulated at lower meta-vanadate concentrations, suggesting that the second strand exchange is inhibited more readily than the first. The apparent K(D) for meta-vanadate is 1.5-2mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions

Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution.

During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to selectively stabilize the "activated" phosphate conformation in order to promote cleavage

Multiple levels of affinity-dependent DNA discrimination in Cre-LoxP recombination.

Cre recombinase residue Arg259 mediates a canonical bidentate hydrogen-bonded contact with Gua27 of its LoxP DNA substrate. Substituting Cyt8-Gua27 with the three other basepairs, to give LoxAT, LoxTA, and LoxGC, reduced Cre-mediated recombination in vitro, with the preference order of Gua27 > Ade27 approximately Thy27 >> Cyt27. While LoxAT and LoxTA exhibited 2.5-fold reduced affinity and 2.5-5-fold slower reaction rates, LoxGC was a barely functional substrate. Its maximum level of turnover was 6-fold reduced over other substrates, and it exhibited 8.5-fold reduced Cre binding and 6.3-fold slower turnover rate. With LoxP, the rate-limiting step for recombination occurs after protein-DNA complex assembly but before completion of the first strand exchange to form the Holliday junction (HJ) intermediate. With the mutant substrates, it occurs after HJ formation. Using an increased DNA-binding E262Q/E266Q "CreQQ" variant, all four substrates react more readily, but with much less difference between them, and maintained the earlier rate-limiting step. The data indicate that Cre discriminates substrates through differences in (i) concentration dependence of active complex assembly, (ii) turnover rate, and (iii) maximum yield of product at saturation, all of which are functions of the Cre-DNA binding interaction. CreQQ suppression of Lox mutant defects implies that coupling between binding and turnover involves a change in Cre subunit DNA affinities during the "conformational switch" that occurs prior to the second strand exchange. These results provide an example of how a DNA-binding enzyme can exert specificity via affinity modulation of conformational transitions that occur along its reaction pathway

Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex.

Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface

Multiple levels of affinity-dependent DNA discrimination in Cre-LoxP recombination.

Cre recombinase residue Arg259 mediates a canonical bidentate hydrogen-bonded contact with Gua27 of its LoxP DNA substrate. Substituting Cyt8-Gua27 with the three other basepairs, to give LoxAT, LoxTA, and LoxGC, reduced Cre-mediated recombination in vitro, with the preference order of Gua27 > Ade27 approximately Thy27 >> Cyt27. While LoxAT and LoxTA exhibited 2.5-fold reduced affinity and 2.5-5-fold slower reaction rates, LoxGC was a barely functional substrate. Its maximum level of turnover was 6-fold reduced over other substrates, and it exhibited 8.5-fold reduced Cre binding and 6.3-fold slower turnover rate. With LoxP, the rate-limiting step for recombination occurs after protein-DNA complex assembly but before completion of the first strand exchange to form the Holliday junction (HJ) intermediate. With the mutant substrates, it occurs after HJ formation. Using an increased DNA-binding E262Q/E266Q "CreQQ" variant, all four substrates react more readily, but with much less difference between them, and maintained the earlier rate-limiting step. The data indicate that Cre discriminates substrates through differences in (i) concentration dependence of active complex assembly, (ii) turnover rate, and (iii) maximum yield of product at saturation, all of which are functions of the Cre-DNA binding interaction. CreQQ suppression of Lox mutant defects implies that coupling between binding and turnover involves a change in Cre subunit DNA affinities during the "conformational switch" that occurs prior to the second strand exchange. These results provide an example of how a DNA-binding enzyme can exert specificity via affinity modulation of conformational transitions that occur along its reaction pathway

Two surfaces of a conserved interdomain linker differentially affect output from the RST sensing module of the Bacillus subtilis stressosome.

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ(B) stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a σ(B)-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix