Strategies that take on a One Health approach to addressing antimicrobial resistance (AMR) focused on reducing human use of antimicrobials, but policy-makers now have to grapple with a different set of political, economic, and highly sensitive trade interests less amenable to government direction, to tackle AMR in the food chain. Understanding the importance and influence of the intergovernmental Codex negotiations underway on AMR in the Food Chain is very weak but essential for AMR public policy experts. National and global food producing industries are already under pressure as consumers learn about the use of antimicrobials in food production and more so when the full impact of AMR microorganisms in the food chain and on the human microbiome is better understood. Governments will be expected to respond. Trade-related negotiations on access and use made of antimicrobials is political: the relevance of AMR ‘evidence’ is already contested and not all food producers or users of antimicrobials in the food chain are prepared to, or capable of, moving at the same pace. In trade negotiations governments defend their interpretation of national interest. Given AMR in the global food chain threatens national interest, both AMR One Health and zoonotic disease experts should understand and participate in all trade-related AMR negotiations to protect One Health priorities. To help facilitate this an overview and analysis of Codex negotiations is provided. A global political consensus has been reached confirming antimicrobials underpin human health security so access and use of these miracle products has to be wound back across all sectors of the economy . One keyThe complex integrated strategies needed to reign-in the use of antimicrobials in the food, agricultural and associated industry sectors have the capacity to transform the somewhat benign and logical ‘AMR  One Health Framework’ into a quagmire of competing interests—as not all producers and users of antimicrobials are prepared to, and some not yet capable of, limiting their use of antimicrobials. But to preserve the AMR One Health global consensus much will depend on how these trade related issues are handled and will require significant leadership and clear recognition of the health security implications of failure.The UK 2016 O’Neil AMR Report ,4. This Such politically sensitive data and revelations from research on AMR in the food chain will inevitably place ‘food trade’ firmly in the spotlight. Trade policy, at its best, can help induce higher safety standards, better quality food, and safer products. But the spread of AMR microorganisms could represent one of the biggest challenges to trade in safe food and may lead to trade disruption and financial loss. Existing trade frameworks and obligations may be capable of addressing this issue but only if sensitized and adapted to prioritize safeguarding health security by providing the flexibility for governments to implement measures to safeguard their food-chain and help preserve antimicrobials, particularly those important for human medicine. AMR is an economic and global trade issue, so criticism of government action or regulatory changes perceived as running counter to concepts of ‘free trade’ will need to be managed. But this is also an opportunity for those who have extolled the benefits of trade to step up and deliver on this crucial AMR agenda. There are after all several precedents where similar large and economically painful transformations have been deemed necessary linked to ‘access and use’—sometimes for the public or global good and often to facilitate trade in new technologies or facilitate new forms of production and accumulation. For example, intellectual property and copyright provisions extended through WTO Trade-Related Aspects of Intellectual Property (TRIPS) ; reducinWith such complex trade agendas, it was inevitably these transformations created economic disruption by altering access and or use provisions which redistributed costs, benefits, investments and profits. All involved high levels of political contestation as access provisions and/or regulations were redefined at the national level or through multilateral negotiations. Interestingly, lessons and tactics used to support or block these transformations are beginning to resonate in the AMR debates and some have been picked up by media .Implementing effective AMR One Health strategies will require a similar level of political commitment and leadership to transform access and use provisions to preserve the efficacy of antimicrobials, particularly for human medicine. National AMR One Health Strategies already focus on altering both access provisions and use made of antimicrobials for human use. The other major area of antibiotic use—food production—is yet to be as systematically adapted to achieve national one health objectives. Unlike reducing human use which is negotiated and conducted entirely at the national level usually by government health authorities, but to influence the access and use of antimicrobials in food production (domestic and imported) is more complex. And involves many more interested parties. The 2006 EU ban on the use of antibiotics as growth promotors is an example of the complex legal, trade and political implications that can flow from such decisions.A fundamental understanding how national trade policies harmonize/comply with international trade rules and obligations is essential. This includes understanding the technical and legal structures that enable and legitimize the use of antimicrobials in the food chain as well as the capacity to exclude them in specific circumstances from imported food. This will require AMR public health experts to be active in setting AMR government priorities in these trade-related negotiations to ensure AMR policy coherence: The international standard setting body for food safety, Codex Alimentarius Commission (Codex); the World Trade Organization (WTO); and also Bilateral and Regional Free Trade Agreements.The rational for engaging in such esoteric areas of trade policy is that any new interpretations, obligations, rules, procedures/guidelines evolving from, for example, Codex negotiations on ‘AMR in the food chain’ have the capacity to impact on the access to and use made of antimicrobials. But of equal importance, such multilateral decisions could also circumscribe the regulatory and legal options available to national governments in implementing their AMR strategies and their domestic export/import policies if national legislation/regulations are not introduced or adapted to reflect One Health priorities. One example to be aware of is introducing the capacity to develop ‘national lists’ of antimicrobials as discussed in Codex TFAMR Report REP 19/AMR. An overview of negotiations currently underway in the Codex food standard setting body may help make transparent the complex political and legal obligations linked to international trade. Understanding the food/trade linkage is critical especially if national inter-agency policy cohesion has not fully integrated these trade-related elements. And introducing new regulations on AMR may be problematic if political commitment or bureaucratic capacity to regulate is weak.For example, the technical and scientific complexity of the AMR/food subject matter and navigating the huge number of Codex standards and guidelines  is challBureaucratically integrating the trade agenda into One Health Action Plans may be difficult but is essential. These trade-related linkages should be comprehensively understood for their effect and appropriately responded to in-line with national AMR One Health security priorities. Code of Practice to Minimize and Contain Antimicrobial Resistance and to develop new Guidelines on integrated monitoring and surveillance of antimicrobial resistance.AMR in the food chain was earlier addressed through the Taskforce on Antimicrobial Resistance (TFAMR) from 2007–2011. In 2016 Governments agreed to re-convene the TFAMR with a broader mandate to address the entire food chain and to report back to the Codex Commission by 2020 . The TerGaining consensus agreement through this Codex/TFAMR process may not be easy, particularly as these two documents will also be directly and indirectly endorsing the use of antimicrobials in the food chain. Which antimicrobials can be used in the food chain and in what circumstances represents a highly contested political agenda, particularly antimicrobials used for growth promotion and those deemed essential for human medicine. Codex Guidelines endorsed by Member States may also provide direct or indirect legitimacy for the use of these antimicrobials. Unlike WTO negotiations, Codex/TFAMR negotiations enable participation and active input from non-government entities. A reading of the open-source negotiating draft documents with input from governments, industry and consumer representatives provide insights into some of the more contentious areas . While fThe question of consistency with WTO rules is often a good excuse for government inaction. And an added factor to be cognizant of—given the ‘standard setting’ role of Codex which links directly into related WTO obligations—is that Codex standards can, and are, often used to justify positions taken in WTO Trade Dispute cases. Or trade disagreements arising when Sanitary and Phytosanitary (SPS) or Technical Barriers to Trade (TBT) agreements are enacted to restrict or place conditionality on imports.The use of antimicrobials in the food chain is a politically and scientifically contested agenda. And, despite the UN General Assembly ‘public health security’ framing and political endorsement of the WHO AMR One Health framework and the Global Action Plan (GAP) , the CodFor example, to date, neither of the TFAMR draft negotiation texts refer to the WHO Guidelines on Use of Medically Important Antimicrobials in Food-producing Animals biocides appear to now be excluded; and, altering Codex Maximum Residue Limits (MRL) to consider MRLs for medically important antimicrobials do not seem to be open for discussion.The WTO along with other international agencies has responded to the United Nations General Assembly Resolution on AMR. WTO Director General, Azevedo, has stated the existing WTO framework provides non-discriminatory measures and flexibility for governments to address AMR One Health policies especially around food safety . AzevedoAMR in the food chain is a new and complex challenge but implementing such legal and regulatory policies referred to by the WTO DG may not be simply. The international trade environment has expanded considerable since the WTO was established and is more legally complex. National trade policy and the governance framework often have to account for both WTO obligations and broader more intrusive obligations imbedded in new FTAs which may limit the scope for independent national based policy development. Also, some important structural and capacity issues may be relevant. For example, governments who have lost some in-house regulatory and governance capacity through adopting neo-liberal market based self-regulation strategies and some regulatory limitations flowing from harmonization and trade facilitation policies. Public health experts are often not sufficiently involved in these trade negotiations. Azevedo’s view that the WTO enables implementation of AMR One Health strategies rests on government’s commitment at the national level to manage/protect the domestic and export food chain in line with WTO obligations. For food-related imports the Sanitary and Phytosanitary (SPS) or Technical Barriers to Trade (TBT) agreements can be activated but have a relatively narrow interpretive space unless backed up by national regulations. This WTO report to Codex/TFAMR also records individual governments’ input on AMR issues linked to SPS reporting and illustrates some sensitive trade access issues yet to be tested, particularly related to proposed EU regulations  . This FAO/WTO ‘toolkit’ is an excellent guide to comprehend these trade rules and obligations for both policy makers and non-WTO experts  . Even in countries with sophisticated governance processes and reliable economic and trade statistics there are considerable gaps in understanding the volume and use made of antimicrobials in animal production and the AMR consequences flowing from this use. Antimicrobials used in agriculture and aquaculture production are not well understood and even less is known of the effects of AMR in the environment, wildlife, water, or soil etc.  AddressiAlways in multilateral negotiations, language, and agreed text describing the terms and definitions of the problem areas and the scope of issues, principles, and definitions that can be legitimately addressed are fundamental. And these definitions will impact on the capacity to agree on meaningful outcomes to address the issues at hand.The formal intergovernmental negotiations remain non-transparent to the broader public and media but the open-source TFAMR working draft texts to revise the AMR Code of Practice (CRD20)  and deveThe current work program of the Codex TFAMR negotiations indicates a considerable amount of work has yet to be undertaken, particularly on the new issues being addressed. It may also be difficult to meet the 2020 deadline. The following three issues are included below as ‘Case Studies’ for those who wish to delve further into the negotiating dynamics. These Case Studies illustrate some of the complex issues yet to be dealt with and deserve the attention and active engagement by governments, consumers, media and public health experts. (1)The scope of the ‘food chain’—new issues to be included;(2)Securing antimicrobials of importance for human medicine;(3)Interpretation attached to evidence—scientific evidence-based versus precautionary principle.The global transition to safeguard antimicrobials is underway but care will have to be taken to ensure that health security is not derailed by narrow interpretations or vested interests. No doubt, particularly at this stage of the negotiations many of the parameters for discussion have ambiguity built-in and while this might placate some concerns there is always the danger these limitations can become in-built into the decisions eventually evolving from the TFAMR   . These dAchieving consensus on a global approach to minimize the spread of AMR is essential but will require significant leadership and incentives to develop the necessary technical capacity to transition away from relying on antimicrobials. But ultimately it is the responsibility of national governments to maintain public confidence in their food chain and to implement governance and regulatory changes needed to address this global health security threat and protect citizens. The TFAMR tasked the Codex Secretariat  . Closing off some issues around biocides the Codex/TFAMR Report now records this agreement: “Antimicrobials used as biocides, including disinfectants, are excluded from the scope of these guidelines”  (p. 10).“Production to consumption continuum including, primary production , harvest/slaughter, packing, processing, storage, transport, and retail distribution to the point of consumption” [Some issues raised in the Expert Group Report  will be umption”  (p. 4). Many very sensitive issues, including defining principles are yet to be settled—use of growth promotors and the introduction of government’s developing ‘national lists’ could be usefully developed   (p. 6).Also worth noting is the WHO/FAO/OIE Report to the TFAMR contains a long list of forthcoming expert meetings to research/analyze outstanding AMR issues including many raised by the Expert Group . But thiGiven the threat to public health of AMR already affecting the food chain and the broader environment, any delay in taking counter-measures to actively protect citizens from such exposure is highly problematic. Especially if reasons for inaction are predicated on the basis that ‘evidence’ is not available when screening and testing has not been actively pursued by governments or the food industries responsible, or, data is not made transparently available for research.List of Critically Important Antimicrobials for Human Medicine (WHO CIA List) [The WHO Guidelines on Use of Medically Important Antimicrobials in Food-producing Animals (WHO Guidelines) [The WHO has already defined the IA List)  which radelines) .Given the logical progression of these two WHO documents, which essentially provides important implementation guidance to help preserve the antimicrobials most important for human health, but this appears to be a step too far for some countries not yet ready or prepared to take these steps. This resistance was reflected in the Codex/TFAMR documents which excludes any endorsement of these WHO Guidelines. This is an important issue that will not simply disappear, so some background may be useful.In 2017 after a two year process the WHO Guidelines were released and immediately drew criticism from the U.S. including in this media release from USDA Acting Chief Scientist questioned the legitimacy of the ‘evidence’ underpinning them as well as the role of the WHO in developing guidelines over subject matter perceived as being the preserve of the Codex and the OIE . This inSome business-focused media coverage provided this commentary on the politics behind this unusual public criticism of the WHO’s mandate to develop such guidance . A laterThis difference in opinion over the WHO Guidelines was carried through to Codex TFAMR negotiations with the WHO representative being asked to clarify the ‘status’ of the WHO CIA List and its Guidelines. A summary of WHO’s response is below but the full explanation should be understood as it clearly defines the WHO’s mandate to develop these two reports, the governance and operational procedures underpinning them, and the political flexibility accorded to governments . The WHO’s statement appears to clarify that both WHO documents have the same status and includes the following points: Both reports are science based, the primary focus is to protect public health and they are not open to negotiations. Their adoption by the World Health Assembly is not required under WHO rules and implementation by Member States is voluntary  (p. 2).With the WHO Guidelines now a source of political contention and questioning the legitimacy of decision making will prove disruptive. But in this important health security agenda questioning the legitimacy of data also has the potential to create a significant fracturing of the global consensus on AMR One Health Policy. Consumer and Health non-government organization’s input to TFAMR indicated their full support for the WHO Guidelines. This dispute over politically endorsing the WHO Guidelines will not be resolved easily or quickly as it signals implementing these WHO Guidelines to preserve the WHO CIA List may be politically problematic or too difficult for some countries. However, in stark contrast, the recommendations outlined in the OIE List of Antimicrobial Agents of Veterinary Importance  and the The two documents may be individually internally consistent according to the guidance for developing them, but not compatible for delivering the objective of preserving the effectiveness of medically important antimicrobials for human use—the WHO CIA List. This, of course, is not the only difference in approach, and it would be naïve to expect that such political differences would not arise when significant economic interests are at stake. But questioning the legitimacy of the WHO Guidelines, particularly by such a powerful player as the U.S. could put a break on measures to reduce using medically important antimicrobials that are currently extensively used in food production. Other interested parties may welcome this dispute to delay transitioning away from antimicrobial use. Worth noting, the TFAMR has not yet substantially focused on antimicrobials important for humans also used in crop production or the broader environment ,37. ThesInterestingly, asset managers of large investments in the global food industry are moving well ahead of the deliberations in Codex (and many governments). Their agenda links into the WHO CIA list and supports many of the implementation elements contained in the WHO Guidelines . These cFor a complex subject such as ‘AMR in the food chain’ the interpretation of what constitutes ‘evidence’ and the legitimacy this conveys matters—particularly in Codex , OIE 4343, and tAMR also shines a light on the need to implement and develop basic hygiene and public health infrastructure. Developing countries’ technical capacity/resources to minimize the dangers of AMR in the food chain are yet to be sufficiently addressed . From anA reading of the many submissions made into the TFAMR negotiations by government, industry and consumer representatives should leave the reader in no doubt of the underlying sensitivities and interpretations of ‘valid’ scientific data and risk. Some of these positions may however be overtaken by other events. For example, the WTO Secretariat’s Report to the TFAMR demonstrates that multilateral dialogue on trade and AMR in the food chain is being opened up to further scrutiny outside of Codex. WTO Members engaged in a substantive dialogue on AMR issues in the SPS Committee for the first time, primarily focused on EU legislative intentions to address AMR in the food chain . This EU regulatory information provided is important and covers a range of issues and given the response from several countries will be politically sensitive and played out in both Codex and WTO forums. Topics worth noting being developed by the EU include legislative measures: Addressing public health risk of AMR; reserving certain antimicrobials for treatment of infections in humans only; misuse of antimicrobials in medicated feed for prophylaxis and limiting treatment duration. The report records interesting responses and questions to the EU representative from several governments. The report also includes a list of ‘regular and emergency’ SPS and TBT Notifications submitted by Member States. The debate opened up in the WTO SPS Committee may not yet have fully registered at the December Codex/TFAMR meeting but is significant. These new inputs now formally expressed to the SPS Committee illustrate further the importance of fully integrating WTO and Codex policy into national AMR One Health strategic planning. For an observer of the Codex/TFAMR negotiations it is interesting to note that national-based AMR One Health implementation policies are actively reducing human access to antimicrobials. And at the global level, governments have politically endorsed the position that antimicrobials need to be protected and treated as a global public good. Contrasting this, reaching agreement on action to stop or reduce the non-therapeutic use of antimicrobials for food-producing animals and also to preserve medically important antimicrobials for humans, seem to require a much higher standard of scientific evidence-based data. As consumers’ understanding of the AMR One Health agenda develops, they may not support such reticence to act on this important health security issue.


Signaling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail of a receptor catalyzes reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered enzyme will contact the substrate in the volume proximal to the membrane when separated by different distances within the membrane plane. In this work, we develop particle-based stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy for slowly diffusing receptors, whereas for rapidly diffusing receptors, increasing molecular reach reduces reaction efficacy. In contrast, if reactions are forced to take place within the two-dimensional plasma membrane instead of the three-dimensional volume proximal to it or if molecules diffuse in three dimensions, increasing molecular reach increases reaction efficacy for all diffusivities. We show results in the context of immune checkpoint receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the importance of the three-dimensional nature of many two-dimensional membrane-confined interactions, illustrating a role for molecular reach in controlling biochemical reactions. Signaling by surface receptors often relies on tethered reactions wherein enzyme binding to a receptor’s cytoplasmic tail catalyzes reactions with nearby substrates. The length and stiffness of the tail, enzyme, and substrate can be summarized by the molecular reach of the reaction. The role of molecular reach in modulating the efficacy of signaling reactions is poorly understood. We show that increasing reach increases reaction efficacy when receptor diffusion is slow but decreases reaction efficacy when diffusion is fast. This switch in efficacy results from the tails of membrane-confined molecules being able to explore the three-dimensional volume proximal to the membrane. The work highlights the three-dimensional nature of two-dimensional membrane interactions, identifying reach as a control parameter for reaction efficacy.The ability of cells to sense their extracellular environment and make decisions relies on a diverse set of biochemical signaling reactions. Common to many of these reactions is the binding or tethering of an enzyme near its substrate before catalysis. Tethered signaling reactions are therefore controlled not only by binding affinities and catalytic specificities but also by the properties of tethers that control the molecular reach of the reaction (defined below). Examples of tethered signaling reactions include those that take place on scaffolds , 2 and tA). This costimulatory receptor is expressed on T cells of the adaptive immune system and is known to initiate signals important for their activation  , cytosoltivation . Phosphotivation , 8. ThisB). Molecular reach determines the probability that the enzyme will contact the substrate when the two receptors are at a defined separation distance on the membrane. The overall molecular reach of the reaction is determined by the reach of the cytoplasmic tail of PD-1 (LPD-1), the reach of SHP-2 (LSHP-2), and the reach of the cytoplasmic tail of CD28 (LCD28). Here, LPD-1, LSHP-2, and LCD28 will, in turn, depend on the respective length and stiffness properties of each component. By using the worm-like chain (WLC) polymer model, the overall molecular reach of the reaction can be defined as the square root of the squared sum of the individual reach parameters: L = 8.5 nm . This model builds on our previous work investigating tethered reactions without diffusion in surface plasmon resonance assays  model for the reaction and diffusion of individual receptors, kinases and phosphatases , 12. Impe assays .Using our particle model, we first study the dephosphorylation of CD28 by PD-1 as the molecular reach of the reaction is varied. We find that the potency of PD-1 increases as the molecular reach increases for slowly diffusing receptors. In contrast, for rapidly diffusing receptors, we find that increases in molecular reach reduce PD-1 potency. We show that this switch in potency as the molecular reach increases also holds in a commonly used biochemical model of reversible phosphorylation by kinases and phosphatases. In both biochemical models, we find that the switch is lost if membrane reactions are modeled using an idealized kernel that forces reactions within the 2D membrane plane. Using a simplified two-particle model that can be solved analytically, we reproduce these results. We then show that the switch arises from the constraint imposed by the molecules diffusing within the plasma membrane, which prevents the tethers from reaching all possible configurations in which a reactive encounter could occur. Consistent with this, the switch is lost if molecules continue to interact using the 3D physiological kernel but are instead allowed to diffuse in 3D. In this case, the region where the molecules diffuse allows the tethers to sample all possible configurations in which a reactive encounter can occur. Our work highlights the 3D nature of 2D membrane-confined reactions and suggests a possible unexpected role for molecular reach in controlling biochemical reactions.kcatσ for separation r, catalytic rate kcat, and molecular reach L; see the next section and  , 12. Hertion and , 14, the16 square voxels in 2D and 224 cubic voxels in 3D. Each curve in B–D, C–H, and To study the first two models in the σ3D determines the probability density that an individual tethered substrate  will come in contact with an individual tethered enzyme  at different separation distances between the membrane tether positions. That is, if the substrate’s tether is at position x in the membrane and the enzyme’s tether is a position y in the membrane, the separation distance between the tether positions is r = |x − y|. By assuming that the substrate and enzyme can be approximated by the WLC polymer model, an analytical expression for the probability density kernel can be obtained σmponents . In the mponents  and 2, LWe note that the original derivation of σ3D by an idealized 2D interaction kernel to artificially force molecules to interact within the plane of the membrane, given byIn a number of simulations, we replace σ3D and σ2D is provided in σ2D arises by restricting the physical diffusion of the tails to the membrane plane. In simulations using σ2D, we still assume that reactions between catalytic sites that are in contact, characterized by catalytic rate kcat, is a distinct process from the probability the sites are in contact (which is determined by σ2D). As such, we still assume that kcat is structurally independent of the reach.A plot of lp is the persistence length and lc is the contour length. The persistence length for unstructured amino acid chains has previously been estimated to be lp = 0.4 nm (lc = (0.4 nm) × N, where N is the number of amino acids between the membrane and the phosphorylated tyrosine and 0.4 nm is the Cα-Cα bond length in a polypeptide chain. The key activator tyrosine in CD28 is the YMNM motif located 11 amino acids from the membrane, and therefore, LCD28 = 1.3 nm. Similarly, the molecular reach parameter for PD-1 is estimated to be LPD-1 = 3.0 nm, where the number of amino acids between the membrane and the tyrosine in the ITSM that binds SHP-2 is N = 56.In the absence of experimental measurements, we approximate the molecular reach parameter for PD-1 dephosphorylating CD28. The molecular reach parameter for CD28 is simply = 0.4 nm , 16. TheThe molecular reach of the enzyme SHP-2 is more difficult to estimate because it is composed of three structured domains with flexible linkers: N-SH2(linker)C-SH2(linker)protein tyrosine phosphatase . Given tL = 8.5 nm.In summary, the molecular reach of the reaction for SHP-2 bound PD-1 dephosphorylating CD28 can be approximated to be A). We explicitly included the effects of molecular reach by modeling the dephosphorylation of CD28 by PD-1 as a second-order reaction whose rate was dependent on the separation distance between the molecules within the membrane (r),L is the molecular reach of the reaction. The function σ3D is the probability density (in units of molecules/nm3 or μM) for finding the enzyme and substrate at the same location when their respective receptors are separated by a distance r within the plane of the plasma membrane. It depends only on the membrane position of the receptors but accounts for the diffusive motion of the tethered enzyme and substrate within the cytosol; see σ3D under the assumption that PD-1 and CD28 can be approximated by the WLC polymer model, obtaining the Gaussian interaction given by To investigate the influence of molecular reach on the ability of PD-1 to inhibit CD28, we developed a CRDME particle model see . The modWe focus on the effects of molecular reach for the dephosphorylation reactions, and therefore, we have introduced two simplifications to the model. First, we do not explicitly include the recruitment of SHP-2 to PD-1. Second, we do not explicitly model LCK molecules but instead model CD28 phosphorylation by a first-order reaction (B). In this case, we found that increasing the molecular reach of the reaction increased the potency of PD-1 so that fewer PD-1 molecules were necessary to achieve the same level of inhibition. Unexpectedly, when using a diffusion coefficient representative of free mobility on the plasma membrane for transmembrane receptors (C).As output of the model, we calculated the steady-state fraction of phosphorylated CD28 as the concentration of PD-1 was increased. We first focused on a situation in which diffusion is minimal, which may be the case when immune receptors bind their ligands , 18, inteceptors , 22, we eceptors C.50). A plot of IC50 over L shows that PD-1 potency increases for small but decreases for large diffusion coefficients, with a transition at intermediate values of the diffusion coefficient (D = 0.00125 μm2/s) at which potency is largely unchanged (D). Taken together, we find a switch in the effect of changing molecular reach, with larger reaches increasing receptor potency when diffusion is slow but decreasing receptor potency when diffusion is fast.We quantified the potency of PD-1 by calculating the concentration of PD-1 required to reduce the phosphorylation of CD28 by 50% . As before, we allowed for diffusion of all chemical species and highlight that the rate of these enzymatic reactions is proportional to the catalytic efficacies  and σ for physiological 3D or idealized 2D interactions). The latter explicitly depends on the separation distance between the molecules in the simulation (r) and on the reaction molecular reach: Le for the kinase phosphorylating the substrate and Lf for the phosphatase dephosphorylating the substrate.A key novelty of our membrane-bound protein reaction model is in accounting for reactions involving sites on molecular tails, which move through the volume proximal to the membrane. This is achieved through the use of the 3D interaction kernel ates see . To dete σ2D see . This 2D σ2D see , as in p σ2D see . To simuA and B; , 24, 25)fication , A and BC and D). When using the idealized 2D kernel, we found that increasing the molecular reach of the reaction increased the potency of the kinase when diffusion was slower (F), but when diffusion was faster, it had no effect on the potency of the kinase (G). As before, we summarized these results by calculating the potency of the kinase as a function of the molecular reach for the physiological and idealized kernels (E and H). We confirmed that using the idealized 2D kernel in the PD-1 model of the last section also led the molecular reach to have a minimal effect in the reaction-limited, i.e., fast diffusion, regime  of tethered signaling reactions depends on molecular reach for large versus small diffusivities when molecules are confined to the 2D plasma membrane. They also suggest that such a switch may not be possible when molecules are forced to interact within the plane of the membrane. To understand what gives rise to this switch and why it is not present when the molecules react in the membrane plane, we developed a simplified two-particle Doi model that could be solved analytically.σ3D), a model in which the B molecule diffuses in 2D but tails are forced to only interact in 2D ), and a model in which the B molecule diffuses in 3D and tails interact in 3D ). In the remainder, we denote these three combinations as the 2.5D, 2D, and 3D models, respectively  of radius R about the origin. R was chosen so that the area (volume) of the circle (sphere) was identical to that of the square (cube) with sides of length 300 nm used in the preceding sections. We replace the Gaussian interaction kcatσ by an approximating indicator function λ1ε][0,(r), defined byIn the Doi model, we assume the B molecule diffuses with diffusivity λ corresponds to the probability per time the molecules react when within a reaction-radius, ε, of each other.Here, w(r) for a diffusing molecule that is initially placed a distance r from the origin then satisfiesd = 2 when the B molecule diffuses within a circular patch of membrane (2.5D and 2D models) and d = 3 when the B molecule diffuses within a spherical volume of cytosol (3D model). A no-flux boundary condition is used to prevent the B molecule from leaving the circle (sphere), and we assume that w(0) is finite (because the MRT should be finite even if the molecules start at the same location).The mean reaction time (MRT) λ and ε are calculated by matching the total volume and the first moment of σ3D for the 2.5D and 3D models. That is, given kcat and L, we choose λ and ε such thatWe find thatσ2D in the 2D model, λ and ε are calibrated by matching the total area and first moment; see ε ∝ L , we again see that when the diffusivity is small, the reaction is most effective (E) and 3D (H) models, increasing the reach always increases the reaction efficacy  and 3D H modelsncreases .As we show in the ε. We therefore see that the well-mixed MRT Here, L increases only arises in the physiological 2.5D model. It is due to the reaction-limited well-mixed MRT, L. L, whereas L for any diffusivity in all three models. The scaling of L results from the use of a 3D Gaussian interaction (with units of inverse volume) in a planar region (with units of area), resulting in an effective well-mixed bimolecular reaction rate kRL that scales like L−1. Because The regime in which r. The probability the reactive sites are in contact is then maximized for L = O(r) in both σ3D and σ2D. If A. If We can interpret the  differences between the diffusion- and reaction-limited regimes as follows. The diffusing molecule is initially placed randomly but, in the limit of very slow diffusion, is effectively stationary. Let the initial separation between the two reactants be keff. keff is given by the product of two factors. The first is the probability the diffusing reactant is sufficiently close to the stationary reactant to react, i.e., within ε = O(L) of the stationary reactant in the Doi model. Because the diffusing reactant is well-mixed, this probability scales like L2 when diffusing within the membrane and like L3 when diffusing in three dimensions. The second factor is the probability per time the molecules can react once sufficiently close, given by λ in the Doi model. For σ3D, the latter scales like L−3  in the 2.5D and 3D models and size O(L2) in the 2D model. keff is therefore constant in the 2D and 3D models while scaling like L−1 in the 2.5D model . We therefore see that in the reaction-limited regime, we can interpret the behavior of the reaction time as being a balance between an exploration effect of the two proteins  and a dilution effect .In the limit of very fast diffusion, we think of the diffusing reaction partner as always existing in a uniform probability cloud. The overall reaction process is like a first-order reaction undergone by the stationary reactant, with effective rate constant  L−3 see , whereas L−2 see . These sL for molecules diffusing and reacting in 3D (3D model) or diffusing and reacting purely in 2D (2D model). In contrast, the reaction time is still dependent on L for molecules diffusing in the membrane but reacting through the 3D interaction kernel (2.5D model). This illustrates how molecular reach in tethered signaling can reduce potency in 2D but not 3D geometries.In summary, we find that for tethered signaling reactions, the reaction time  when diffusion is slow but decrease reaction rates (or receptor potency) when diffusion is fast. This switch is critically dependent on molecules diffusing in 2D but explicitly allowing them to react in the 3D volume proximal to the membrane using a 3D reaction kernel. The work underlines the importance of the 3D nature of 2D membrane-confined reactions.It is an open problem to understand how membrane confinement modulates receptor-ligand binding and biochemical reactions. Mathematical models of membrane reactions commonly restrict molecules to not only diffuse in 2D but to react through 2D interactions , 28, 29.We have explored the molecular reach of the reaction primarily using a stationary Gaussian reaction kernel inspired by the WLC polymer model. It is likely that in some biological situations, the polymer does not equilibrate quickly (stationary assumption) and/or the kernel is not Gaussian. We calculated that the stationary assumption is valid in our simulations see . HoweverIt should also be noted that we have not considered biological contexts in which all reactants involved in a tethered signaling reaction are present at high densities. For sufficiently large concentrations, our general observations concerning the influence of molecular reach on reaction statistics could potentially change. Such density-dependent results were recently observed in a model for transport through the nuclear pore, in which a continuum of elastic tethers was shown to potentially hinder diffusive particle motion for small numbers of molecules while enhancing particle motion at sufficiently large densities . At highThe ability of receptors within the NTR or immunoreceptor group  to regulTethered signaling depends on binding, catalysis, and the molecular reach of the reaction. Although standard assays are available to study binding  and catIn this work, we have focused on the role of molecular reach in modulating reaction efficacy or potency. Given that phosphorylation reactions, and noncovalent post-translational modifications more generally, have been shown to give rise to a variety of information processing phenotypes, it would be interesting to examine the impact of molecular reach in these contexts , 40. For


Kounis syndrome (KS) is defined as the occurrence of acute coronary syndrome (ACS) associated with an anaphylactic reaction, and there have only been a few reports of its occurrence under general anesthesia.A 69-year-old woman underwent transurethral resection of a bladder tumor under general anesthesia. Cefazolin was administered intravenously after induction of general anesthesia. During the operation, we suspected ACS from sudden ST segment depression on electrocardiogram. The delayed onset of an erythematous rash reminded us of the anaphylactic reaction of KS. Coronary artery spasm of type 1 KS was diagnosed based upon the findings of coronary computerized tomography. Eleven days after the first surgery, the patient underwent nephroureterectomy uneventfully by a change in antibiotics. Finally, cefazolin proved to be the trigger drug by the intradermal test.When electrocardiogram changes suggesting ACS occur during general anesthesia, it is necessary to take KS into consideration as a differential diagnosis. Kounis syndrome (KS), also known as allergic angina, has been described as an acute coronary syndrome (ACS) associated with an anaphylactic reaction. In KS, a spastic contraction of coronary smooth muscle cells that could progress to acute myocardial damage or immediate coronary thrombosis is caused by the release of inflammatory mediators from mast cells during the allergic activation process . After tHerein, we describe a patient who showed ST segment depression on electrocardiogram (ECG) and hypotension that suggested ACS and was subsequently diagnosed as KS after the appearance of a cefazolin-induced anaphylactic erythematous rash under GA.A 69-year-old woman  was scheduled to undergo transurethral resection of bladder tumor (TUR-BT) and laparoscopic left nephroureterectomy under the diagnosis of bladder cancer and ureteral cancer. She was treated with trichlormethiazide for hypertension and did not have any history of allergy or ACS. Preoperative ECG showed a flat ST segment. Routine laboratory investigations were normal. Within the past year, the patient had undergone TUR-BT twice under spinal anesthesia and once under GA using propofol, desflurane, fentanyl, remifentanil, and rocuronium. Cefazolin was administered intravenously during every surgery.2) decreased from 100 to 94% with a fraction of inspired oxygen of 40% without wheeze. Sixty minutes after induction of GA, the ST segment in lead II of the ECG decreased to − 1.0 mV and isosorbide dinitrate was administered intermittently under the suspicion of ACS. Because of further progression of ST segment depression, we initiated continuous intravenous infusion of nitroglycerin and nicorandil. As the systolic blood pressure (SBP) dropped to 70 mmHg, continuous phenylephrine and intermittent ephedrine were administered to maintain SBP at 80 mmHg. Seventy-three minutes after induction of GA, TUR-BT was finished and the ST segment decreased to − 3.4 mV maximally. When the surgical drape was removed, an anaphylactic erythematous rash was observed on the abdomen, chest, arms, and legs. Then, we suspected anaphylactic reaction to an unknown agent of KS. Corticosteroid and chlorpheniramine were administered immediately.After placement of a thoracic epidural catheter, GA was induced with propofol, rocuronium, and fentanyl, and it was maintained with desflurane, remifentanil, and rocuronium. After the trachea was intubated, cefazolin was administered intravenously and TUR-BT started. Forty-five minutes after induction of GA, the resistance of the respiratory tract increased gradually under sufficient effect of rocuronium without any mechanical respiratory problem. Tidal volume in the mechanical ventilator decreased approximately from 350 to 250 ml under pressure-controlled ventilation. Percutaneous oxygen saturation  4 days after TUR-BT, the cardiologist diagnosed the case as type 1 variant KS. Five days after TUR-BT, intradermal tests with all of the drugs used during GA were negative.Transthoracic echocardiography findings were within normal limits. The 12-lead ECG revealed ST depression in I, II, aVF, and V3–6. The laboratory findings for troponin and CK-MB were normal. As the patient responded well to the treatment for anaphylaxis, we were able to stop continuous phenylephrine administration 101 min after induction of GA and the ST depression began to return gradually. Subsequent left nephroureterectomy was postponed and ureteral stent insertion was carried out. When the ureteral stent insertion ended 166 min after induction of GA, blood pressure and the ST depression returned to almost normal with nitroglycerin and nicorandil support. The cutaneous symptoms diminished and SpOEleven days after TUR-BT, laparoscopic left nephroureterectomy was performed under GA and epidural anesthesia. Since cefazolin was one of suspected causes of KS at that time, ciprofloxacin was administered in the ward before surgery and no reaction was observed. Preventative intravenous corticosteroid and chlorpheniramine were administered before GA, and continuous intravenous nicorandil was administered during surgery. GA was induced by thiamylal and suxamethonium and maintained with desflurane, fentanyl, and remifentanil. The minimum dose of rocuronium was administered intermittently to keep T1 of train-of-four under neuromuscular monitoring throughout the surgery. The surgery was completed uneventfully. The patient was discharged on the 18th day after the first operation. The intradermal test performed 8 months later proved that cefazolin was a trigger drug.KS has three variants , 5; typeKS might result in severe conditions, including death, usually as a consequence of myocardial infarction , 6. TherSeveral causes have been reported to induce KS, including drugs, food, environmental exposures, and drug-eluting stents. The case reports of KS under GA have described rocuronium , cisatraIt has been reported that antibiotics are the most frequently involved drugs in KS and that rocuronium is associated with a high incidence of anaphylaxis , 13. Thu2 without any special causes 45 min after induction of GA, which might have been initial symptom of KS, did not immediately remind us of anaphylaxis.The time from exposure to trigger to the onset of KS has been reported to be within 1 h in 80% of KS patients . As manyWhen ECG changes suggestive of ACS occur during GA, it is important to monitor the respiratory and cutaneous symptoms carefully, in consideration of anaphylactic manifestation of KS as a differential diagnosis.


Currently, millions of people are living with human immunodeficiency virus type 1 (HIV-1), which causes acquired immunodeficiency syndrome. However, the spread of the HIV-1 resistance to antiviral agents is the major problem in the antiretroviral therapy and medical management of HIV-infected patients. HIV-1 reverse transcriptase (RT) is one of the key viral targets for HIV-1 inhibition. Therefore, the studies on the combatting the HIV resistance that occurs due to the structural changes in RT, are in great demand. This work aims to provide an overview of the state-of-the-art molecular docking approaches applied to the studies of the HIV-1 resistance, associated with RT structure changes. We have reviewed recent studies using molecular docking with mutant forms of RT. The work discusses the modifications of molecular docking, which have been developed to find the novel molecules active against resistance mutants of RT and/or recombinant strains of HIV-1. The perspectives of the existing algorithms of molecular docking to the studies on molecular mechanisms of resistance and selection of the correct binding poses for the reverse transcriptase inhibitors are discussed. Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is an important target for design of new anti-HIV drugs. HIV-1 RT inhibitors are used both for treatment of HIV infection and for prevention of mother-to-child transmission of HIV-1 ,4,5,6,7.Molecular modeling allows explaining the molecular mechanisms of resistance associated with a particular three-dimensional complex of RT and a small molecule. This method may also be used for revealing new compounds that are active against the mutant forms of HIV-1 RT . DockingHIV-1 reverse transcriptase is a heterodimer consisting of two subunits: p66  and p51 (440 aa) ,15. It hThe functions of p51 were studied earlier ,19. AlthPolymerization starts with the interaction of reverse transcriptase with tRNA primer and RNA/DNA substrate, which in turn leads to the changes in the p66 conformation. To perform polymerization RT needs to bind primer (human tRNA) and template (virus DNA/RNA). Unbounded deoxynucleotide triphosphate (dNTP) molecules are also necessary to build DNA chains.P66 subunit contains two active sites responsible for (1) interaction with the nucleoside triphosphates (dNTP) resulting in the single-strain DNA synthesis (polymerase activity) and (2) the RNA-DNA hybrid cleavage . It is known that interaction of the polymerase domain with both a template and a primer is necessary to perform DNA polymerization. The polymerase domain contains four subdomains shaped like a human right hand: thumb (positions 237–318), fingers (1–85 and 118–155), palm (86–117 and 156–236) and connection (319–426). P51 subunit contains the same subdomains as p66 does, however, the spatial orientation of the domains in p51 differs from those in p66. The binding site of p66 with RNA/DNA formed with fingers, palm and connection of polymerase domain and the whole ribonuclease domain.The other highly conserved regions of polymerase domain include (1) (YXDD) motif  that incThere are two main types of RT inhibitors. The first ones, nucleoside reverse transcriptase inhibitors (NRTIs) are the structural analogs of the nucleosides used by RT to build DNA chain and interact with RT in its active site . ClinicaAny set of HIV-1 mutations can be classified according to the main mechanism leading to reverse transcription disruption. For example, several studies describe two distinct possible mechanisms of RT-NRTI interaction disruption: exclusion and excision .There are several mutations and its combinations leading to the resistance to NRTIs see . Some ofOther mutations that can potentially cause resistance may be classified as the thymidine analog mutations. They include the following ones: M41L, D67N, K70R, L210W, T215F/Y, K219E/Q . The preEarlier, we have mentioned the general set of mutations associated with the resistance to at least one antiretroviral drug. Some of them may be prevalent for a particular nucleoside/non-nucleoside RT inhibitor. Each drug can be characterized by its own pattern of mutations leading to HIV-1 resistance to that drug  have been described previously . The autThere are many attempts to classify the mutations leading to HIV resistance to NRTIs according to their molecular mechanisms, which might be very complicated and involve multiple pathways. Also, novel and under-investigated mutations may occur resulting in new potential mechanisms of resistance.Non-nucleoside inhibitors (NNRTIs) bind with reverse transcriptase in the allosteric site located about 10 Å away from the active site of RT at the subunits interface biochemical and X-ray data enabled to reveal that NNRTIs are non-competitive inhibitors of RT . ClinicaMutations causing resistance to NNRTIs are around the binding pocket of the NNRTIs and contain the following single replacements K103N, Y181C, L100I, K101E, V106A, V179D, Y188L, G190A, P236L see . CombinaIn particular, the most frequent mutations often resulting in HIV-1 drug resistance are regularly considered in the molecular docking studies: K103N, Y181C, Y188L/C/H/F, L100I. π-π stacking between the changed amino acids residues and small molecules [A probable mechanism leading to the resistance to RT in position Y181C and Y188L/C/H/F is associated with the loss of olecules . The losolecules . The L10olecules . The mutolecules .There are several mutations located in the connection subdomain and in RNA H active site, which increase resistance to both NRTIs and NNRTIs . For insSome relatively new mutations and their effect on HIV-1 RT-associated resistance to both NRTIs and NNRTIs are described ,50. The Molecular docking requires three-dimensional (3D) structure of RT with the amino acid changes occurring due to mutations. The data on the molecular mechanisms of resistance to NRTIs and NNRTIs can be obtained either from various biochemical methods or based on the data on three-dimensional complexes of inhibitors with RT obtained using X-ray techniques. Knowledge about the three-dimensional structures of enzyme-inhibitor complexes is especially important for the molecular modeling.Molecular docking is one of the broadly used computational procedures in the computer-aided drug design ,12,51,52http://ambermd.org/), GROMACS  or other packages [To obtain the three-dimensional structure of mutant RT, the general molecular modeling software may be used. The 3D structure of mutant RT optimization can be done by energy minimization or, preferably, by simulation of molecular dynamics using AMBER   is the mThere are a lot of studies on HIV-1 RT inhibitors where molecular docking is used for one purpose or another. Most studies are directed to the molecular docking of potential inhibitor with the wild type RT. In our review we consider the approaches where molecular docking was performed with mutant forms of RT. Some recent approaches, providing new insights on the mechanism of the HIV-1 resistance to both NRTIs and NNRTIs are presented in Usually, molecular docking is used in the HIV-1 resistance studies for the two main purposes. First, using this procedure, the authors try to explain the interaction of the experimentally tested inhibitors with a protein. Currently, such studies are widely used in tandem with experimental findings and seem to be important in the understanding of the ligand-protein interactions. Second, molecular docking is used in virtual screening to find new reverse transcriptase inhibitors. The latter often requires the combination with other computational and/or experimental methods. Here, we discuss both applications of molecular docking.In a publication by Fourati et al. the influence of the rare K65E replacement on the resistance of HIV-1 to NRTIs is discussed . We suppAn impact of A98S polymorphism on the response to the combinations of tenofovir (TDF), and emtricitabine (FTC) was studied earlier . First, Most studies were devoted to molecular docking of NNRTIs. Some recent examples of molecular docking of NNRTIs to resistant HIV RT variants are considered further.A set of diarylpyridine derivatives (DAPYs) were synthesized and tested against wild-type RT and five RT mutant variants . Twenty-The diarylpyrimidine analogs were tested and the mechanism of their activity was studied using molecular docking  In partiSeveral experiments were performed to identify the binding mode of a new (+)-calanoide analog, F18, with RT, indicating that the binding modes of F18 and nevirapine (NVP) can be different . Using mThen, the clusterization of binding poses allowed to reveal the similar stable conformations of F18 and NVP in NNRTIs . But the orientation of the F18 in Y181C variant of RT is completely different from that of wild-type and L100I variants. The molecular docking results along with the experimental studies demonstrated several differences in the binding mode of F18 with p51 of RT and NVP with p51, respectively.http://autodock.scripps.edu/) and ArgusLab . These programs were applied to investigate the binding modes between catechol diether based NNRTIs and RT wild-type, as well as Y181C and Y181C/K103N mutants [π-π stacking interactions between Tyr181 and the small molecules are lost in both the mutated variants RT (Y181C) and RT (K103N/Y181C). These results are partially in agreement with earlier suggestions about the mechanisms of resistance for Y181C and K103N mutants.In the study of Samanta et al. several molecular docking programs were considered, including Autodock  derivatives, which probably act as NNRTIs and their activity against K103N/Y181C mutant RT are described . A set oThe mechanisms of action of rilpivirine were investigated using molecular modeling methods . The autThere are several approaches leading to the selection of small compounds that are active against HIV RT using molecular docking. However, docking is not as widely used for this purpose as for the explanation of molecular mechanisms. Also, molecular docking is often employed for wild-type RT rather than for mutant RT variants.It may be concluded that the existing application of the molecular docking to HIV-1 RT resistance studies lead mainly to the discovery of new molecules targeted at well-investigated mutant RT forms ,70,71,72Summarizing the results of the studies on the application of molecular docking to the development of new HIV RT inhibitors we can make several conclusions. Molecular docking is used for designing NRTIs and NNRTIs. However, the number of studies dedicated to the NNRTIs design is significantly higher. This fact can be explained by the difficulties to properly estimate the binding energy between NRTIs and RT, due to the mechanism of NRTIs action related to competitive binding.While several possible mechanisms for HIV-1 resistance to NRTIs have been studied, described, and classified, there are still many controversial hypotheses about the mechanisms of the resistance, and a role of each mutation in the level of resistance is not known with high confidence . Thus, tMolecular docking studies with mutant RT forms require the data about the possible binding site(s) and certain binding poses of the compounds. Such investigations are restricted if there are no crystallographic complexes of mutant resistant variants of RT with known inhibitors. Such cases are typical for the recently discovered mutations.While virtual screening of compounds that are active against mutant RT forms is not widely used, an application of molecular docking to virtual screening of compounds, active against wild-type of RT can be performed ,79,80,8150) were highly dependent on the ligand set. Nevertheless, these findings support the hypothesis about the general possibility of the application of docking procedure for selection of the compounds that can be potentially active on HIV RT. There is a study , where aIn the study of Vadivelan et al., several computational methods including docking were used to screen a library of 10,000 structures of chemical compounds . MoleculIn particular, in the study of Tintori et al. , the molTherefore, the applications of the molecular docking algorithms to automated selection of the compounds active against the mutant variants of RT are highly in-demand. As have already been mentioned, the number of ligand and protein complexes is rather high, if a set of ligands should be docked into a set of proteins, where each protein contains a set of mutations. Therefore, the systematic application of molecular docking for virtual screening of RT inhibitors requires a significant amount of computing resources and thus is related to big data approaches and their application in the field of cheminformatics. We suppose that these new approaches, libraries or application programming interfaces directed to the molecular docking with the enzyme with multiple mutations and their combinations may be one of the possible ways of evolution for molecular docking approaches.While detailed enumeration of all mutation combinations requires the development of new software, libraries, and technical support, there is a need for new approaches aimed at automatic selection of favorable conformations.The development of approaches, which might improve prediction of the RT inhibition using molecular docking ,87,88,89New approaches aiming at the improvement of the predictive value of the molecular docking with mutant forms of RT are also highly in-demand. We propose that the development of various machine learning methods for improvement of docking scoring functions may lead to significant enhancement of the molecular docking predictive value. In particular, there is a number of studies where various machine learning methods were applied for prediction of binding affinity of various ligand sets ,91,92,93In this mini-review, we have considered recent approaches directed to the molecular docking of both nucleoside and non-nucleoside inhibitors to the resistant variants of RT, published in the last decade. We also discussed common mutations leading to resistance to NRTIs and NNRTIs, which are typically used in computer-aided drug design. Application of molecular docking, as well as the limitations of this approach, were considered. Molecular docking is a powerful and useful procedure for understanding the binding mode of newly synthesized and tested compounds.


Two elderly patients with poorly controlled type 2 diabetes mellitus had difficulty self‐managing their medications. Exenatide long‐acting release (LAR), with an extended administration interval of 1 month, maintained hemoglobin A1c (HbA1c) level in the 7% range. Monthly administration of exenatide‐LAR may be considered for use in carefully selected elderly individuals. There was no problem with liver function, AST: 24 (10‐40) IU/mL, and renal function, creatinine: 0.68 (0.45‐0.82) mg/dL. Diabetic complications were not observed. Initially, hemoglobin A1c (HbA1c) level was controlled at 6% by glimepiride (3 mg) and metformin (250 mg); however, she visited our clinic before the scheduled date claiming that “the medicine is gone.” Due to cognitive decline, an overdose of glimepiride and metformin was suspected, and the drugs were discontinued.A 90‐year‐old woman was diagnosed with type 2 diabetes mellitus more than 10 years ago. Her body weight was 41.7 kg (BMI: 20.1 kg/mThe patient was prescribed teneligliptin (20 mg), a dipeptidyl peptidase 4 (DPP‐4) inhibitor with low hypoglycemia risk, but HbA1c levels increased to 10.2% within 6 months. Although resuming glimepiride treatment was considered, we opted to administer 2 mg exenatide‐LAR once weekly to minimize the risk of hypoglycemia caused by overdosing. Despite declining cognitive function, she visited our clinic weekly for exenatide‐LAR injections. HbA1c level rapidly decreased; after 4 months, it reached 7.1% . There was no problem with liver function, AST: 24 (10‐40) IU/mL, and renal function, creatinine: 0.54 (0.45‐0.82) mg/dL. Diabetic complications were not observed. She was treated with teneligliptin (20 mg), but blood glucose control gradually worsened and HbA1c level increased to 9.7%. Her family informed us that she often forgot to take her multiple medications, which we assumed accounted for the poor blood glucose control.An 85‐year‐old woman was diagnosed with type 2 diabetes mellitus several years ago. Her body weight was 61.0 kg (BMI: 31.1 kg/mAs the patient did not wish to receive at‐home injections, weekly injections of 2 mg exenatide‐LAR were administered at our clinic. After switching from teneligliptin to exenatide‐LAR, HbA1c level rapidly decreased and was maintained in the 6% range (Figure 3We presented two cases of type 2 diabetes mellitus in elderly patients who could not self‐manage medications. Weekly administration of exenatide‐LAR reduced blood glucose levels, which were maintained by monthly administration. This is the first report of successful monthly exenatide‐LAR treatment.Exenatide‐LAR is encapsulated in microspheres made of biodegradable polymers, which enables gradual, long‐term release via diffusion and subsequent erosion of the polymers. Blood concentration of exenatide‐LAR peaks 7 weeks and remains high for 12 weeks by single administration.Since DPP‐4 inhibitors are highly effective, especially in East Asians,Elderly patients are often incapable of self‐managing their medications due to cognitive decline and require assistance from caregivers. Extending the interval between exenatide‐LAR injections from 1 week to 1 month after achieving blood glucose control is beneficial, as it reduces the number of clinic visits and medications required per month, improving self‐management, and lessens the workload of caregivers and clinic staff. Thus, even when self‐management is challenging, monthly exenatide‐LAR provides reliable medical treatment, with minimal intervention by outsiders. Monthly exenatide‐LAR is cheaper than DPP‐4 inhibitors; thus, monthly exenatide‐LAR treatment reduces medical costs.We described the effectiveness, free of hypoglycemia, and relative convenience of weekly exenatide‐LAR in elderly patients with type 2 diabetes mellitus who had difficulty self‐managing medications. We showed that extending the administration interval to 1 month did not diminish drug effectiveness. This reduces the burden on patients, caregivers, and clinic staff, as well as medical costs. As few drugs are effective and safe in elderly patients, exenatide‐LAR is potentially useful for the treatment of elderly patients with type 2 diabetes mellitus.None declared.KT: is the primary physician, treated the patients, and drafted the manuscript. RN: reviewed, revised, and supervised the drafting of the manuscript. All authors read and approved the final manuscript.


E. coli strains that maximize transcription of metagenomic DNA. Our results show that colony-lysis on plates allows detection of an endogenous intracellular amylase activity naturally present in E. coli and clearly increased detection of clones coding for cellulase activities in a metagenomic screening, while allowing recovery of survivor positive clones from the lysed colonies in all cases. Therefore, this tool represents an important step towards the effective access to the extraordinary potential of the uncultivated bacteria genetic resources.Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline. The lytic cluster is tightly regulated to prevent impaired bacterial growth in absence of the inducer and produced very efficient though not complete bacterial lysis upon induction, which allowed the recovery of live bacteria. The system can be used in combination with specialised fosmids and  This is because the great majority of them cannot be cultivated and characterised in a laboratory. Therefore, this extraordinary source of genetic information remains hidden even after extensive screening based on standard cultivation methods. To explore these sources, the metagenomic technologies2 involving direct isolation and cloning of DNA from environmental samples into suitable vectors have been developed. This offers ways of gene identification independent of the microorganism cultivation, either by sequence-based or function-based analysis, thus allowing more extensive analyses of the real diversity in nature.Culture-independent molecular analyses have revealed that most of the existing microorganisms in nature are still unknown to us3.While sequence analysis based on comparison with previously known sequences, has the inherent limitation that the genes identified are unlikely to be strongly divergent from the genes already known, functional metagenomic analysis, which is based on the detection of phenotypic changes in a bacterial host due to the acquisition of a metagenomic library clone, has the advantage of allowing the identification of new genes. However, functional metagenomic has two major limitations3. Of particular interest are those that incorporate viral components of the transcriptional machinery, which support transcription even across transcriptional terminators potentially present in the environmental DNA4.On the one hand, the possibility of expression of metagenomic DNA in the surrogate host that maintains the DNA library is low. To circumvent this limitation a number of broad host-range vectors for functional screening in a number of different gram negative bacteria have been developed, along with expression systems to allow transcription of long stretches of environmental DNA from vector promoters3. Therefore, the selection of clones depends on the detection of visible phenotypes, not always easy to see, among the myriad of negative colonies. Detection is also limited by the ability of the substrate of intracellular activities to enter the bacterial cells, or the ability of the surrogate host to export extracellular enzymes.On the other hand, the identification of clones with the activity of interest requires the development of highly sensitive and cost-effective screening strategies to detect clones displaying even low levels of activity among the many thousands screened. Direct detection on agar plates has the advantage that it can be carried out without the need for any special equipment. Usually, the identification of positive clones consist of a phenotypic screening based on changes in the morphology or colour of the colony, or the presence of clear halos around colonies grown on particular differential mediaE. coli has been the most frequently used host for functional screening, efficient secretion of metagenomic enzymes of this type by E. coli is unlikely. Thus, so far, the identification of these hydrolysing activities directly from E. coli colonies probably relies on the spontaneous lysis of a small percentage of cells within the colony to release their contents to the medium. Accordingly, any procedure increasing cell permeability without compromising enzyme activity and compatible with the recovery of live bacteria from the positive colony is expected to increase the frequency of positive hits for such non-secreted extracellular enzymes.For example, most of the polymer-hydrolysing enzymes are extracellular enzymes and, although 4. The plasmid pMPO579 is a fosmid vector that contains an oriT to be transferred by conjugation, along with a phage T7 RNA-polymerase-dependent promoter and a salicylate-inducible psal promoter followed by a nutL site, whose transcription is subjected to transcription antitermination by the lambda phage antitermination protein N. Besides its own ori2 replication origin of F plasmids, the fosmid also has a second broad-host range RK2 replication origin engineered for arabinose-inducible copy number. The specialised E. coli strain also produces the N antitermination protein in response to salicylate, thus allowing processive heterologous gene transcription across the metagenomic DNA fragment. This system has already been successful in identifying antibiotic resistance determinants4 and other functions of interest5 that would not have been detected with other methods available.In this work we have used a metagenomic library constructed using fosmid pMPO579 and the specialized strain MPO554Salmonella and E. coli cells in response to the addition of anhydrotetracycline (AHT). Despite massive lysis causing the release of the bacterial cell contents to the growth medium, a small fraction of surviving bacterial cells consistently remains after the induction of lysis6. Here we have adapted this system for its use in functional metagenomic analyses and have evaluated its usefulness as a tool for increasing detection of positive clones in agar plate assays. The results corroborate that the inducible autolysis system does not affect bacterial viability in the absence of induction, but efficiently lyses cells upon induction. Release of cell contents by this method permits the detection of intracellular enzymatic activities not identified otherwise and improves detection of extracellular enzymatic activities while still allowing the recovery of live positive clones.To circumvent the aforementioned limitation in the detection of activities that require substrate transport or protein secretion we have used a previously developed inducible autolysis system based in the lysis operon of the lambda phage. This device provokes lysis of 6, the set of plasmids available is not compatible for their use in combination with the fosmid of the metagenomic library due to antibiotic resistance marker incompatibility. In order to make both (fosmid and autolytic plasmids) compatible, we transferred the lysis system to a multi-copy vector that confers resistance to ampicillin. To that end the SRRz cluster of lambda phage was cloned under the Ptet promoter control in pMPO1070 (a derivative of pASK-IBA43) generating plasmid pMPO1077. The lysis gene cluster consists of four genes: S encoding the holin that produces a collapse of the membrane potential and permeabilisation of the cell membrane7; R encoding the endolysin degrading the peptidoglycan layer; and Rz and Rz1 whose products form a complex that bridges the cell and outer membranes, causing the disruption of the latter8.Although an AHT-inducible lysis system was developed in a previous workE. coli DH5α and bacterial culture lysis was characterized.Both set of plasmids, the previously developed low copy vectors pMPO1632 and its control pMPO1631 (lacking the lysis system), and the new high copy version pMPO1077 and its control pMPO1070 were transferred to 600 of bacterial cultures carrying the lysis systems decreased after AHT induction in both, high and low-copy vectors. Interestingly, in the high copy vector the lysis began earlier (apparently at the time of induction) and was faster than the lysis observed from low-copy vector. The number of colony forming units (cfu) on LB plates decreased 100-fold when compared to the cfu at the time of induction for the low-copy vector and more than 1000-fold for the high-copy vector. However, in both cases a fraction of the bacterial population survived  containing 1:10 lysis vs control strains, were plated on LB plates containing starch. After colony growth, lysis was induced and plates were incubated overnight at room temperature. A lugol solution11 was subsequently poured over the plates to reveal amylase activity. Pale blue, purplish or colourless halos surrounding colonies (in contrast to the blue colour of negative controls) indicated the presence of the activity. As shown in Fig. To determine if lysis on solid media is sufficient to allow detection of an intracellular activity, we tested if the lysis system promoted detection of the endogenous amylase activity naturally present in The expression of the metagenomic DNA in our specialised fosmid (pMPO579) and strain (MPO554) is induced by salicylate. For the use of the autolytic system in combination with this fosmid and strain it is necessary that the lysis can be induced while the heterologous DNA is being expressed. To test this, we measured lysis induction in cultures of MPO554 bearing plasmids pMPO579 and the lysis (pMPO1077) or control plasmid (pMPO1070), after 90 minutes of growth in the presence or absence of salicylate. As shown in Fig. 5. The constructed library consists of 185,000 independent clones maintained in the E.coli strain EPI300-T1.To evaluate the system in a real screening, we decided to look for cellulase activity in a metagenomic library previously constructed in our laboratory. The metagenomic library consists of 6.5 Gb of metagenomic DNA of a petroleum-contaminated soil and was constructed in a fosmid vector that promotes heterologous expression of metagenomic DNA after salicylate induction in the specialized strain MPO55412 to the MPO554 NalR strain4 carrying the lysis plasmid (pMPO1077). The mating mixture was diluted on LB selective medium containing 1% of carboxymethyl cellulose (CMC) to obtain 102 to 103 cfu of transconjugant cells per plate (15-cm diameter). The plates also contained salicylate to induce heterologous transcription and arabinose to increase the fosmid copy number. After colony growth, we induced lysis by overlaying the plates with soft agar containing AHT, as above. The plates were further incubated overnight at room temperature. Subsequently, cellulase activity was detected by Congo red staining13. Radial diffusion of enzyme produces zones of hydrolysis, which can be visualized by staining the non-degraded substrate with Congo red dye. Approximately 200.000 colonies distributed across 150 plates were screened.For the screening, the metagenomic library was transferred by triparental matingAfter detection of putative positive clones, survivors from the colonies were picked out through the soft agar layer and cellulase activity was confirmed as before: firstly, by growing the positive colonies on LB plates supplemented with CMC, salicylate, arabinose and antibiotics; secondly, by inducing subsequent lysis with AHT and Congo red staining. Finally, we confirmed 13 positive clones out of the 50 initially detected.R/pMPO1077 to MPO554 GmR /pMPO1077 and MPO554 GmR /pMPO1070 as control. Mixed cultures of both strains (MPO554 GmR with lysis or empty vector) were diluted and spread in ratio 1:1 onto LB plates containing CMC, arabinose, salicylate and the appropriate antibiotics. As before, after colony growth, lysis was induced and cellulase activity was revealed with Congo Red. As shown in Fig. versus small or no halo) were picked through the soft agar layer and lysis was tested in EBU plates  intracellular activity present in E. coli, such as the amylase activity.We have shown that this autolytic system induced by AHT works in However, in spite of the high efficiency of lysis, the survival percentage was sufficiently high to allow recovery of live bacteria from every single colony tested. Interestingly, survival was not due to loss of the plasmid or mutations in the lytic system since the survivors maintained their sensitivity to lysis in the presence of AHT. Whether this stochastic possibility of survival is due to the regulatory system by tetracycline or to the intrinsic dynamics of the lysis system it has not been explored, but for its application in functional metagenomic screening, this supposes an added advantage given that, after the initial screening, we can reuse the lytic system present in the positive hits to confirm the phenotype. The confirmation of putative positive clones is very important to rule out false positives that may occur during the screening.5. We tested the system in combination with specialised fosmid and strains developed in our laboratory4. We transfer by conjugation the metagenomic library to the specialised strain in which we had previously introduced the autolytic system.We have tested the system in a real screening for cellulases using a metagenomic library constructed previously in our laboratory from a petroleum-contaminated soil4 (data not shown) and allows the growth of colonies in the presence of arabinose to increase the fosmid copy number and salicylate to promote heterologous transcription. Even under these conditions of induction of metagenomic DNA expression, the addition of AHT induces the production of sufficient amount of lytic protein to lyse bacterial cells in liquid cultures and colonies on agar plates. The lysis of the colonies results in the release of the bacterial content to the agar medium and significantly improves the ability to detect positive clones.Our results suggest that the presence of the lysis system does not adversely affect the mating frequency compared to the previous data3 and could be combined with previously constructed metagenomic libraries simply by transferring the library to recipient strains carrying the lysis plasmid or alternatively by electroporation of the lysis plasmid to cells hosting the metagenomic library. This new system is therefore a powerful tool for functional metagenomic analyses, less expensive and more efficient than others described before.Our autolytic system combined with fosmid and strains that maximize metagenomic DNA expression improves significantly the sensitivity of the screening procedures based on phenotypic changes of colonies on agar plates for the search of extracellular activities. This system is also compatible with many of the fosmids and vectors normally used for the construction of metagenomic libraries21. The SRRz (1917 pb) cluster was obtained by PCR amplification of lambda DNA using DNA primers lisFw (5′-ACGGATGGCAACATATTAAC-3′) and lislabdrev (5′-ATATGGGCAACTCTATCTGC-3′) and cloned under the Ptet promoter of pMPO1070 derivative of pASKIBA43. The plasmid pMPO1070 derived from pASKIBA43 to which the strong RBS sequence and the 6xhistidine tag was removed by digestion with NdeI and XbaI and subsequent filling of 5′-protruding ends with Klenow and religation. Plasmid pMPO1070 containing the SRRz under the Ptet promoter control was named pMPO1077.DNA manipulations were performed according to standard procedures22 was used. The gene was amplified by PCR from pBBR1MCS523 using the previously phosphorylated primers semiFRTGmFw (5′-GAAAGTATAGGAACTTCTGGACGCACACCGTGG-3′) and semiFRTGmRev (5′- TAGAGAATAGGAACTTCCTGGCGGCGTTGTGAC-3′). The product of PCR was religated and subsequently electroporated into MPO554 bearing the plasmid pCP2024. pCP20 has temperature-sensitive replication and thermal induction of the FLP recombinase. MPO554 contains a FRT sequence into the chromosome recognized by FLP recombinase allowing site-specific recombination. MPO554/pCP20 was transformed with the religated product of PCR indicated above and GmR transformants were selected at 37 °C. After colonies growth we tested loss of pCP20 and selected GmR, ApS and CmS colonies. Integration of the gentamicin resistance gene into the FRT sequence was confirmed by PCR amplification using primers SacP1 and trgEC-E24.To integrate the gentamicin resistance gene into the chromosome of MPO554 a modification of the Datsenko procedure−1 chloramphenicol, 15 µg mL−1 nalidixic acid, 10 µg mL−1 gentamicin and 100 µg mL−1 ampicillin. The copy number of the fosmids in EPI300TM-T1 and derivative strains was increased by the addition of 1 mM arabinose. 1 mM salicylate was used as an inducer of heterologous expression for functional screenings in plates.The bacterial strains and plasmids used in this study are described in in Supplementary Table For amylase activity screenings, bacteria were grown in LB containing starch 0,5% and supplemented with appropriated antibiotics. To reveal the amylase activity after colonies growth and lysis (see below) plates were stained with lugol solution (Sigma) and the development of colour was observed. Pale blue, purplish or colourless halos indicated the positive activity while blue colour indicated negative colonies.−1 Congo red for 30 minutes13. The stain was then poured off and the plate washed with 1 M sodium chloride (30 minutes). A clear halo surrounding colonies indicates a cellulase producing clone.For cellulase activity screenings, bacteria were grown in LB containing 1% of carboxymethyl cellulose (CMC) and supplemented with appropriated antibiotics and arabinose and salicylate (see below). After colonies growth and lysis (see below) plates were stained with an aqueous solution of 10 mg mLet al.6. For lysis of liquid cultures, bacteria were grown at 37 °C in LB supplemented with the appropriate antibiotics and supplements. After growth, the saturated cultures were diluted in LB and allowed to grow until reaching an optical density (A600) of 0.2, at this point each culture was divided into two groups. In one of the duplicate groups, lysis was induced with the addition of AHT at a final concentration of 0.2 μg mL−1 while the other set of cultures was the non-induced control group. Both sets of cultures were incubated at 37 °C and the A600 were measured at regular time intervals. To quantify the number of viable bacteria, cultures dilutions were spread on LB plates without antibiotic and colony-forming units (CFU) were counted.For lysis induction we used the protocols previously described by Camacho R cultures of MPO554 NalR carrying the appropriate combination of plasmids were grown at 37 °C in LB supplemented with antibiotics. Once grown, cultures were diluted in LB supplemented with arabinose 1 mM and, and once an optical density (A600) of 0.2 was reached, each culture was divided into two groups. One of the duplicated groups was induced with salicylate while the other set of cultures were the non-induced group. Both sets of cultures were incubated at 37 °C and the A600 was measured at regular time intervals. After 90 minutes of growth at 37 °C, autolysis of each culture was induced with AHT to a final concentration of 0.2 μg mL−1.For induction of expression with salicylate and subsequent lysis induction cultures of MPO554 Nal−1 in a layer of soft agar (LB with agar 8 mg mL−1) and plates were incubated at 37 °C until lysis was observed or o/n at room temperature.For lysis of colonies in solid media, once the bacteria are grown on the appropriate plates, autolysis was induced by pouring AHT to a final concentration of 0.2 µg mL12 with DH5α/pRK2013 as the helper strain. Conjugative matings were performed on LB-agar without antibiotic selection overnight at 30 °C. The mating mixtures were then plated on LB-agar with the necessary antibiotics for the transconjugants selection, salicylate for inducing heterologous expression and arabinose for increasing the copy number of the fosmid, and incubated at 30 °C for 24 h.Vectors and metagenomic libraries were transferred by triparental matingsMating frequencies were estimated as the ratio of transconjugant clones of the recipient strain  to total clones of the recipient strain .A more detailed protocol for the use of the lysis system in a metagenomic screening of cellulases is provided in Supplementary Methods.Supplementary material


A 64-slice CT scanner was used at 100-kVp tube voltage and 150-mA tube current, and images were reconstructed using IMR techniques. Image noise, attenuation at the aorta, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) at the proximal right and left main coronary arteries (pRCA and LMCA) were calculated. CCTA images were qualitatively evaluated using a four-point scale  and analyzed using a non-inferiority test with a pre-defined non-inferiority margin of -0.2. The mean CCTA radiation dose  and mean aortic root CT attenuation values  did not differ significantly between the two groups. The mean noise in groups A and B was 26.0 ± 4.8 HU and 29.2 ± 4.4 HU, respectively (p = 0.005). The noise reduction ratio in the groups, compared to filtered back projection, was 65.0% and 68.1%, respectively. The mean grade of image quality did not differ significantly . Group B CCTA image quality was non-inferior  to that of Group A. We concluded that low-dose CCTA with prospective ECG-triggering and IMR might be applied to overweight subjects, as well as to normal-weight subjects, by using a fixed tube current without an increase in tube current based on the patient’s body size.We investigated the feasibility of low-dose coronary computed tomography angiography (CCTA), using a prospective electrocardiogram (ECG)-triggered axial scan protocol, knowledge-based iterative model reconstruction (IMR), and fixed tube current, in overweight subjects. Forty non-overweight (group A; body-mass index [BMI] < 25 kg/m Reducti2 . When th2 . Advance2 . Advance2 , 10. In 2 . Several2 [2 , 12. In 2 [2 , it is eWe assumed that with IMR, tube current increase according to body size might not be necessary, even if the subject is overweight and the tube voltage is 100 kVp, because IMR could reduce the image noise significantly. However, no previous report has investigated image quality and radiation dose in relation to BMI, using CCTA with IMR, a low tube voltage, and a fixed tube current. The purpose of this study was to evaluate the feasibility of using low-dose CCTA, implementing a prospective electrocardiogram (ECG)-triggered axial scan protocol, 100 kVp tube voltage, IMR, and fixed tube current, in overweight subjects.This study was reviewed and approved by the Institutional Review Board of our institution . Because this study was a retrospective observational study, the need for obtaining informed consent from participants was waived.2 and 5 with BMI = 25–30 kg/m2) who were eventually not included in this study. The margin of non-inferiority for the qualitative image quality, which served as the basis for the sample size calculation, was set as -0.2 [The sample size was derived from preliminary image quality assessments of 10 subjects (5 with BMI < 25 kg/m2) and an overweight  group were enrolled retrospectively and consecutively; participants underwent CCTA for coronary artery disease screening using prospective ECG-gating CCTA. For subjects whose heart rates exceeded 65 bpm before examination, a β-blocker  was administered orally, 1 hour prior to CCTA. The exclusion criteria were as follows: (i) a heart rate exceeding 65 bpm even after oral administration of a β-blocker, (ii) arrhythmia, (iii) known hypersensitivity to contrast media containing iodine, (iv) decreased renal function (serum creatinine > 150 μmol/L), (v) hemodynamic instability, and (vi) congestive heart failure.Forty participants each from a non-overweight , in the craniocaudal direction, during a single breath-hold at end-inspiratory suspension. The scan range captured the heart from the carina level to the diaphragm. A step-and-shoot technique was used with a prospective ECG-gated protocol. The scanning parameters were as follows: (i) step-and-shoot axial scanning, (ii) 400-ms gantry rotation time, (iii) 100-kVp tube voltage, and (iv) 150-mAs tube current without a tube current increase based on the patient’s body size. Through an 18-gauge intravenous catheter placed in the antecubital fossa, Ioversol, containing a 350 mg/mL iodine solution , was injected at a rate of 4–5 mL/s. Thereafter, 50 mL of 0.9% saline was administered by a power injector  at a speed of 5 mL/s. The body weight was used to determine the total dose of contrast (1 mL/kg). A real-time bolus-tracking method was used for imaging. The region of interest (ROI) was drawn at the proximal descending aorta. After 7 seconds, the scanning process proceeded only when the attenuation at the ROI exceeded 130 HU. In all scans, participants successfully executed the breath-hold maneuver. The subjects underwent simultaneous ECG recordings in each study. The dose-length product (DLP) was multiplied by 0.014 mSy / (mGy × cm), the conversion coefficient, in order to calculate the effective radiation dose [All CCTA images were reconstructed by knowledge-based iterative reconstruction . The parameters for reconstruction were: (i) 0.9-mm slice thickness, (ii) 0.45-mm increments, (iii) 512 × 512-pixel image matrix, (iv) XCC kernel, and (v) 15–23-cm field of view. We fed the images through a picture archiving and communication system . Post-processing for CCTA was achieved using commercial software .Image quality was quantitatively analyzed in CCTA reconstructed with IMR in both non-overweight and overweight patients. On axial CT images, a round ROI was placed on the ascending aorta, proximal right coronary artery (RCA), and left main coronary artery (LM) to calculate the vascular attenuation values. To ensure that all three series of axial images were obtained at the same level, the cross-reference function on PACS was utilized. The image noise of CCTA was defined as the standard deviation of the attenuation values measured at the ascending aorta. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated as follows.Two radiologists with 10 and more than 20 years of experience in cardiac CT, who were blinded to the patient’s medical records, independently performed qualitative assessment of the image quality of CCTA reconstructed with IMR in both groups A and B. They used a 4-point grading system at the four main coronary arteries  as follows .Grade 1 (poor/non-diagnosable): severely degraded image, inability to evaluate vessel lumenGrade 2 (adequate): moderately degraded image, minor difficulty in evaluating vessel lumenGrade 3 (good): marginal image degradation, no difficulty in evaluating vessel lumenGrade 4 (excellent): no detectable degradation of imaget-tests. Differences in sex distribution between two groups were assessed for statistical significance using a chi-square test. For analyzing differences between the groups in terms of CT attenuation, image noise, SNR, CNR, and radiation dose, independent two-sample t-tests were used. Interobserver reproducibility of CCTA attenuation and noise was verified by the Intraclass correlation coefficient (ICC). ICCs of <0.40, 0.40–0.75, and 0.76–1.00 indicated poor agreement, fair to good (moderate) agreement, and excellent agreement, respectively. Qualitative image quality was evaluated by using linear mixed model analysis, considering multiple vessels per patient. The 95% confidence interval (CI) was estimated to test the image quality differences between the two groups, qualitatively. The non-inferiority of the qualitative image-quality in group B compared to group A was demonstrated if the lower limit of the two-sided 95% CI lies above the non-inferiority margin. The non-inferiority margin for image quality differences among the two subject groups was set as -0.2 [Categorical variables were represented as numerical values of frequencies and/or percentages, while continuous variables were noted as mean ± standard deviation (SD). Data distribution was evaluated using the Shapiro–Wilk test and Q-Q plots. Demographic differences between the two allocated groups, such as age, height, weight, BMI, and heart rate, were analyzed using independent two-sample The overall data exhibited a normal distribution. Excellent interobserver reliability was proven in the quantitative analysis of CCTA image quality . Cohen’s kappa test showed good interobserver agreement in the qualitative assessment of the image quality of CCTA .2) and another 40 were allocated to group B . The mean radiation dose of CCTA was not significantly different between the two groups . The clinical characteristics of the two groups are summarized in Eighty patients  who underwent CCTA for coronary artery disease screening were retrospectively enrolled. Forty individuals were allocated to group A . The mean noise in Group A was lower than that in Group B . Group A showed significantly higher SNR at the RCA and LM than Group B . The CNR of the RCA and LM were also significantly higher in Group A than in Group B   . The noi the LM) .p = 0.478). The non-inferiority of the CCTA image quality in Group B was proven, as the lower limit of the 95% CI of the image quality difference exceeded -0.2, which is the pre-set non-inferiority margin   , Fig 2.Our study showed that CCTA with prospective ECG-triggering and IMR can be applied to overweight subjects as well as to normal weight subjects, using a fixed tube current, without the need for tube current modulation based on the patient’s body size.In 2009, Hausleiter et al.  reportedWith existing reconstruction based on FBP, the radiation dose needs to be doubled to attain the same level of image noise in patients with high BMI as for patients with standard BMIs . Recentl2, and showed that IMR reduced image noise in CCTA to 56–67%, as compared with FBP techniques [In previous studies, we evaluated the feasibility of lowering tube voltage for CCTA in non-overweight patients, with BMI < 25 kg/mchniques , 15. Odachniques  reportedchniques , and IMRchniques , 15. On This study had a few limitations. First, IMR is one of many reconstruction algorithms and the results in this study could not be applied to other algorithms from other vendors, such as the ADMIRE and MBIR algorithms. Second, this was a single-center, retrospective study. Multi-center prospective clinical trials are needed to confirm our results. Third, we compared the quantitative or qualitative image qualities of CCTA, without evaluating the diagnostic accuracy for coronary artery disease. Fourth, non-inferiority test was conducted to compare qualitative image qualities. Statistical non-inferiority was found in a pre-defined non-inferiority margin, but clinical non-inferiority can not be guaranteed. Last, we did not perform a phantom study for spatial resolution assessment of our CT system. We focused on clinically evaluating the quantitative and qualitative image quality of CCTA with low radiation dose, based on previous reports , 15. FutIn conclusion, low-dose CCTA with 100 kVp, prospective ECG-triggering, and IMR might be applied to overweight subjects as well as to normal weight subjects, using a fixed tube current, without increasing tube current based on the patient’s body size.S1 FileAttached files are data of 40 non-overweight  and 40 overweight individuals , who underwent CCTA with prospective ECG-triggering and IMR for coronary artery disease screening.(XLSX)Click here for additional data file.