To assess fidelity of the Healthier You: NHS Diabetes Prevention Programme (NHS‐DPP), a behavioural intervention for people in England at high risk of developing type 2 diabetes, to the specified programme features.Document analysis of the NHS‐DPP programme specification, including National Institute for Health and Care Excellence (NICE) PH38 diabetes prevention guidance. This was compared with the intervention design  from all four independent providers delivering the NHS‐DPP. Documents were coded using the Template for Intervention Description and Replication framework (describing service parameters) and the Behaviour Change Technique Taxonomy v1.Providers demonstrated good fidelity to service parameters of the NHS‐DPP. The NHS‐DPP specification indicated 19 unique behaviour change techniques. Framework responses for the four providers contained between 24 and 32 distinct behaviour change techniques, and programme manuals contained between 23 and 45 distinct behaviour change techniques, indicating variation in behaviour change content between providers’ intervention documents. Thus, each provider planned to deliver 74% of the unique behaviour change techniques specified, and a large amount of behaviour change content not mandated.There is good fidelity to the specified service parameters of the NHS‐DPP; however, the four providers planned to deliver approximately three‐quarters of behaviour change techniques specified by the NHS‐DPP. Given that behaviour change techniques are the ‘active ingredients’ of interventions, and some of these techniques in the programme manuals may be missed in practice, this highlights possible limitations with fidelity to the NHS‐DPP programme specification at the intervention design stage. The National Health Service (NHS) Diabetes Prevention Programme (NHS‐DPP) is a behavioural intervention for adults in England at risk of developing type 2 diabetes.This is the first fidelity evaluation and first known assessment of the behaviour change technique content of a national intervention.The NHS‐DPP intervention design demonstrated good fidelity to the service parameters itemized in the programme specification, but programme manuals included only 74% of the specified behaviour change techniques.As behaviour change techniques are active ingredients that produce behaviour change, this highlights possible limitations with fidelity to the NHS‐DPP programme specification and dilution of likely effect in NHS‐DPP delivery. The National Health Service (NHS) Diabetes Prevention Programme (NHS‐DPP) is a behavioural intervention for adults in England at risk of developing type 2 diabetes.This is the first fidelity evaluation and first known assessment of the behaviour change technique content of a national intervention.The NHS‐DPP intervention design demonstrated good fidelity to the service parameters itemized in the programme specification, but programme manuals included only 74% of the specified behaviour change techniques.As behaviour change techniques are active ingredients that produce behaviour change, this highlights possible limitations with fidelity to the NHS‐DPP programme specification and dilution of likely effect in NHS‐DPP delivery.The Healthier You: NHS Diabetes Prevention Programme (NHS‐DPP) is a behavioural intervention focused on encouraging lifestyle behaviour change for adults in England with elevated blood glucose levels The required NHS‐DPP intervention content is outlined within a published Service Specification Given that the Service Specification An evaluation of the NHS‐DPP at the formative stage This prior evaluation Previous reviews have focused mostly on fidelity of intervention delivery  NICE PH38 public health guideline A comparison was made of the programme specification and the intervention design. The full programme specification (describing what should be present within the NHS‐DPP) consisted of:four framework response documents (one per provider organization) describing the proposed service delivery, submitted in providers’ bids for service procurement;six programme manuals .The intervention design (describing what providers plan to deliver) consisted of:The 12 documents were examined using two frameworks. Planned programme content for the full programme specification and each of the providers’ intervention designs was reviewed using the Template for Intervention Description and Replication (TIDieR) framework For each of the 12 documents reviewed, data extraction and coding was carried out independently by two researchers (EC and RH). TIDieR variables were extracted and behaviour change techniques were coded using author‐developed data collection forms (see Doc. S1 detailing coding procedures). Researchers underwent training in the use of the BCTTv1 Interrater reliability (IRR) was calculated using Cohen's kappa coefficient This study analysed only written documents and did not include data collected from human participants, and therefore falls outside the remit of NHS Research Ethics Committees. However, the wider programme of research of which this study is a part was reviewed and approved by the North West Greater Manchester East NHS Research Ethics Committee .The service parameters specified by NHS England The intended behaviour change technique content from the full programme specification  compared with their framework responses. Providers A and D stated plans to deliver more techniques according to their framework responses , which did not track through to their programme manuals. Given that providers’ manuals are more proximal to the planned delivery, this suggests dilution in planned behaviour change content. Across the programme manuals and framework responses combined, a total of 41, 38, 47 and 39 behaviour change techniques were identified in providers’ intervention designs respectively.Behaviour change technique content was compared between the full programme specification and providers’ intervention designs to assess fidelity of the NHS‐DPP design. Of the 19 specified behaviour change techniques in the full programme specification, providers included 13 (68%), 15 (79%), 13 (68%) and 18 (95%) specified techniques respectively in their framework responses  behaviour change techniques specified by NHS England The current analysis provides a detailed understanding of the whole NHS‐DPP intervention design; it utilized more recent documentation compared with a previous fidelity analysis of the NHS‐DPP intended to be the key ‘active ingredients’ in their intervention designs. Similarly, authors coded all identifiable techniques in the full programme specification that were recognized as being required by NHS England, and thereby determined the ‘active ingredients’ of the programme specification. It is noted that the NHS Service Specification All techniques that could result in behaviour change, according to the BCTTv1 This article provides a reliable method for assessing fidelity of intervention design using behaviour change technique coding; kappa values demonstrated high agreement between coders. However, providers were considered to demonstrate fidelity when a behaviour change technique stated in the full programme specification was present in providers’ intervention design documents, although there is no compelling evidence that use of a technique once is sufficient.Finally, Hawe and colleagues , which reported good fidelity of the intervention design. However, the current analysis has identified a 26% loss of fidelity by behaviour change technique omission according to providers’ programme manuals. The programme manuals describe a session‐by‐session protocol for delivering the NHS‐DPP in practice, therefore likely providing the most accurate representation of the planned techniques in each providers’ intervention design. Programme manuals were not available for the previous fidelity assessment of the NHS‐DPP Providers generally demonstrated good fidelity to the service parameters of the NHS‐DPP design, but according to their programme manuals only planned to deliver 74% of the specified behaviour change techniques in the programme specification This is the first known assessment of behaviour change technique content of a national diabetes prevention programme intervention and one of the first fidelity evaluations of a national intervention. Previous diabetes prevention trials Sixty‐four per cent of planned behaviour change techniques across all of the programme manuals were not specified by NHS England or NICE. However, we do not yet know whether this is problematic as the NHS‐DPP is evidence‐inspired rather than evidence‐based. That is, there is not yet any effectiveness analysis of the NHS‐DPP, only expert opinion guided by evidence from related research 1c in people with type 2 diabetes The impact of both absent and additional non‐specified behaviour change techniques on the effectiveness of the NHS‐DPP is yet to be established. Previous systematic reviews suggest the provision of social support and ‘self‐regulatory’ techniques such as goal‐setting, self‐monitoring and feedback are effective in interventions targeting diet, physical activity and weight loss Already as a result of this document analysis, NHS England have stated requirements for providers to be more explicit regarding behaviour change strategies in their framework response documents which track through to their programme manuals; this requirement is stated in the new NHS Service Specification This article provides a method for measuring fidelity of a national programme's intervention design, extending on previous fidelity research which, to date, has focused more on evaluating fidelity of intervention delivery. This method builds on suggestions set out by the NIH‐BCC The present research has identified all key behaviour change techniques in the NHS‐DPP intervention design, providing a baseline description for evaluating later steps in the NIH‐BCC model The current document analysis of the NHS‐DPP intervention design has identified generally good fidelity to the service parameters of the NHS‐DPP programme specification. However, according to their programme manuals, providers planned to deliver only 74% of behaviour change techniques specified by NHS England This work is independent research funded by the National Institute for Health Research . The views and opinions expressed in this manuscript are those of the authors and do not necessarily reflect those of the National Institute for Health Research or the Department of Health and Social Care.None declared.Table S1. Cohen's kappa values for each specification document and provider document.Doc. S1. Behaviour change technique coding procedures.Click here for additional data file.


Biodiversity declines threaten the sustainability of global economies and societies. Acknowledging this, businesses are beginning to make commitments to account for and mitigate their influence on biodiversity and report this in sustainability reports. We assessed the top 100 of the 2016 Fortune 500 Global companies' (the Fortune 100) sustainability reports to gauge the current state of corporate biodiversity accountability. Almost half (49) of the Fortune 100 mentioned biodiversity in reports, and 31 made clear biodiversity commitments, of which only 5 were specific, measureable, and time bound. A variety of biodiversity‐related activities were disclosed , but only 9 companies provided quantitative indicators to verify the magnitude of their activities . No companies reported quantitative biodiversity outcomes, making it difficult to determine whether business actions were of sufficient magnitude to address impacts and were achieving positive outcomes for nature. Conservation science can advance approaches to corporate biodiversity accountability by helping businesses make science‐based biodiversity commitments, develop meaningful indicators, and select more targeted activities to address business impacts. With the biodiversity policy super year of 2020 rapidly approaching, now is the time for conservation scientists to engage with and support businesses in playing a critical role in setting the new agenda for a sustainable future for the planet with biodiversity at its heart. Article impact statement: Science‐based biodiversity commitments, meaningful indicators, and activities need development to address environmental impacts of business. Biodiversity underpins and sustains ecosystems globally, and declines in biodiversity threaten the resilience of nature, global economies, and societies  CBD  sets a vCorporate biodiversity accountability  is an important aspect of organizational stewardship and legitimacy, which an increasing number of businesses are undertaking  Fortune  through,sustainability” and company name). The most recent reports (dated up to 2016 and searched for in September 2017) were collated. Sustainability reports can also be referred to as environmental, corporate social responsibility, sustainability, registration reports, or financial reports that contain non‐financial information. Companies made up of multiple subsidiary companies  were assessed only when sustainability reporting was done for the Fortune‐listed company as a whole, not subsidiary companies. Websites were not included in our analysis when the year of biodiversity commitments or activities were not stated; only dated interactive online sustainability reports were analyzed. Reports were searched for “biodiversity” OR “nature” OR “species” OR “ecosystem” in acknowledgment of the broad definition of biodiversity  that are commonly used in relation to nature‐based commodities without mention of biodiversity‐related terms.Online searches for the Fortune 100 sustainability reports were conducted using the Global Reporting Initiative (GRI) sustainability disclosure database b ; measurable ; and time bound .Reports were searched for concise biodiversity commitments, which were commonly associated with a dedicated chapter or subchapter in the sustainability report or were listed as a commitment in disclosure or materiality tables of reports  . We evalGRI” OR “global reporting initiative” OR “sustainable development goal” OR “SDG” OR “convention on bio” OR “convention for bio” OR “CBD.”When biodiversity was mentioned in reports, we recorded whether this was in line with the GRI  of the Fortune 100 mentioned biodiversity or related terms, and an additional 16 companies mentioned sustainable forestry or fishing  . CompaniThe 49 companies that mentioned biodiversity all used a typical format of sustainability disclosure, which included a predominantly qualitative narrative explaining the importance of biodiversity and what actions they take regarding biodiversity. Their treatment of biodiversity ranged from a brief single mention in the context of other environmental issues  to a dedicated biodiversity chapter, with clear biodiversity commitments and disclosure of biodiversity‐related activities.Twenty‐four of the 49 companies that mentioned biodiversity made links with the biodiversity‐focussed SDGs. This is far greater than the 6 companies that acknowledged the CBD. Although not intended as a reporting framework, the SDGs resonate with the private sector and are being used to frame their sustainability commitments and activities.Only 31 of Fortune 100 companies had clearly stated commitments relating to biodiversity . CommitmOf the 23 finance sector companies, 12 were banks, and 9 of these are Equator Principles Financial Institutions (EPFIs). Eight EPFIs mentioned their adherence to the Equator Principles (which have requirements to ensure impacts on biodiversity are minimized [Equator Principles Only 5 businesses of 31) had commitments that could be classified as specific, measurable, and time bound   Fig. . These aThe lack of standardized quantitative performance indicators creates challenges for comparing performance both between companies and for individual companies through time. Although the GRI suggests performance indicators for use alongside qualitative disclosures for biodiversity, this is a voluntary framework a, and nThe most commonly disclosed qualitative information concerned habitats protected or restored and partnerships formed (disclosed by 37 companies respectively) Fig. . ExampleThe most commonly disclosed quantitative biodiversity information also concerned habitats protected or restored (9 companies) Fig. . For exaOur assessment of the 2016 Fortune 100 Global companies has revealed that big businesses are giving biodiversity limited treatment in sustainability reports. These empirical findings support accounting and accountability research suggesting that corporate biodiversity accountability is in its infancy  and outputs (activities undertaken) to outcomes (changes in biodiversity)  can, anScientists must not underestimate the private sector's focus on risk as a reason to drive action on social and environmental issues, rather than the misconception that only companies that stand to benefit directly from the environment will take action   and agriculture, have direct impacts on biodiversity and will require approaches that focus business understanding of risks and impacts at site‐level operations when developing commitments, actions, and performance measures. Other high biodiversity risk sectors, like food retailers, will require approaches that trace the biodiversity impacts of commodities through sometimes long supply chains. Finally, medium biodiversity risk sector companies, like finance and insurance firms, will require approaches that can capture indirect biodiversity impacts  to address biodiversity performance .Now is a critical time for conservation scientists to engage in order to generate a science‐driven field of biodiversity mainstreaming. Although our analysis highlights that the world's biggest businesses have a long way to go in developing and reporting on such commitments, the scene is set for rapid improvements. If these were set in place prior to the biodiversity policy super year of 2020, when the international biodiversity conservation strategy will be revisited, then businesses could truly start to play a part in the new agenda for a sustainable future for the planet, which has biodiversity at its heart.The percentage of high‐, medium‐, and low‐risk Fortune 100 Global companies with biodiversity and sustainable forestry or fishery goals by sector (Appendix S1), the 2016 Fortune 100 companies, their ranking, and their latest sustainability reports and source (Appendix S2), the 2016 Fortune 100 companies with biodiversity or biodiversity‐related commitments (Appendix S3), and examples of biodiversity‐related activities disclosed in sustainability reports by the 2016 Fortune 100 Global companies (Appendix S4) are available online. The authors are solely responsible for the content and functionality of these materials. Queries  should be directed to the corresponding author.Click here for additional data file.


Children and pregnant women use 75% of the blood supply in sub‐Saharan Africa (SSA) but face widespread blood shortages. To increase safe blood supply, Africa‐specific evidence and strengthened capacity for transfusion research are needed. Our study analysed seven years of SSA transfusion publications, compared researched topics against priorities and enumerated SSA transfusion research collaborations.Data on research topic, journal type, authors’ institutions and country were extracted from transfusion‐related SSA articles published between 2008 and 14 and used to construct a quantitative, graphic visualization of collaborations. Research topics were compared to those identified as priorities for SSA blood services in 2008 and 2015.Of the 2176, 267 articles (average 38/year) met criteria for analysis. They involved 1245 authors, 673 institutions, 59 countries (35 SSA) and 1375 collaborations. About 41% were on transfusion‐transmitted infections. About 34% were published in specialist transfusion journals. Only 7% involved exclusively collaborations within SSA. Two of the top fifteen institutions by publication quantity were from outside SSA.Despite a general paucity of SSA‐relevant transfusion research, Francophone SSA was well‐represented. Published research topics are not well matched to SSA research priorities; research on supply, distribution, financing and systems is particularly neglected. The study provides a baseline against which to track any refocusing of research activity to better meet SSA’s needs. Transfusion research hubs within and beyond SSA have been identified as a springboard network for expanding SSA transfusion research capacity. Children and pregnant women use 75% of SSA’s blood supply, predominantly for emergencies Blood transfusion services are critical for achieving the Sustainable Development Goals particularly for children and pregnant women with severe anaemia and haemorrhage Blood services are expected to provide universal access to safe and effective blood and blood products for transfusion to meet the needs of their national population Building research capacity of blood transfusion services in Africa programme (T‐REC), which involved the African Society for Blood Transfusion, and the Francophone Africa Blood Transfusion Research Network Globally, the vast majority of evidence used to guide blood services’ practices and policies has been generated by wealthy countries where the organization of blood services and the clinical use of blood are very different from those in poorer countries. SSA‐specific transfusion research is scarce. In 2008, a workshop for blood service leaders, policymakers, blood users and industry was held in Mombasa, Kenya, to agree a list of priority transfusion research topics for SSA An effective way to address the chronic and critical lack of priority‐focused transfusion research capacity within SSA would be to expand existing research collaborations within (i.e. south–south) and beyond, Africa (i.e. south–north). The objectives of this study were to use bibliographic analysis of publications as a novel approach for documenting existing collaborations and networks for blood transfusion research in SSA and to explore researchers’ productivity, the type and frequency of topics that have been researched.Data were drawn from published articles on blood transfusion topics involving SSA between January 2008 and December 2014. This period covered the seven years between the two workshops. We obtained information from the publications concerning which countries, institutions and researchers in SSA were involved in blood services research and created a quantitative visualization of these research collaborations. The aim was to create a baseline against which subsequent changes in the profile and extent of transfusion research collaborations and networks could be compared. A secondary aim was to identify successful collaborations that could be expanded and modelled elsewhere. In addition, by matching the transfusion research topics covered in the global literature against the stated priorities of SSA’s transfusion community, research topics that were priorities, but which had been neglected, could be identified, and become targets for future research efforts and investments.A comprehensive search of the Medline, Web of Science and EBSCO Global Health databases using English search terms was conducted to identify published articles on blood transfusion and blood services using a set of consensus search words. This strategy was able to identify papers written in French if they had an English abstract. Articles were selected if they were peer‐reviewed, involved primary research on blood services and/or blood transfusion (excluding plasma exchange and haemodilution), had been conducted in SSA or used transfusion‐related biological or clinical data from human participants in SSA and were published between 1 January 2008 and 31 December 2014. The World Health Organization definition for SSA was used so the literature search covered all African countries south of the Sahara.Terms relating to blood transfusion and blood banking (with wildcards when necessary) and individual names for countries in SSA were used to search the electronic databases  The initial literature search yielded 2176 articles, and after removal of non‐relevant articles, duplicates and non‐primary research, 267 articles were included in the final analysis Fig. . Three nOn average, 38 articles had been published on transfusion in SSA each year over the 7 years. There had been little change in the annual number of transfusion publications or research topics since 2008 Table  apart fr2https://www.worldatlas.com/articles/the-regions-of-africa.html produced the most publications , followed by east Africa  , southern Africa   and central Africa  . Thirty per cent (79/267) of all articles were from Nigeria, and five countries  accounted for 58% (156/267) of the single country articles. The median h5‐index of authors from five countries was 50 or above. These were as follows: Namibia , Zambia , South Africa , Mozambique  and Cote d’Ivoire . Of the top five countries ranked by number of articles, South African researchers had the highest median h5‐index (50).The authors’ institutions were located in 35 countries in SSA Table ; eleven Articles were published in both specialist transfusion journals 91/267, 34%) and non‐transfusion journals  . The h5‐index of the authors in these journals ranged from 2 to 148. A total of 239 authors in these publications had h5‐indices between 0 and 50, and 26 had H5‐indices between 51 and 150. The h5‐index was not found for 2/267 articles. The top three journals by authors’ h5‐index were all non‐transfusion journals – PLOS One  and Clinical Infectious Diseases .Forty per cent (106/267) of articles were published jointly from institutions in Africa and in the global north (i.e. south–north collaboration) and 7% (18/267) were collaborations between institutions exclusively in different African countries (i.e. south–south collaboration). When ranked by the number of publications, two of the top fifteen institutions were from outside SSA Table . Komfo AThe data from the 267 articles used to construct the graph of the network collaborations Fig.  and S4.This study identified 267 articles on blood transfusion and blood services in SSA published during the 7‐year period. Almost half of the articles focused on just one of the eight themes , and of these, over 80% focused on transfusion‐transmitted infections. Very little had been published on other research topics identified by SSA transfusion stakeholders as priorities, such as blood sparing, supply and distribution of blood, and systems and financing Articles on transfusion in SSA were distributed between specialist and non‐specialist transfusion journals. By country, South African authors had both the highest number of articles and h5‐index ≥50. The h5‐index varies between different research disciplines and grows as citations accumulate so it depends on the ‘academic age’ of a researcher. The higher values of h5‐index found in our study  which provides training in research skills with much material delivered remotely through webinars to facilitate access, combined with small group interactions and mentorship There are no conflicts identified.European Commission Grant FP7/98388. The funders did not have any role in the writing of the manuscript or the decision to submit it for publication.Supplementary File S1 Terms used for literature search.Supplementary File S2 Database of articles used in literature review. Supplementary File S3 The top 25 most connected institutions–the highest value indicates the most ‘connectedness’ . Supplementary File S4 List of the top 26 institutions with the broadest range of different collaborations in transfusion articles published 2008–14, ranked by the number of collaborations. Supplementary File S5 Coding used to classify institutions according to their African regional location.Click here for additional data file.


The b‐type cytochromes are commonly cytoplasmic, or are within the cytoplasmic membrane, while c‐type cytochromes are always found outside of the cytoplasm. The mechanism of heme attachment allows for complex c‐type multiheme complexes, having the capacity to hold multiple electrons, to be assembled. These are increasingly being identified as secreted into the extracellular environment. For organisms that respire using extracellular substrates, these large multiheme cytochromes allow for electron transfer networks from the cytoplasmic membrane to the cell exterior for the reduction of extracellular electron acceptors. In this review the structures and functions of these networks and the mechanisms by which electrons are transferred to extracellular substrates is described.Heme containing proteins are involved in a broad range of cellular functions, from oxygen sensing and transport to catalyzing oxidoreductive reactions. The two major types of cytochrome ( Phototropic organisms utilize light energy to move an electron to a low potential redox center from a high potential redox center. The subsequent movement of the electron through a chain of increasingly positive potential redox centers then occurs spontaneously. The energy released at each stage is used to generate a proton motive force, which then drives the formation of adenosine triphosphate (ATP). Chemotrophic organisms also couple the energy released by the oxidation of reduced substrates to generate a proton‐motive force. The electrons released again make their way through a succession of redox proteins that ultimately conserve energy through ATP, either through substrate level phosphorylation or through the generation of a proton motive force that ultimately powers ATP synthase. These catabolic processes all require the transfer of electrons from one redox center to another, either through inter‐ or intra‐protein electron transfer, or by both processes.Typically, metals are required for biological redox reactions. A range of redox‐active metals can function as the catalytic sites on redox enzymes, most commonly iron, molybdenum, copper and tungsten, but also including cobalt and manganese. As iron is the most abundant transition metal in the earth's crust it is unsurprising that proteins containing multiple redox centers are abundant in iron. The redox centers that allow electrons to move between these centers, or store electrons for reactions requiring multiple electrons are typically composed of iron, either in the form of iron–sulfur clusters or heme groups. Iron–sulfur clusters are a more ancient form of redox center and are typically oxygen sensitive, so organisms must either retreat to anaerobic environments, or bury the cofactors deep inside proteins that are expressed under aerobic conditions, such as the mitochondrial NADH dehydrogenases.a‐, d1‐, o‐, and siroheme but the most common forms are bound as part of b‐ and c‐type cytochromes.Heme cofactors are a more recent evolution of redox center that are more oxygen tolerant than iron–sulfur clusters. There are several different types of heme cofactor, including heme b‐ and c‐type cytochromes lies in the way in which the protoporphrin IX molecule is associated with the polypeptide chain. In the former it is a noncovalent interaction, while in the latter it is a covalent interaction. The most common form of heme found in biological proteins is the b‐type heme, which is typically found in globin‐like and cytoplasmic heme proteins. In a b‐type cytochrome the heme inserts into a hydrophobic crevice of the protein structure with the propionates facing the protein surface and the axial ligands helping to position the heme within the hydrophobic cavity. b‐type cytochromes include electron transporters such as cytochrome b5, catalytic enzymes such as such as cytochrome P450 or globins that function as oxygen carriers or sensors.The key difference between c‐type cytochromes commonly function as electron transfer proteins or as oxidoreductive enzymes that catalyze the reduction of reactive substrates such as nitrite, sulfite, peroxide, or hydroxylamine. After translation of a c‐type cytochrome polypeptide, the unfolded apocytochrome is transported across the cytoplasmic membrane by the Sec system, after which CXXCH motifs within the amino acid sequence are recognized by one of four different cytochrome maturation systems that covalently attach a heme group to the two cysteine residues via thio‐ether linkages between the cysteine sulfur and the vinyl groups of the heme.Wollinella succinogenes includes a CX15CH heme binding site.Kuenenia stuttgartiensis.ccm maturation system to covalently attach the heme to the active site of cytochrome c nitrite reductase NrfA. This allows the coordination of a lysine to the distal side of the active site heme.15CH motif of Wolinella succinogenes MccA also required a dedicated heme attachment apparatus.Agromonas oligotrophica S58 that contains 86 CXXCH motifs. Other examples include a protein from Desulfuromonas soudanensis and Thermincola ferriacetica predicted to contain 69 hemes and 58 hemes respectively.Genomic and metagenomic sequencing has revealed many genes that would encode for cytochromes containing multiple covalently attached hemes. The largest identified so far is a hypothetical protein from b‐type and c‐type cytochromes are similar,b‐ and c‐type hemes is unlikely to be catalytic, but could be to do with the increased stability and higher capacity for “heme loading” in c‐type cytochromes. To better understand the differences between b‐ and c‐type cytochromes we performed a bioinformatic analysis of proteins containing either noncovalently or covalently attached hemes. While it is possible to identify possible c‐type cytochromes based on the CXXCH motif and signal peptides for membrane translocation, there is no clear motif that unambiguously assigns a protein as a b‐type cytochrome. To compare the different classes of b‐ and c‐ type heme containing cytochrome we therefore searched the RCSB Protein Data Bank for structures of proteins known to contain either b‐type (residue code HEM) or c‐type (residue code HEC) heme ligands. Previous studies have reported such ratios in terms of amino acid number per heme,The range of redox potentials achievable by both b‐type or c‐type by checking hemes for covalent cysteine ligands. b‐type cytochromes comprise approximately 70% of the total number of structures, while c‐type cytochromes comprise the remaining 30% of heme containing proteins. Of these heme‐containing structures approximately 68% are predicted to contain a single heme, 15% to contain two hemes and the remaining 16% contain 3–16 hemes per monomer. We split the b‐type hemes into three classes, general b‐type, globin, and cysteine ligated  and then determined the relative molecular weight per heme group of each protein structure in the protein data bank , with a molecular weight (Mw) of 8.6 kDa . These peroxygenases are secreted by fungi to degrade lignocellulose using peroxide secreted from fungi. The active site contains several characteristics of conventional P450s, but do not require interaction sites for electron transfer partners, which may explain their smaller size.The smallest 1.2b‐type, with the remaining 60% belonging to the c‐type cytochrome family. There are no proteins with two hemes having cysteine ligation, and only one globin, which is a sensor from Bordetella pertussis that is proposed to allow allosteric responses to oxygen binding.c‐type cytochrome, called DHC2 from Geobacter sulfurreducens, which has a monomer weight/heme ratio of 4.6 kDa/hemeb‐type cytochrome is a transmembrane superoxide oxidase from Esherichia coli with a much higher ratio of 11.1 kDa/hemeb‐type cytochromes. These are transmembrane cytochromes that contain two bis‐histidine coordinated hemes packed within the hydrophobic transmembrane helical bundles. These are located near opposing sides of the membrane interface and are often part of electrogenic ET systems.There are substantially fewer proteins that contain two hemes, and of these only 40% are b‐type cytochrome larger than the small monoheme b‐type cytochromes that are responsible for single electron transfers to electron donors. The remaining di‐heme b‐type proteins are heme binding proteins involved in cytochrome c maturation, where the hemes lie stacked parallel inside the protein.The transmembrane location consequently makes the di‐heme 1.3c‐type and 1 b‐type cytochromes. The smallest tri‐heme protein is cytochrome c7 from Desulfuromonas acetoxidans with a ratio of 2.4 kDa/heme. This peptide/heme ratio appears to be at the minimum limit as structures with increasing numbers of hemes are either at or above this ratio. Consequently, the trimeric cytochrome c7 heme configuration represents a structurally minimized form of heme packing. Cytochrome c7 variants are found in other bacteria, including the Ppc group of cytochromes from G. sulfurreducens, which contains five homologous c7 proteins PpcA, PpcB, PpcC, PpcD, and PpcE, that are reported to be involved in electron transfer between the cytoplasmic and outer membrane.There are few representative structures of cytochromes containing three hemes in the database, namely the 4 b‐type cytochrome structure with three hemes currently known is the flavohemoglobin from Alcaligenes eutrophus, a globular cytoplasmic protein containing three b‐ hemes and an FAD molecule.The only 1.4c‐type cytochromes. This indicates that proteins containing multiple hemes are incorporated as covalently attached hemes, and that cytochromes with four or more hemes are all located outside of the bacterial cytoplasm. This appears logical as hemes can be packed within a polypeptide chain more efficiently if covalently bound when compared to noncovalently bound hemes. This allows for effective use of polypeptide to build a cytochrome containing multiple hemes with active sites and interfaces for electron exchange. This compartmentalization also means that, for bacteria that use networks of electron transfer proteins, electrons are kept out of the cytoplasm in order to minimize production of radical species and prevent damage to DNA and regulatory proteins.The only structurally characterized proteins with more than three hemes are prokaryotic c‐type cytochromes can be divided into two different groups depending on their relative protein weight /heme ratio ,There are currently no structures available for cytochromes that contain 7, 13, 14, 15, or more than 16 hemes per monomer, despite examples of amino acid sequences containing up to 85 CXXCH motifs being predicted in the genome of cytochrome replete iron‐oxidizing bacteria.c‐type heme HAO complex made of three octaheme cytochromes,A. oligotrophica S58 in an operon with a porin. This cytochrome has a predicted peptide Mw of 255 kDa, and would have a peptide weight/heme ratio of 2.86, grouping it in the peptide minimized cytochrome family. Based on the linear correlation between heme chain length and heme number from Figure The biologically relevant conformations of peptide minimized cytochromes are monomeric and proposed to be involved in electron transfer, electron scavenging, or electron storage. In contrast, many of the peptide replete cytochromes are catalytic and consist of dimeric or higher‐order complexes, such as the 24 2c‐type cytochromes and typically only express these under very specific conditions, some bacteria are capable of expressing a large number of different cytochromes with high numbers of hemes. In particular, bacteria of the genera of Shewanellacea and Geobacteracea have been the subject of study for the past three decades due to their ability to reduce a broad range of extracellular substrates, including insoluble Fe(III) and Mn(IV) minerals. They are linked to the broad range of c‐type cytochromes that are expressed within the periplasmic compartment and on the surface of the cell. The interactions between the different cytochromes have been studied for over a decade and substantial progress has been made in elucidating the electron transfer networks involved in coupling cytoplasmic respiration to the reduction of extracellular substrates. These systems have evolved different mechanisms to overcome the challenges of extracellular electron transfer: namely 1) electron transfer from the quinol pool in the cytoplasmic membrane to electron carriers in the periplasm 2) electron transfer across the outer membrane or cell wall and then 3) electron transfer from cytochromes on the cell surface into extracellular substrates. For the remainder of this review we shall look at how progress has been made into understanding how different organisms undertake extracellular electron transfer.While eukaryotes and many bacteria contain genes for only a few 3Shewanella is able to transfer electrons from the quinol pool in the inner membrane to solid‐phase electron acceptors located outside of the cell. In order to achieve this, a tetraheme quinol dehydrogenase, CymA, oxidizes reduced quinols and transfers these electrons to periplasmic cytochromes, such as the small tetraheme cytochrome (STC) and fumarate reductase (FccA). These soluble cytochromes can act as electron shuttles, moving electrons across the periplasm to the MtrCAB complex embedded in the outer membrane .G. sulfurreducens and Anaeromyxobacter dehalogenans.Sideroxydans lithotrophicus and Rhodopseudomonas palustris.Porin‐cytochrome complexes with similar modular configurations to MtrCAB have been identified in several bacterial species including Shewanellacea genus express a range of additional cell surface localized cytochromes that are capable of supporting extracellular electron transfer.Shewanella are localized to the cell surface though secretion by the type II secretion system and anchor to the cell surface through an acylated N‐terminal cysteine.In addition to MtrC, the mineral respiring species within the Shewanella to possess multiple porin‐cytochrome complexes is unclear; however, expression of MtrDEF has been shown to be upregulated in Shewanella biofilms.Shewanella genomes.MtrF forms part of an operon encoding MtrA and MtrB homologues (MtrD and MtrE respectively). Subsequently, MtrF is proposed to form a MtrDEF porin‐cytochrome complex. The requirement for Shewanella have been resolved.c‐type hemes within the structure. Each cytochrome is comprised of four domains that consist of an N‐terminal split β‐barrel (domain I), a penta‐heme domain (domain II), a second split β‐barrel (domain III) and then either a penta‐ or hexaheme (UndA) C‐terminal domain (domain IV). The four domains are arranged in such a way that the two multiheme domains are in the middle of the structure with the two β‐barrel domains flanking each side and the hemes within the two multiheme domains form a continuous network with the iron atoms of adjacent hemes all being within 10 Å of each other.The structures of the outer membrane cytochromes of Despite low sequence homology, the overall conformation of the 10 core hemes is well conserved across the four different OMC clades, with only the terminal hemes at opposite ends of the staggered cross showing variability in their positions. The role of the β‐barrel domains is unclear, but it may be to do with conferring rigidity on the structure, preventing the tightly packed multiheme chain from collapsing on the surface of the cell.Shewanella secretes small redox active molecules, flavins, into the extracellular environment where they are proposed to either serve as redox shuttles between cytochromes on the cell surface and electron acceptors located too distant for direct electron transfer, or where they potentially serve as redox cofactors of the outer membrane cytochromes.4Geobacter to transfer electrons into solid surfaces is still poorly understood, in part because there are a broad range of cytochromes expressed that have overlapping functions. Most recently these have been proposed to allow Geobacter to respond more efficiently to a range of different redox potentials.Despite intensive research, the mechanism that is used by Shewanellacea typically use a single quinol dehydrogenase, CymA, during anaerobic respiration there are at least two quinol dehydrogenases in Geobacter involved in anaerobic respiration. Both are constitutively expressed and one, ImcH, is required for respiration of extracellular substrates with a potential > −100 mV versus Standard Hydrogen electrode (SHE). The other, CbcL, is involved in respiration of substrates at lower potentials.While the Geobacter there are no obvious flavocytochomes like the fumarate reductase FccA of S. oneidensis, or tetraheme electron shuttles like STC. Instead there are five tri‐heme c7 type cytochromes that have high homology to one another, these are PpcA, PpcB, PpcC, PpcD, and PpcE. The members of the Ppc cytochrome family have highly conserved structures and cover a similar potential range, but have subtle differences in the redox potentials of their individual heme groups.The two quinol dehydrogenases then transfer electrons into the periplasmic cytochrome pool. In Shewanella a porin‐cytochrome complex has been identified that is predicted to transfer electrons across the outer membrane, but there are at least five other gene clusters encoding distinct porin‐cytochrome complexes in Geobacter sulfurreducens PCA.S. oneidensis porin cytochrome complex, these are comprised of an 8‐heme periplasmic cytochrome, a transmembrane porin and a 12‐heme cytochrome on the cell surface. These cytochromes share no homology with any structurally resolved cytochromes, suggesting that the arrangement of hemes passing through the outer membrane will be different.Like Shewanella, the Geobacter species do not appear to use soluble electron shuttles but rely on direct contact between cells or long distance contact generated by nanowires to deliver electrons.Geobacter to extracellular acceptors that include Fe(III) minerals and carbon electrodes. The first outer membrane cytochrome to be structurally characterized from Geobacter was OmcF, an analogue of cytochrome c6 from photosynthetic algae.6. Normally cytochrome c6 is involved in photosynthesis, where it functions as an electron shuttle between cytochrome b6f and photosystem 1 in cyanobacteria. However, in Geobacter the extracellular OmcF is tethered to the outer membrane by a lipid anchor and has redox midpoint potential of +100 mV versus SHE.6, but higher than the midpoint potential of most other characterized Geobacter cytochromes, making it unlikely to be an electron shuttle involved in extracellular electron transfer. The most compelling evidence for a role for OmcF is that its expression is linked to the expression of two other outer membrane cytochromes, making it either a regulator of extracellular expression, or is involved in cytochrome transport across the outer membrane.In contrast to G. sulfurreducens.bis‐His coordinated, preventing ligand association and allowing for rapid electron transfer between adjacent hemes by limiting conformational rearrangement on reduction. The histidine for heme 5 is provided from the peptide chain of an adjacent OmcS monomer, making heme ligand assembly part of the polymerization process  and perpendicular (T‐junction) heme pairs. This arrangement of hemes is often seen in multiheme cytochromes where electron transfer is necessary. The distances between the porphyrin rings, approximately 3.5 Å for parallel hemes and 6 Å for T‐junction heme pairs allow for rapid transfer of electrons between adjacent hemes. It is unclear how, if at all, the two ends of the OmcS pili are capped to allow for electron exchange between the periplasm and extracellular substrate, but the OmcS nanowire appears optimized for electron exchange between adjacent hexaheme monomers without a clear electron ingress/egress site for cytochromes within the polymeric chain.Geobacter surface that connects the polymeric wire to the extracellular interface.The relative molecular weight:heme ratio is 7.5 kDa/heme, which is higher than that of simple electron transporters such as STC and NrfB, and similar to catalytic enzymes such as HAO (7.5) As the OmcS hemes are buried within the structure this suggests that the OmcS polymers are not optimized for nonspecific electron transfer through the sides of the OmcS polymer but are insulated, perhaps to allow for effective conduction away from the cell surface, rather than reduction of nearby extracellular substrates. The unusual coordination of heme 5 means that the terminal monomer of the OmcS heme chain will have a pentacoordinate heme, giving directionality to the OmcS polymer Figure . This te5Shewanella and Geobacter, Gram‐positive bacteria are characterized by possessing a cell wall composed of peptidoglycan, sugars, phospholipids, peptides, amino acids, and glycoprotein.Thermincola potens strain JR in a current‐producing MFC operating at high temperature altered this view.T. ferriacetica, that shares 99% similarity with T. potens, was also shown to be capable of transferring electrons from acetate to the anode of an MFC to generate electric current.T. potens has several genes coding for multiheme cytochromes,In contrast to Gram‐negative organisms, such as T. potens and Tfer_0070 in T. ferriacetica) that is anchored to the inner membrane and is proposed to receive electrons from the menaquinone pool; a periplasmic decaheme cytochrome (TherJR_0333 in T. potens and Tfer_1887 in T. ferriacetica) proposed to transfer electrons within the periplasmic space of Thermincola; a hexaheme cytochrome, proposed to be embedded in the peptidoglycan (TherJR_1122 in T. potens and Tfer_0075 in T. ferriacetica), and a nonaheme cytochrome proposed to be the terminal reductase localized at the cell surface of the bacteria (TherJR_2595 in T. potens and Tfer_3197 in T. ferriacetica) , one heme with histidine‐methionine coordination (heme 9), and two hemes with a histidine as the proximal ligand and an open coordination side as the distal position (hemes 2 and 5). It was shown that these two high‐spin hemes, located at opposite ends of the nine heme arrangement may work as the putative active sites for substrate binding, which is a novelty within the family of multiheme cytochromes.Shewanella, it clearly follows a different design. The heme arrangement is similar to that of NrfA family of proteins. While heme 1 to 4 align with the hemes of the tetraheme cytochrome c554 from Nitrosomonas europaea with heme 2 as the active site, hemes 5 to 9 can be superimposed to the NrfA heme core structure with heme 5 of OcwA as the active site. Furthermore, hemes 1 to 4 and 6 to 9 align to the heme core structure of the sulfite reductase MccA, with heme 2 acting as the active site. OcwA was shown to reduce nitrite and hydroxylamine, as well as iron oxides.Thermincola spp., allowing these Gram‐positive bacteria to grow and survive in environments with various terminal electron acceptors.Although the organization of the hemes in OcwA vaguely resemble the “staggered cross” of the four major clades of outer membrane cytochromes from 6Geobacter extracellular electron transport system, and a range of different extracellular cytochromes in the Shewanella extracellular electron transport systems. We prefer to think in terms of “overlapping function”, rather than “redundancy,” as the energetic costs of maintaining these systems in a genome must provide selective advantage to an organism. The challenge is for researchers to strive to understand the overlapping roles of these proteins. While these are not yet clear they may, for example, allow the bacteria to access a greater range of substrates by shifting the redox potentials of the electron transfer network in response to the potential at both the cytoplasmic membrane and extracellular surface. Piecing together how electrons are distributed across intermediate distances within the periplasm or intermediate space will help to shed further light on these complex, multicomponent systems.Over the last two decades, the substantial increase in structural and biochemical data on multiheme cytochromes has allowed a much greater understanding of their role in facilitating electron transfer within the cell. Substantial in vitro and in vivo research on the model organisms detailed earlier reveal that multiheme cytochromes with low monomer weight: Heme ratios do not form stable electron transfer networks but instead rely on transient interactions for electron transfer, thereby generating dynamic networks of electron transfer proteins that move electrons from the quinol pool to the cell surface. There is a large amount of apparent redundancy in these systems, with multiple quinol dehydrogenases and highly homologous periplasmic cytochromes in the This research was supported by the Biotechnology and Biological Sciences Research Council grants BB/P01819X/1, BB/K009885/1, BB/L023733/1 and by Fundação para a Ciência e a Tecnologia (FCT) Portugal , by Project LISBOA‐01‐0145‐FEDER‐007660  funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by the European Union's Horizon 2020 research and innovation program under grant agreement No 810856.


Addressing malnutrition requires strategies that are comprehensive and multi‐sectoral. Within a multi‐sectoral approach, the health system is essential to deliver 10 nutrition‐specific interventions, which, if scaled up, could substantially reduce under‐5 deaths in high‐burden countries through improving maternal and child undernutrition. This study identifies the health system components required for the effective delivery of these interventions, highlighting opportunities and challenges for nutrition programmes and policies. We reviewed implementation guidance for each nutrition‐specific intervention, mapping the delivery process for each intervention and determining the health system components required for their delivery. We integrated the components into a single health systems framework for nutrition, illustrating the pathways by which health system components influence household‐level determinants of nutrition and individual‐level health outcomes. Nutrition‐specific interventions are typically delivered in one of four ways: (i) when nutrition interventions are intentionally sought out, (ii) when care is sought for other, unrelated interventions, (iii) at a health facility after active community case finding and referral, and (iv) in the community after active community case finding. A health system enables these processes by providing health services and facilitating care seeking for services, which together require a skilled and motivated health workforce, an effective supply chain, demand for services and access to services. The nutrition community should consider the processes by which nutrition‐specific interventions are delivered and the health system components required for their success. Programmes should encourage the delivery of nutrition interventions at every client–provider interaction and should actively generate demand for services—in general, and for nutrition services specifically. The health system plays an essential role as the primary delivery platform for nutrition‐specific interventions that reduce maternal and child malnutrition. Efforts to improve nutrition should include investments in and strengthening of the health system.Understanding the processes within the health system that contribute to the delivery of these nutrition‐specific interventions is necessary to improve coverage of these interventions by identifying bottlenecks to the delivery and uptake of services.The nutrition community should consider the processes and health system components required for successful nutrition programmes, namely, a skilled and motivated health workforce, an effective supply chain, demand for services and access to services.1Malnutrition is a complex issue requiring coordinated efforts across sectors to address its underlying causes. The multi‐sectoral nature of nutrition is increasingly being recognized, with most global and national initiatives now including representation from diverse actors and ministerial departments, including health, education, food and agriculture and social protection . We chose to focus the scope of this work on these 10 nutrition‐sensitive interventions for which there is strong evidence of effect. However, many nutrition sensitive interventions delivered through the health system require the same elements.In this paper, we identify and describe the health system components required for the delivery of nutrition‐specific interventions and explore how the framework could be used to identify opportunities for increasing coverage of nutrition interventions. Understanding the health system as a complex adaptive system that includes path dependence processes, non‐reversible processes that lead to other outcomes, is integral to the increased and sustained coverage of these nutrition interventions  and the activities required to promote care seeking or active case finding so that a community member receives the intervention when needed. This led to the development of four service component diagrams depicting the health system components required for the delivery process. Finally, we integrated the service components into a single health systems framework for nutrition. We used evidence from both the health systems literature and nutrition literature to identify the ‘upstream’ building blocks required for each ‘downstream’ service component. Using an iterative process, we expanded the health systems framework to include all components required to deliver the 10 nutrition‐specific interventions.2.1This study did not involve any human subjects research.33.1Table Figure Each of the nutrition‐specific interventions is delivered through at least one of the delivery processes Figure –d; howev3.2We overlayed information from the implementation guidance documents onto the four delivery processes to create four complementary diagrams of service components required to achieve intervention coverage Figure –d.providing health services and, second, by promoting and facilitating care seeking for health services . These pathways, in turn, depend on four common health systems components:A skilled and motivated health workforceThese service component diagrams highlight two pathways by which health systems contribute to intervention delivery: first, by providing health services and for promoting and facilitating care seeking. The delivery of all nutrition‐specific interventions requires a direct interaction between a health worker and a patient (if we consider folic acid supplementation in lieu of fortification). Facility‐based interactions are necessary both for direct service provision and for promoting care seeking for future services. Community health workers (CHWs) are needed for active case finding at village level, without which, demand and care seeking for certain interventions , would likely be lower than current levels. Further, quality of the intervention delivered, with particular emphasis on those requiring counselling , depends on the skills and motivation of the health workforce.An effective supply chainHealth workers are needed for both providing health services is a well‐functioning supply chain, to make available the necessary drugs or supplements needed for intervention delivery. Vitamin A, MMS, balanced energy, maternal calcium supplementation and management of SAM and MAM each require either a nutrition supplement or a ready‐to‐use therapeutic food (RUTF). The availability of these products is contingent on the supply chain moving the products from a central purchasing unit or storage facility to individual health facilities, local retailers and CHWs. Stockouts are a barrier to care and can diminish subsequent care seeking by community members.Demand for servicesA second component needed for promoting care seeking, through health education, messaging campaigns and direct counselling of patients who come to facilities for unrelated services. Community health workers, when appropriately trained, equipped and supported, can bolster demand generation at the village or community level.Access to servicesSix of the eight nutrition‐specific interventions in Table willingness to seek care but also of their ability to seek care. Various constraints can hinder care‐seeking ability, including poor geographical access, financial barriers and restrictive socio‐cultural forces. The health system plays an essential role in promoting and facilitating care seeking by ensuring that there are adequate numbers of well‐positioned health facilities, by adopting close‐to‐community strategies whenever possible, and through financing strategies that enable all community members to access care when needed.Care seeking is a product not only of an individual's Figure 4A strong health system is essential for the delivery of nutrition‐specific interventions WHO, . Many nuFuture analyses could use implementation research or statistical modelling to identify the health system components that are responsible for the greatest bottlenecks and thus the greatest opportunities for impact. While we should aim to generate more evidence on the implementation of nutrition programmes, we should also draw on the broader health systems literature and literature from other health domains. Lessons could be learned from non‐nutrition interventions that operate through similar delivery mechanisms.The four identified health systems components reflect immediate outputs of the health system; however, it is important to note that health systems literature highlights many other elements beyond the four highlighted in this paper, necessary for successful provision of care, for example, health information systems, financing or leadership/governance WHO, . More brIn addition to strengthening health systems, we should also consider adjusting delivery processes to make better use of existing health systems as they are now , to provide counselling on the need to adhere to the nutrition supplementation they have already been given , or to provide continued support and address current challenges with breastfeeding or complementary feeding. As long as the health worker is skilled and has the material required for the intervention, each interaction with the health system is an opportunity. The relevant strategies here thus include (a) improving demand for, and access to, health services in general and (b) enhanced pre‐service and in‐service training of health workers to ensure they use every patient interaction to assess, treat and counsel on nutrition‐related issues.First, programme implementers could work with other health‐sector actors in low‐ and middle‐income countries (LMICs) to increase the number and frequency of interactions—Second, for interventions that are currently delivered when people seek care for unrelated services Figure , programLastly, while the health system plays an important role in the delivery of nutrition‐specific interventions, it is only one of the sectors that should be considered within a multi‐sectoral strategy to address malnutrition Appendix . Educati5and promote and facilitate care seeking for those services. Programmes should encourage the delivery of nutrition interventions at every client–provider interaction and should actively generate demand for services—in general, and for nutrition services specifically. To reach an entire population, an active community workforce as part of the health system is likely needed for all nutrition interventions.Nutrition is a multi‐sectoral problem, but for nutrition‐specific interventions, the health sector is critical. The nutrition community should consider the processes by which nutrition‐specific interventions are delivered and the health system components required for their success, namely, a skilled and motivated health workforce, an effective supply chain, demand for services and access to services. A strong health system will both provide health services The authors declare that they have no conflict of interest.SEK, TR and TSL performed the research. RB contributed essential insights and experiences to the work. SEK and TR drafted the first version of the manuscript. All contributed to multiple revisions of the manuscript and read and approved the final version submitted.


In the dark path of tumorigenesis, the more carefully the cancer biology is studied, the more brilliant answers could be given to the countless questions about its orchestrating derivers. The identification of the correlation between Toll‐like receptors (TLRs) and different processes involved in carcinogenesis was one of the single points of blinding light highlighting the interconnection between the immune system and cancer. TLRs are a wide family of single‐pass membrane‐spanning receptors that have developed through the evolution to recognize the structurally conserved molecules derived from microorganisms or damaged cells. But this is not everything about these receptors as they could orchestrate several downstream signalling pathways leading to the formation or suppression of cancer cells. The present review is tempted to provide a concise schematic about the biology and the characters of TLRs and also summarize the major findings of the regulatory role of TLRs and their associated signalling in the pathogenesis of human cancers. Upon exposure to bacteria, viruses, protozoa and fungi, innate immune cells including neutrophils, monocytes, macrophages, dendritic cells (DCs), natural killer (NK) cells and the complement system are activated. The response of the innate immunity to microbial pathogens relies on the specific host‐receptor detection of pathogen‐ and danger‐derived molecular signatures, known as PAMPs and DAMPs, respectively. When PAMPs and DAMPs are recognized by germline‐encoded pattern‐recognition receptors (PRRs), different types of cytokines would be released, which in turn attract secondary defensive immune cells. In the long list of PRRs, Toll‐like receptors (TLRs) are the most important ones. TLRs are one of the largest and most well‐studied families of PRRs, which were first recognized in the fruit fly, Drosophila melanogaster.2From the immunological point of view, the characteristics of these molecules have been well‐studied in different reports to unveil the structure, signalling and functions of TLRs in cells. The results of high‐resolution X‐ray crystallography analysis have revealed that TLRs deviate considerably from the canonical LRR structure and thereby they could interact with a wide range of ligands in a highly divergent fashion. This different structure gives TLRs the ability to be activated in a different manner from other LRRs, resulting in the involvement of these receptors in other biological processes rather than only regulating innate immunity.2.1All the TLRs are synthesized from their mRNAs into functional configurations in the endoplasmic reticulum (ER) and are translocated to the Golgi complex followed by trafficking to either the plasma membrane or endosomes. The localization of TLRs is a complicated process, which is controlled by a group of ER‐associated proteins. Thus far, ten functional TLRs have been identified in humans according to their subcellular localization. Although TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are expressed on the cell surface and migrate to phagosomes after activation, the expression site of TLR3, TLR7, TLR8 and TLR9 is in intracellular compartments, in particular the endosomes and the endoplasmic reticulum. Among all, the subcellular localization of TLR4 is unique as this TLR could be found either at the plasma membrane or at endosomal vesicles.A member of the ER‐resident HSP90 protein family, gp96, serves as a general chaperone for the surface‐expressed TLR1, TLR2, TLR4 and TLR5, and intracellular TLR7 and TLR9. Deficiency in gp96 leads to the loss of expression of TLR1‐TLR5 and TLR7 or improper TLR9 protein folding.2.2TLRs belong to type I transmembrane glycoproteins and contain three major domains, ectodomain, single‐spanning transmembrane domain and cytoplasmic TLR domain. The ectodomain is oriented towards extracellular space or luminal space  and contains multiple2.3Although each TLR recognizes distinct ligands, the mechanisms of TLR activation and signal transduction are somehow the same. Several microbial ligands have been enumerated for TLRs, which their lists are summarized in Table 2.4Given the importance and the key roles of TLRs in regulation of immune responses, it is not surprising that their emitted signals are a succession of complex events in the cells that lead to activation or suppression of a wide range of downstream signalling axes. Generally speaking, the TLR signalling pathway could be mediated through either MYD88‐dependent or MYD88‐independent manner. The TLR signalling has been well reviewed in an article provided by Luo et al2.52.5.1When it comes to TLRs, the first thing that comes to mind is its bona fide action in response to specific molecules derived from bacteria and viruses.TLRs are indeed the main players of the innate immunity system; however, their central role in regulation of host protective adaptive immune responses should not be underestimated. It has been suggested that TLRs could stimulate both T cell‐ and B cell–mediated immune responses upon exposure to adjuvants containing microbial lysates or products.2.5.2Apart from participation in the regulation of immune responses, which is well‐described in the previous reports, TLRs have some non‐canonical functions in tissue repair, regulation of apoptotic cell death and autophagy. Any aberrancy in the regulation of these receptors could orchestrate the initial step of tumour development, endangering normal human cells to form cancer cells Figure .Several mechanisms have been enumerated for TLRs through which they could induce tissue repair and regeneration upon an injury. Generally, participation of TLRs in wound healing occurs at both early and delayed stages. Although at the early phase of wound healing TLRs recruit inflammatory cells to the injured site to produce cytoprotective signals through up‐regulating the expression of anti‐apoptotic genes, these receptors could modulate fibroblast recruitment and regenerative responses at the delayed phase. TLR4 could also increase epithelial cell life span and prevent the migration of keratinocytes.Listeria monocytogenes through ERK‐mediated activation of autophagy. The more carefully the association of TLRs with the process of autophagy is studied, the more molecular mechanisms have been found to explain the precise role of this cascade in the activation of this self‐devouring event. It turns out that TLRs could recruit Beclin‐1, which in turn interacts with TRAF6, inducing K63‐polyubiquitination of the BH3 domain for its activation and subsequent formation of autophagosomes.When it became evident that activated TLR4 could enhance autophagic elimination of phagocytosed mycobacteria in macrophages via VPS34‐dependent formation of cytoplasmic LC3 aggregates, a new chapter has been opened in the biology of TLRs in the regulation of autophagy. It did not take long that the role of other TLRs in the regulation of autophagy has been established. TLR7 can eliminate bacillus Calmette‐Guerin (BCG) through up‐regulation of ATG5 and Beclin‐1.3___that are responsible for the regulation of immune responses against unknown antigens, such as cancer cells___could play such a controversial mechanism of action? The answer to this question may be in the amplitude and length of receptor activation. Although chronic low‐grade TLR activation favours a tumour‐promoting pro‐inflammatory state, high‐dose TLR activation induces antitumour response.The first evidence supporting the involvement of TLRs in the tumorigenesis event has stemmed from a report indicated that the risk of cancer development is significantly higher in organs that are directly or indirectly exposed to bacterial TLR ligands. This finding is also reflected in the low incidence of cancer development in germ‐free animals.3.1The idea behind the involvement of TLRs and their related signalling in the formation of human cancers has originated from the considerable number of studies reported the abnormal expression of TLRs on tumour cells, where they may influence tumour growth and immune responses.3.1.1Each TLR, either as a single‐player or as a team, could participate in the development of a specific type of tumour. When it comes to oral and gastrointestinal cancers, the first TLR that comes to mind is TLR2.Helicobacter pylori gastritis.So far, we have looked at the association between the individual TLRs and the induction of human cancers; however, in most cases, these receptors act as a group to enhance the survival and proliferative capacity of the tumour cells. TLR4 and TLR9 could endow prostate cancer cells the ability to proliferate more aggressively.Another critical complication that is associated with grouped TLRs expression is the regulation of drug‐resistance phenotype, which eventually lead to the poor outcome of patients and even increase the risk of cancer recurrence. Co‐expression of TLR1, TLR7 and TLR9 in myeloma cells has shown to be involved in induction of drug resistance.3.1.2The aberrant overexpression of TLRs on malignant cells and their well‐established association with the promotion of human cancers has put an end to the multiple questions wondering the role of TLRs in the regulation of tumorigenesis, especially at the early stage of the event. This causes the new stream of attempts to be accomplished for proposing the probable mechanisms through which TLRs could promote carcinogenesis Figure .In 2000, Hanahan and Weinberg proposed a model to define ‘six hallmarks’ that a tumour requires for maintaining its survival.As mentioned earlier, the TLR network has a tight cross‐talk with the NF‐κB axis through either MYD88‐dependent or MYD8‐independent manner.In a recent study, we also examined the combination of TAK‐242 and four well‐known chemotherapeutic agents: paclitaxel, cisplatin, doxorubicin and arsenic trioxide in breast and ovarian cancer cells. Interestingly, we realized that the inhibition of TLR4 boosted the cell cytotoxicity of all drugs, which indicates the fact TLR4 would confer chemo‐resistance to a broad range of anti‐cancer agents.Having established the role of TLR‐associated signalling pathways in multiple steps of tumorigenesis, intense interest has been attracted to investigate whether this network could also participate in tumour cell invasion and metastasis. In similarity with other stages in tumorigenesis, the results of two studies showed that TLR activation in tumour cells increases the tendency of tumour cells to adhere to the extracellular matrix and endothelial cells, which ultimately elevates the risk of tumour metastasis. It has been shown that LPS promotes tumour invasion through the TLR4‐mediated activation of the NF‐κB pathway, resulting in the up‐regulation of matrix metalloproteinase 2 (MMP2) and the β1‐integrin subunit.H .pylori–associated gastric cancer, in which it has shown that H .pylori–induced cyclooxygenase‐2 (COX‐2) and prostaglandin E2 (PGE2) expression enhanced tumour angiogenesis via interacting with TLR2 and TLR9.Since vascular endothelial growth factor (VEGF), the main factor involved in tumour angiogenesis can be induced by activation of TLRs, and it is not surprising to bring up TLRs as potent regulators of tumour angiogenesis. The first study that introduced the role of TLRs in tumour angiogenesis was conducted on Although TLRs are the main player in the regulation of immune responses, their aberrant expressions on tumour cells could confer the resistant phenotype to malignant cells against cytotoxic effects of lymphocytes through production of some pro‐inflammatory compounds. During cancer progression in the setting of chronic inflammation, integration of TLRs on the tumour cells or tumour‐infiltrating immune cells and their ligands leads to the secretion of cytokines and chemokines from these cells into the tumour microenvironment. The released cytokines basically suppress the immune response, either by impairing the function of APCs, T cells and TAA‐specific immunity or by recruiting more immune suppressive cells to the tumour nidus, leading to the production of more inflammatory cytokines that activate cancer‐associated fibroblasts (CAFs).Malignant tumour cells selectively reprogramme their metabolism to meet the rapid energy requirements for proliferation, survival and metastasis as well as for sustaining the tumour‐suppressive microenvironment.3.2The complexities of TLRs and their associated signalling pathways have portrayed another picture for this cascade as a defender against human cancers. It has been suggested that the activation of TLRs in the cells could allay immune responses to trigger an anti‐cancer signal Figure .3.2.1th century when Deidier (1725) reported that infection in cancer patients could be concomitant with the remission of malignant diseases. In the 1890s, William B. Coley observed that repeated injections of a mixture of bacterial toxins were effective for the treatment of cancer patients. Although Coley could not explain the mechanism through which the bacteria induced anti‐tumour activity, the results of this observation introduced bacterial toxins as an effective therapeutic strategy against human cancers. In 1943, Shear and Turner discovered that LPS had anti‐cancer properties.Evidence of antitumour effects of microbial products can be dated back to the early 183.2.2The results of deep molecular investigations proposed that TLRs could exert their anti‐tumour activity either by regulation of the apoptotic pathway or through modulating the activity of the immune cells, such as DCs, NK cells and T lymphocytes.Although multiple lines of evidence emphasized the anti‐apoptotic effects of TLRs, in many cases, there are conflicting results. TLR9 is the best example of this controversy as the apoptosis‐inducing capacity of this receptor is mediated through the mitochondrial‐dependent pathway. It has been shown that CpG‐DNA–mediated activation of TLR9 not only increased the expression of Bax but also induced caspase‐dependent apoptotic cell death in murine macrophages.Through inducing the cytotoxic signalling, mobilizing NK and T cells, and producing the anti‐tumour antibodies, the immune system could conveniently take hold of cancer cells. Being the most important components of the immune system and being expressed in most, if not all, immune cells, it is not surprising that TLRs may play a part in the anti‐cancer effects of the immune system. As professional APCs, which express a large number of TLRs, DCs are at the interface of innate and adaptive immune responses. Activated DCs amplify immune system‐induced anti‐tumour responses through presenting tumour antigens to cytotoxic T lymphocytes. The activation of DCs could be mediated through several mechanisms, but one of the most important ones is the stimulation of the TLR signalling pathway. TLR5, TLR7 and TLR9 are the best examples of TLRs that could reinforce the anti‐tumour activity of DCs.Converting tumour‐supporting macrophages to tumour suppressors that produce inflammatory cytokines and promote M1 polarization is another suggested mechanism that has been attributed to the anti‐tumour activity of poly I:C‐activated TLR3. This response is mediated by TNF‐α through a MyD88‐independent pathway.4In many cases, cancer cells mimic many characteristics of immune cells. So, under the mask of these cells, neoplastic cells communicate and modulate the immune system for their own survival and growth. Despite several reports shedding light on the association between TLRs and the incidence of tumorigenesis, a considerable number of questions remained unanswered about the precise mechanisms of these receptors in cancer development. Given this, further exploration and more precise understanding concerning the role of TLRs in the maintenance of cancer cells are required to increase the current knowledge about their participation in tumour biology. These findings could also shed more light on the molecular basis of innate immunity, tumorigenesis and cancer biology, which all together could start a new chapter in the modern understating of cancer and its treatment strategies.The authors declare that they have no conflict of interest.Yazdan Mokhtari : Conceptualization ; Data curation ; Writing‐original draft ; Writing‐review & editing . Atieh Pourbagheri‐Sigaroodi : Conceptualization ; Data curation ; Visualization ; Writing‐original draft . Parisa Zafari : Conceptualization (supporting); Data curation ; Validation . Nader Bagheri : Conceptualization (supporting); Validation ; Writing‐review & editing . Seyed H Ghaffari: Conceptualization (supporting); Supervision ; Writing‐review & editing . Davood Bashash: Conceptualization (lead); Project administration (lead); Supervision (lead); Validation (lead); Writing‐review & editing (lead).