Computational Exploration of Potential CFTR Binding Sites for Type I Corrector Drugs.

Cystic fibrosis (CF) is a recessive genetic disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The recent development of a class of drugs called "correctors", which repair the structure and function of mutant CFTR, has greatly enhanced the life expectancy of CF patients. These correctors target the most common disease causing CFTR mutant F508del and are exemplified by the FDA-approved VX-809. While one binding site of VX-809 to CFTR was recently elucidated by cryo-electron microscopy, four additional binding sites have been proposed in the literature and it has been theorized that VX-809 and structurally similar correctors may engage multiple CFTR binding sites. To explore these five binding sites, ensemble docking was performed on wild-type CFTR and the F508del mutant using a large library of structurally similar corrector drugs, including VX-809 (lumacaftor), VX-661 (tezacaftor), ABBV-2222 (galicaftor), and a host of other structurally related molecules. For wild-type CFTR, we find that only one site, located in membrane spanning domain 1 (MSD1), binds favorably to our ligand library. While this MSD1 site also binds our ligand library for F508del-CFTR, the F508del mutation also opens a binding site in nucleotide binding domain 1 (NBD1), which enables strong binding of our ligand library to this site. This NBD1 site in F508del-CFTR exhibits the strongest overall binding affinity for our library of corrector drugs. This data may serve to better understand the structural changes induced by mutation of CFTR and how correctors bind to the protein. Additionally, it may aid in the design of new, more effective CFTR corrector drugs.

Carotenoids promote lateral packing and condensation of lipid membranes.

Carotenoids are pigment molecules that protect biomembranes against degradation and may be involved in the formation of functional bacterial membrane microdomains. Little is known on whether different types of carotenoids have different effects on the membrane or if there is any concentration dependence of these effects. In this work, we present results from molecular dynamics simulations of phospholipid bilayers containing different amounts of either ß-carotene or zeaxanthin. Both ß-carotene and zeaxanthin show the ability to laterally condense the membrane lipids and reduce their inter-leaflet interactions. With increasing concentrations, both carotenoids increase the bilayer thickness and rigidity. The results reveal that carotenoids have similar effects to cholesterol on regulating the behavior of fluid-phase membranes, suggesting that they could function as sterol substitutes and confirming their potential role in the formation of functional membrane domains.

CAMERRA: An analysis tool for the computation of conformational dynamics by evaluating residue-residue associations.

A computational method which extracts the dominant motions from an ensemble of biomolecular conformations via a correlation analysis of residue-residue contacts is presented. The algorithm first renders the structural information into contact matrices, then constructs the collective modes based on the correlated dynamics of a selected set of dynamic contacts. Associated programs can bridge the results for further visualization using graphics software. The aim of this method is to provide an analysis of conformations of biopolymers from the contact viewpoint. It may assist a systematical uncovering of conformational switching mechanisms existing in proteins and biopolymer systems in general by statistical analysis of simulation snapshots. In contrast to conventional correlation analyses of Cartesian coordinates (such as distance covariance analysis and Cartesian principal component analysis), this program also provides an alternative way to locate essential collective motions in general. Herein, we detail the algorithm in a stepwise manner and comment on the importance of the method as applied to decoding allosteric mechanisms. © 2018 Wiley Periodicals, Inc.

Effects of carotenoids on lipid bilayers.

Carotenoids have been found to be important in improving the integrity of biomembranes in eukaryotes. However, the molecular details of how carotenoids modulate the physical properties of biomembranes are unknown. To this end, we have conducted a series of molecular dynamics simulations of different biologically-relevant membranes in the presence of carotenoids. The carotenoid effect on the membrane was found to be specific to the identity of the carotenoid and the composition of the membrane itself. Therefore, different classes of carotenoids produce a different effect on the membrane, and different membrane phases are affected differently by carotenoids. It is apparent from our data that carotenoids do trigger the bilayer to become thinner. The mechanism by which this occurs depends on two competing factors, the ability of the lipid tails of opposing monolayers to either (1) compress or (2) interdigitate as the bilayer condenses. Indeed, carotenoids directly influence the physical properties via these two mechanisms, thus compacting the bilayer. However, the degree to which these competing mechanisms are utilized depends on the bilayer phase and the carotenoid identity.

Characterizing protein conformations by correlation analysis of coarse-grained contact matrices.

We have developed a method to capture the essential conformational dynamics of folded biopolymers using statistical analysis of coarse-grained segment-segment contacts. Previously, the residue-residue contact analysis of simulation trajectories was successfully applied to the detection of conformational switching motions in biomolecular complexes. However, the application to large protein systems (larger than 1000 amino acid residues) is challenging using the description of residue contacts. Also, the residue-based method cannot be used to compare proteins with different sequences. To expand the scope of the method, we have tested several coarse-graining schemes that group a collection of consecutive residues into a segment. The definition of these segments may be derived from structural and sequence information, while the interaction strength of the coarse-grained segment-segment contacts is a function of the residue-residue contacts. We then perform covariance calculations on these coarse-grained contact matrices. We monitored how well the principal components of the contact matrices is preserved using various rendering functions. The new method was demonstrated to assist the reduction of the degrees of freedom for describing the conformation space, and it potentially allows for the analysis of a system that is approximately tenfold larger compared with the corresponding residue contact-based method. This method can also render a family of similar proteins into the same conformational space, and thus can be used to compare the structures of proteins with different sequences.

Pressure-induced conformational switch of an interfacial protein.

A special class of proteins adopts an inactive conformation in aqueous solution and activates at an interface (such as the surface of lipid droplet) by switching their conformations. Lipase, an essential enzyme for breaking down lipids, serves as a model system for studying such interfacial proteins. The underlying conformational switch of lipase induced by solvent condition is achieved through changing the status of the gated substrate-access channel. Interestingly, a lipase was also reported to exhibit pressure activation, which indicates it is drastically active at high hydrostatic pressure. To unravel the molecular mechanism of this unusual phenomenon, we examined the structural changes induced by high hydrostatic pressures (up to 1500 MPa) using molecular dynamics simulations. By monitoring the width of the access channel, we found that the protein undergoes a conformational transition and opens the access channel at high pressures (>100 MPa). Particularly, a disordered amphiphilic α5 region of the protein becomes ordered at high pressure. This positive correlation between the channel opening and α5 ordering is consistent with the early findings of the gating motion in the presence of a water-oil interface. Statistical analysis of the ensemble of conformations also reveals the essential collective motions of the protein and how these motions contribute to gating. Arguments are presented as to why heightened sensitivity to high-pressure perturbation can be a general feature of switchable interfacial proteins. Further mutations are also suggested to validate our observations. Proteins 2016; 84:820-827. © 2016 Wiley Periodicals, Inc.

DMSO enhanced conformational switch of an interfacial enzyme.

Interfacial proteins function in unique heterogeneous solvent environments, such as water-oil interfaces. One important example is microbial lipase, which is activated in an oil-water emulsion phase and has many important enzymatic functions. A unique aprotic dipolar organic solvent, dimethyl sulfoxide (DMSO), has been shown to increase the activity of lipases, but the mechanism behind this enhancement is still unknown. Here, all-atom molecular dynamics simulations of lipase in a binary solution were performed to examine the effects of DMSO on the dynamics of the gating mechanism. The amphiphilic α5 region of the lipase was a focal point for the analysis, since the structural ordering of α5 has been shown to be important for gating under other perturbations. Compared to the closed-gorge ensemble in an aqueous environment, the conformational ensemble shifts towards open-gorge structures in the presence of DMSO solvents. Increased width of the access channel is particularly prevalent in 45% and 60% DMSO concentrations (w/w). As the amount of DMSO increases, the α5 region of the lipase becomes more α-helical, as we previously observed in studies that address water-oil interfacial and high pressure activation. We believe that the structural ordering of α5 plays an essential role on gating and lipase activity.

A 2015 global update on folic acid-preventable spina bifida and anencephaly.

BACKGROUND: Spina bifida and anencephaly are two major neural tube defects. They contribute substantially to perinatal, neonatal, infant, and under-five mortality and life-long disability. To monitor the progress toward the total prevention of folic acid-preventable spina bifida and anencephaly (FAP SBA), we examined their global status in 2015. METHODS: Based on existing data, we modeled the proportion of FAP SBA that are prevented in the year 2015 through mandatory folic acid fortification globally. We included only those countries with mandatory fortification that added at least 1.0 ppm folic acid as a fortificant to wheat and maize flour, and had complete information on coverage. Our model assumed mandatory folic acid fortification at 200 µg/day is fully protective against FAP SBA, and reduces the rate of spina bifida and anencephaly to a minimum of 0.5 per 1000 births. RESULTS: Our estimates show that, in 2015, 13.2% (35,500 of approximately 268,700 global cases) of FAP SBA were prevented in 58 countries through mandatory folic acid fortification of wheat and maize flour. Most countries in Europe, Africa, and Asia were not implementing mandatory fortification with folic acid. CONCLUSION: Knowledge that folic acid prevents spina bifida and anencephaly has existed for 25 years, yet only a small fraction of FAP SBA is being prevented worldwide. Several countries still have 5- to 20-fold epidemics of FAP SBA. Implementation of mandatory fortification with folic acid offers governments a proven and rapid way to prevent FAP SBA-associated disability and mortality, and to help achieve health-related Sustainable Development Goals. Birth Defects Research (Part A) 106:520-529, 2016. © 2016 Wiley Periodicals, Inc.

Mapping allostery through computational glycine scanning and correlation analysis of residue-residue contacts.

Understanding allosteric mechanisms is essential for the physical control of molecular switches and downstream cellular responses. However, it is difficult to decode essential allosteric motions in a high-throughput scheme. A general two-pronged approach to performing automatic data reduction of simulation trajectories is presented here. The first step involves coarse-graining and identifying the most dynamic residue-residue contacts. The second step is performing principal component analysis of these contacts and extracting the large-scale collective motions expressed via these residue-residue contacts. We demonstrated the method using a protein complex of nuclear receptors. Using atomistic modeling and simulation, we examined the protein complex and a set of 18 glycine point mutations of residues that constitute the binding pocket of the ligand effector. The important motions that are responsible for the allostery are reported. In contrast to conventional induced-fit and lock-and-key binding mechanisms, a novel "frustrated-fit" binding mechanism of RXR for allosteric control was revealed.

Investigation of Carbohydrate Recognition via Computer Simulation.

Carbohydrate recognition by proteins, such as lectins and other (bio)molecules, can be essential for many biological functions. Recently, interest has arisen due to potential protein and drug design and future bioengineering applications. A quantitative measurement of carbohydrate-protein interaction is thus important for the full characterization of sugar recognition. We focus on the aspect of utilizing computer simulations and biophysical models to evaluate the strength and specificity of carbohydrate recognition in this review. With increasing computational resources, better algorithms and refined modeling parameters, using state-of-the-art supercomputers to calculate the strength of the interaction between molecules has become increasingly mainstream. We review the current state of this technique and its successful applications for studying protein-sugar interactions in recent years.

Solvent-induced α- to 3(10)-helix transition of an amphiphilic peptide.

The amphiphilic peptide of the triacylglycerol lipase derived from Pseudomonas aeruginosa plays a critical role in guarding the gate for ligand access. Conformations of this peptide at several water-oil interfaces and in protein environments were compared using atomistic simulations with explicit solvents. In oil-containing solvents, this peptide is able to retain a folded structure. Interestingly, when the peptide is immersed in a low-polarity solvent environment, it exhibits a "coalesced" helix structure, which has both α- and 3(10)-helix components. The observation that the 3(10)-helical conformation is populated in a highly nonpolar environment is consistent with a previous report on polymethylalanine. Frequent interconversions of the secondary structure (between α-helix and 3(10)-helix) of the peptide are also observed. We further studied how this solvent-induced structural transition may be connected to the trigger mechanism of lipase gating and how the lipase senses the hydrophobic-hydrophilic interface.

2012 Update on global prevention of folic acid-preventable spina bifida and anencephaly.

BACKGROUND: Folic acid was proven in 1991 to prevent most cases of spina bifida and anencephaly. In 2008, less than 10% of folic acid-preventable spina bifida and anencephaly (FAPSBA) was prevented through folic acid fortification programs. This study updates the global estimates of the proportion of FAPSBA prevented with various types of folic acid fortification as of 2012. METHODS: For each country, we estimated the annual birth prevalence of FAPSBA and the daily amount of folic acid consumed from mandatory folic acid fortification programs. Assuming in Model I (our original Bell and Oakley model) that it required 400 µg, and in Model II (a new model), 200 µg of folic acid daily for total prevention of FAPSBA, we estimated the percentage of FAPSBA being prevented in each country by fortification. RESULTS: Using the original model, we estimate that 15% of FAPSBA is being prevented in 2012, compared with 2006 (6.8%) and 2008 (9.1%). We estimate in our new model that 25% of FAPSBA is being prevented. CONCLUSION: We estimate an increasing prevention of FAPSBA in the world through folic acid fortification, yet the pace is slow. Our new model estimates that only 25% prevention and reminds us that there remains a lot of work to do in countries that do not implement mandatory fortification, which is key to achieving global and total prevention. If we are to prevent all FAPSBA, there is an urgent need to build the global political will to find sufficient resources to aid in this effort.

Regulatory monitoring systems of fortified salt and wheat flour in selected ASEAN countries.

BACKGROUND: Considerable efforts have been made over the past decade to address vitamin and mineral deficiencies. An increasing number of countries in the Association of Southeast Asian Nations (ASEAN) are adopting mandatory food fortification as one of the primary strategies to overcome these deficiencies. Experience shows that fortified foods can reach large parts of the population, including the poor, if the fortification is done on a mandatory rather than a voluntary basis and if the food vehicle is widely consumed. OBJECTIVE: To review the importance of regulatory monitoring as an essential component of food fortification efforts in selected ASEAN countries, with special focus on the available information on regulatory monitoring systems for iodized salt and fortified wheat flour. METHODS: The role of regulatory monitoring in strengthening food fortification programs was discussed during a joint regional meeting of the World Health Organization, UNICEF, the Flour Fortification Initiative, the Global Alliance for Improved Nutrition, the Micronutrient Initiative, and the World Bank on regulatory monitoring of salt and wheat flour fortification programs in Asia, which took place in Manila, Philippines, on 27-29 September 2011. This paper reviews the regulatory monitoring systems of selected ASEAN countries that participated in this meeting. RESULTS: Problems and challenges in regulatory monitoring systems for iodized salt and fortified wheat flour in selected ASEAN countries are identified, and a description of the role of regulatory monitoring in strengthening food fortification initiatives, particularly of salt and flour, and highlights of areas for improvement are presented. CONCLUSIONS: Regulatory monitoring consists of monitoring activities conducted at the production level, at customs warehouses, and at retail stores by concerned regulatory authorities, and at the production level by producers themselves, as part of quality control and assurance efforts. Unless there are appropriate enforcement and quality assurance mechanisms in place to stimulate compliance by food producers, i.e., regulatory monitoring, having national legislation will not necessarily lead to increased coverage of fortified products and associated outcomes.

Solvent-dependent gating motions of an extremophilic lipase from Pseudomonas aeruginosa.

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.

Fortification of wheat flour and maize meal with different iron compounds: results of a series of baking trials.

BACKGROUND: Wheat and maize flour fortification is a preventive food-based approach to improve the micronutrient status of populations. In 2009, the World Health Organization (WHO) released recommendations for such fortification, with guidelines on the addition levels for iron, folic acid, vitamin B12, vitamin A, and zinc at various levels of average daily consumption. Iron is the micronutrient of greatest concern to the food industry, as some believe there may be some adverse interaction(s) in some or all of the finished products produced from wheat flour and maize meal. OBJECTIVE: To determine if there were any adverse interactions due to selection of iron compounds and, if differences were noted, to quantify those differences. METHODS: Wheat flour and maize meal were sourced in Kenya, South Africa, and Tanzania, and the iron compound (sodium iron ethylenediaminetetraacetate [NaFeEDTA], ferrous fumarate, or ferrous sulfate) was varied and dosed at rates according to the WHO guidelines for consumption of 75 to 149 g/day of wheat flour and > 300 g/day of maize meal and tested again for 150 to 300 g/day for both. Bread, chapatti, ugali (thick porridge), and uji (thin porridge) were prepared locally and assessed on whether the products were acceptable under industry-approved criteria and whether industry could discern any differences, knowing that differences existed, by academic sensory analysis using a combination of trained and untrained panelists and in direct side-by-side comparison. RESULTS: Industry (the wheat and maize milling sector) scored the samples as well above the minimal standard, and under academic scrutiny no differences were reported. Side-by-side comparison by the milling industry did indicate some slight differences, mainly with respect to color, although these differences did not correlate with any particular iron compound. CONCLUSIONS: The levels of iron compounds used, in accordance with the WHO guidelines, do not lead to changes in the baking and cooking properties of the wheat flour and maize meal. Respondents trained to measure against a set benchmark and/or discern differences could not consistently replicate perceived difference observations.

Enhancing processes for introduction, production, quality assurance, and delivery of US Title II food aid products.

BACKGROUND: Enacted in 1950, Public Law 480 (PL480) dramatically increased the volume of U.S.food aid and the scope of interventions it supports. Billions of dollars have been invested, both to enhance the diets of chronically undernourished people in development settings, and to support nutritional needs during conflicts and natural disasters. OBJECTIVE: Review the institutional processes-from procurement to delivery-that support this programming. METHODS: We examined the systems that govern and oversee the many components of food aid programming and the extent to which they support a whole-of-government, multi-agency food aid agenda. We conducted consultations with US government employees and contractors, academics, industry representatives, donor agency staff United Nations personnel, and field-level food aid programming technical staff from many countries. A survey of USAID implementing partners conducted among 64 responding offices in 40 countries provided data on the use and effectiveness of enriched, fortified, or blended Title II commodities, the use of new commodities, and related procurement or logistics aspects. Expert panels provided input and feedback throughout the process. RESULTS: We identified potential improvements to overall delivery and cost-effectiveness of USAID programming to better meet the nutrition needs of beneficiaries. Options include changes in product formulation, the range of products provided, and/or the modes of product approval, processing, procurement, and distribution. This research points to several improvements in processes related to food aid: 1) Establish an interagency committee to oversee all government interests in the food aid agenda through an ongoing review process. 2) Enhance processes and quality assurance along the product value chain including the importance of effective interaction with the private sector to incorporate industry best practices and create public-private partnerships to promote product innovations. 3) Strengthen the evidence base for innovations in products, programming approaches, and institutional processes. CONCLUSION: Successful programming requires knowledge and understanding of the unit cost of impact, not simply tonnage and "numbers of hungry people fed". Empirical rigor is essential; any significant program changes, including those recommended here, should be tested and monitored.

Nutritional enhancement of US Title II food aid products.

BACKGROUND: Food aid provided by the United States has saved the lives of the vulnerable for many years. Recognizing the need for a thorough review of product formulations and specifications, the US Agency for International Development (USAID) commissioned a 2-year assessment of quality issues relating to Title II food aid products. This article presents findings and recommendations of that review relating to product enhancements. OBJECTIVE: The core question addressed was: Are current commodity specifications for enriched FBFs appropriate in light of evolving nutritional science and food fortification technology, or do they need to be updated? METHODS: Empirical data were derived from a number of sources, including a survey of Title II implementing partners focusing on procurement and logistics, and uses of FBFs and other foods. Input of implementing partners, civil society, and donor organizations was obtained through individual consultations, international and small group meetings. More than 400 individuals accessed the project's website. The project convened a panel of experts in food technology and science, food policy, law, industry, medicine, development and humanitarian work, and the maritime industry, and held regular joint meetings with USDA and USAID. The draft report was widely disseminated and posted on the website. RESULTS AND CONCLUSIONS: The findings of this research led to the following broad areas of improvement in US Title II food aid products: 1) Improve the formulation of existing FBF products used in Title II programming. This includes the addition of a dairy source of protein to products targeted to children 6 to 24 months of age, pregnant and lactating women, wasted children, and wasted individuals undergoing HIV/AIDS treatment. 2) Upgrade the vitamin and mineral mixes used and diversify approaches to addressing micronutrient needs. Enhance the composition of premixes used to fortify blended foods as well as milled grains and vegetable oil; facilitate shipping offortificant premix with bulk cereals for in-country fortification; and develop micronutrient powders (sachets) and other point-of-use fortification options. 3) Develop or adopt non-cereal-based (e.g., lipid-based) products for the management of nutritional deficiencies. This is an argument for more choice among appropriate tools, not for discarding products that have already shown their value over many years. It also does not reduce the need to maintain a focus on supplying high volumes of quality grains as the main staple in food aid baskets.

Antitumor T-cell Immunity Contributes to Pancreatic Cancer Immune Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T-cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune resistance in pancreatic cancer was induced in response to antitumor T-cell immune responses and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide evidence that T-cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune resistance. Three KPC mouse models of pancreatic cancer were used: the mT3-2D (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) subcutaneous and orthotopic models, as well as the KP1 (p48-CRE/LSL-Kras/Trp53 flox/flox ) subcutaneous model. KPC cancer cells were grown in immunocompetent and immunodeficient C57BL/6 mice and analyzed to determine the impact of adaptive immunity on malignant epithelial cells, as well as on whole tumors. We found that induced T-cell antitumor immunity, via signal transducer and activator of transcription 1 (STAT1), stimulated malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and altered intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA-approved drug ruxolitinib overcame these tumor-protective responses and improved anti-PD-1 therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.

Miller's best/enhanced practices for flour fortification at the flour mill.

BACKGROUND: Cereal flour fortification has been identified as an effective mass fortification intervention as part of a national public health strategy to overcome micronutrient deficiencies and improve the health status of populations, especially women and children. OBJECTIVE: The effectiveness of cereal flour fortification programs requires the use of micronutrient premixes that provide the desired health benefits. METHOD: The Miller's Best/Enhanced Practices for Flour Fortification at the flour mill has been developed to provide specific guidance to millers and government officials to ensure that flour fortification practices are carried out in a way that results in the anticipated public health impact. RESULTS: The paper provides information specific to the use of micronutrient premixes, feeders, the fortification process, and quality control systems to ensure that both minimum and enhanced practices can be followed by the millers. CONCLUSIONS: Guidelines for basic and best/enhanced practices to be followed for each stage of the flour fortification process at the flour mill are presented. The paper is designed to be a companion to the Recommended Practices for the Production and Procurement of Premix used in Cereal Fortification Programs and supplementary to existing food quality manuals and systems, such as Good Manufacturing Practices, Food Quality Systems, and Hazard Analysis and Critical Control Points. Other stakeholders involved in flour fortification programs have the responsibility to follow best practices of their own to ensure optimum effectiveness.

Revised recommendations for iron fortification of wheat flour and an evaluation of the expected impact of current national wheat flour fortification programs.

BACKGROUND: Iron fortification of wheat flour is widely used as a strategy to combat iron deficiency. OBJECTIVE: To review recent efficacy studies and update the guidelines for the iron fortification of wheat flour. METHODS: Efficacy studies with a variety of iron-fortified foods were reviewed to determine the minimum daily amounts of additional iron that have been shown to meaningfully improve iron status in children, adolescents, and women of reproductive age. Recommendations were computed by determining the fortification levels needed to provide these additional quantities of iron each day in three different wheat flour consumption patterns. Current wheat flour iron fortification programs in 78 countries were evaluated. RESULTS: When average daily consumption of low-extraction (< or = 0.8% ash) wheat flour is 150 to 300 g, it is recommended to add 20 ppm iron as NaFeEDTA, or 30 ppm as dried ferrous sulfate or ferrous fumarate. If sensory changes or cost limits the use of these compounds, electrolytic iron at 60 ppm is the second choice. Corresponding fortification levels were calculated for wheat flour intakes of < 150 g/day and > 300 g/day. Electrolytic iron is not recommended for flour intakes of < 150 g/day. Encapsulated ferrous sulfate or fumarate can be added at the same concentrations as the non-encapsulated compounds. For high-extraction wheat flour (> 0.8% ash), NaFeEDTA is the only iron compound recommended. Only nine national programs (Argentina, Chile, Egypt, Iran, Jordan, Lebanon, Syria, Turkmenistan, and Uruguay) were judged likely to have a significant positive impact on iron status if coverage is optimized. Most countries use non-recommended, low-bioavailability, atomized, reduced or hydrogen-reduced iron powders. CONCLUSION: Most current iron fortification programs are likely to be ineffective. Legislation needs updating in many countries so that flour is fortified with adequate levels of the recommended iron compounds.