Vascular fibrinolytic balance is maintained primarily by interplay of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1). Previous research has shown that polymorphisms in genes from the renin-angiotensin (RA), bradykinin, and fibrinolytic systems affect plasma concentrations of both t-PA and PAI-1 through a set of gene-gene interactions. In the present study, we extend this finding by exploring the effects of polymorphisms in genes from these systems on incident cardiovascular disease, explicitly examining two-way interactions in a large population-based study.ACE I/D polymorphism for both dominant and additive coding schemes. There were significant interactions between the following polymorphism pairs even after adjustment for known risk factors: ACE I/D & PAI-1 4G/5G (p = 0.012), BDKRB2 C181T & ACE I/D (p = 0.016), BDKRB2 C58T & ACE I/D (p = 0.025), BDKRB2 exon 1 I/D & AT1R A1166C (p = 0.017), and BDKRB2 C58T & AT1R A1166C (p = 0.015).Data from the population-based PREVEND study in Groningen, The Netherlands  were analyzed. The effects of the polymorphisms and their interactions on cardiovascular events were analyzed via Cox proportional hazards models. There was no association between five of the six polymorphisms singly and risk of cardiovascular disease. There was a significant main effect for the This study suggests possible interactions between genes from the RA, bradykinin, and fibrinolytic systems on the risk of cardiovascular disease, extending previous research that has demonstrated that interactions among genes from these systems influence plasma concentrations of both t-PA and PAI-1. Further explorations of these interactions are needed. Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) are involved in thrombus formation and degradation. Vascular fibrinolytic balance is determined by the competing effects of t-PA and PAI-1 Given their role with respect to regulating vascular fibrinolytic balance, it is possible that variations in polymorphisms in genes from these systems may ultimately influence the risk of cardiovascular disease. Some, but not all, studies have shown such associations Data analyzed in this study were obtained from the Prevention of Renal and Vascular End-stage Disease (PREVEND) study. The PREVEND study is a large observational study currently underway in the city of Groningen, the Netherlands (started in 1997–1998). The PREVEND cohort consists of a random sample of subjects with less than 10 mg/L of urinary albumin and a selected sample of subjects with greater than 10 mg/L of urinary albumin. A total of 8,592 subjects completed the total screening program and were followed over time. Details of the study have been described previously The combined endpoint for the present analysis was incident cardiovascular disease, defined as death from CVD , hospitalization for MI , percutaneous transluminal angioplasty (PCTA), or coronary artery bypass graft (CABG). The first well-defined cardiovascular event of each participant was used for analysis. Event-free survival time for each participant was defined as the period from the date of the outpatient clinic baseline assessment to the date of death, MI, PCTA, or CABG or death from any cause or until December 31, 2005, the latest date on which information regarding specific causes of death follow-up information was available.Only subjects with a negative history of coronary artery disease (CAD) at baseline were included in the present analysis. Previous documented CAD was defined as history of myocardial infarction, revascularization procedure, or obstructive coronary artery disease prior to inclusion in the PREVEND study. A history of myocardial infarction was based on subjects' medical history, including a structured questionnaire, and information on previous CAD was complemented by review of the medical report. This approach has been used by other published reports from the PREVEND study ACE I/D polymorphism . About 89% of the subjects had complete data on all six polymorphisms . To evaluate the potential impact of missing genotype information, six new variables were created to identify cases that had missing data on each of the polymorphisms and another variable to indicate whether a case was missing data on any of the polymorphisms (composite variable). Times to first well-defined cardiovascular event between subjects with missing data and without missing data on each of the six polymorphisms (and the composite variable) were compared using Kaplan-Meier estimates. None of the log-rank tests revealed significantly different survival curves . Based on these findings, for each subsequent analysis, a complete-case approach was utilized . A similar approach was used by Borggreve et al. Demographic and clinical information was collected for each participant at baseline. In addition, genotyping was conducted on six polymorphisms as described by Asselbergs et al. SAS 9.1  and STATA SE 9.1  were used for data analysis. The effects of the polymorphisms on cardiovascular events were analyzed via Cox proportional hazards models. The proportional hazards assumption for the polymorphisms and potential confounders  were assessed by graphing log-log survival functions and by evaluation interactions between each variable with time. These examinations suggested that there were no substantial violations of the proportional hazards assumption.n = 5,661 with UAC ≥10 mg/L and n = 2,477 with UAC <10 mg/L at baseline in the present analysis sample). To overcome this selection bias in the present study, design-based analyses were performed using sampling probability weights. Due to this weighting method, our conclusions can be generalized for the general population In the screening phase of the PREVEND study, subjects with an elevated UAE were over-sampled to acquire sufficient subjects with microalbuminuria  to capture additive effects,  to capture dominant effects, and  to capture recessive effects .As an initial step, the unadjusted, unmoderated effects of each polymorphism were examined. Prior to this analysis, all genotypes were coded . We used an uncorrected significance level of α = 0.10 as suggestive evidence for interaction and a more conservative significance level of α = 0.025 as strong evidence for interaction. A similar approach was used by Asselbergs et al. The third step of the analysis was to adjust the estimates by including the following demographic and clinical variables collected at baseline: sex, age, body mass index, presence of diabetes, systolic and diastolic blood pressure, HDL cholesterol, total cholesterol, c-reactive protein, current smoking status, and waist-to-hip ratio. Prior to interpretation, for pairs of polymorphisms where there were multiple significant interactions , we selected the model that provided the most parsimonious fit to the data using Akaike's information criterion (AIC) from the covariate-adjusted model.Finally, because there is some evidence that the prediction of plasma t-PA/PAI-1 levels by these polymorphisms may differ by sex BDKRB2 C181T polymorphism (TT) was rare (1.3%). Therefore, neither recessive coding  nor additive coding  were used for this polymorphism in any analysis. The symbol “N/A” reflects these models in subsequent tables.Baseline demographic and clinical characteristics of the sample can be found in n = 463 events were detected among the 8,138 subjects in the dataset (5.7%). ACE I/D polymorphism was significant at the 0.05 level for the dominant  as well as the additive coding scheme . The findings suggest a smaller hazard associated with the presence of each ACE D allele  to 1 (ACE ID genotype) to 2 (ACE DD genotype)). This effect is opposite of what one would expect to find based on previous research. The ACE DD genotype has been found to be associated with increased levels of PAI-1 in some During a median follow-up of 7.52 years (range 1 day to 8.3 years), a total of P values for the statistical tests for unadjusted two-way interactions among the polymorphisms are provided supporting information tables S1a-S1f. Significant results with strong or suggestive evidence for interaction are in bold). A total of 6 gene-gene interactions were detected out of 15 possible pairs of polymorphisms. Of the 9 possible interactions for the combination of ACE I/D and PAI-1 4G/5G, 8 were significant (the ACE I/D x PAI-1 4G/5G (recessive-dominant) was the only non-significant interaction for this pair).We found evidence for RAS-fibrinolytic and bradykinin-RAS interaction effects on incident CVD (AT1R A1166C x PAI-1 4G/5G), it appears that adjusting for known risk factors attenuates the suggestive significance observed in the unadjusted analysis . Hence, this interaction is not further explored. Statistical significance was maintained for the other five interactions following adjustment for potential confounders.A summary of the 16 gene-gene interactions with strong or suggestive evidence can be found in BDKRB2 C58T x ACE I/D interaction shows non-significant subgroup effects in the unadjusted model and one significant subgroup effect only after adjustment for covariates. Given these findings and the exploratory nature of this study, we do not provide further interpretations of this interaction. For the four remaining interactions, the interaction appears to be due to significant effects of one genetic variation only at certain levels of other genetic variation . For those with the PAI-1 4G4G genotype, the effect of the ACE I/D polymorphism was in the opposite direction, but not statistically significant .Among those with at least one ACE I allele, individuals with the BDKRB2 C181T TT or BDKRB2 C181T CT genotypes had a significantly higher hazard of cardiovascular disease as compared to those who carried the BDKRB2 C181T CC genotype . For those with the ACE DD genotype, the effect of the BDKRB2 C181T polymorphism was in the opposite direction, but was not statistically significant in the adjusted model  .Among those with at least one AT1R A1166C AA genotype, individuals with the BDKRB2 exon 1 I/D +9/+9 genotype had a significantly lower hazard of cardiovascular disease as compared to those who carried either the BDKRB2 exon 1 I/D −9/+9 or −9/−9 genotypes . Among those with at least one AT1R A1166C C allele, the effect of the BDKRB2 exon 1 I/D polymorphism was in the opposite direction, but was not statistically significant .For those with the AT1R A1166C AA genotype, individuals with the BDKRB2 C58T TT or BDKRB2 C58T CT genotypes had a significantly higher hazard of cardiovascular disease as compared to those who carried the BDKRB2 C58T CC genotype . Among those with at least one AT1R A1166C C allele, the effects of the BDKRB2 C58T polymorphism were in the opposite direction, but not statistically significant.For those with the ACE I/D x PAI-1 4G/5G (additive-additive) interaction, the three-way interaction was significant in the unadjusted (P = 0.028) and the adjusted models (P = 0.028). Thus, sex may play a role in the ACE I/D x PAI-1 4G/5G interaction. Preliminary examination of the results suggests that the findings described earlier for the ACE I/D x PAI-1 4G/5G interaction may be stronger for females than for males. There was strong evidence of an ACE I/D x PAI-1 4G/5G interaction effect for females (adjusted P = 0.001), while the evidence was less convincing for males (adjusted P>0.10) (data not shown). Subsequent research is necessary to validate this finding.To assess whether the four significant gene-gene interactions varied by sex, three-way interaction terms were added to each model (sex x genotype1 x genotype2), along with the appropriate lower order effects . For three of the four interactions, none of the three-way interactions were statistically significant at the 0.10 level of significance in the unadjusted or adjusted models. However, for the ACE I/D and PAI-1 4G/5G polymorphisms, the BDKRB2 C181T and ACE I/D polymorphisms, the BDKRB2 exon 1 I/D and AT1R A1166C polymorphisms, and the BDKRB2 C58T and AT1R A1166C polymorphisms on the risk of cardiovascular disease. The interaction between the ACE I/D and PAI-1 4G/5G polymorphisms appears to be robust to the choice of genotype coding scheme. Interestingly, several of these interactions were noted in a study of the epistatic effects of these polymorphisms on plasma t-PA and PAI-1 levels BDKRB2 C181T x ACE I/D interaction).Vascular fibrinolytic balance is maintained primarily by an interplay of t-PA and PAI-1 ACE I/D and risk of cardiovascular disease appears to be limited to those who carry the PAI-1 5G allele and suggests a protective effect of the ACE D allele. While an unexpected finding, a protective effect of ACE DD in the presence of the PAI-1 5G allele is consistent with the multifactor dimensionality reduction analysis findings of Coffey et al. ACE DD group in the presence of PAI-1 5G5G, which would be expected considering the observed protective effect in the present study (data not shown). Although not significant in the present study, there was a suggestion of increased risk of the ACE D allele among those with the PAI-1 4G4G genotype in the present study. This is consistent with previous basic science and human clinical studies. Both the ACE DD genotype and the PAI-1 4G allele are associated with increased PAI-1 levels The association between RAS and bradykinin genes showed epistatic effects on both PAI-1 as t-PA levels among females and males, similar to the results in the current study.The other three interactions involve the bradykinin and RA systems. Bradykinin plays an important role in determining t-PA levels. In addition to its role in the inflammatory process, bradykinin is a potent endothelial cell stimulant that has been shown to induce the acute release of t-PA from the endothelium through a B2 receptor mechanism BDKRB2 C181T polymorphism), one can estimate that under the null hypothesis, one would expect 2.6 and 10.5 interactions to be nominally significant at α = 0.025 and α = 0.10, respectively, by chance alone . We found a total of 16 significant gene-gene interactions (P<0.10), most of which remained significant after adjusting for known risk factors. The findings of significant interactions between genes from the RA, bradykinin, and fibrinolytic systems on the risk of cardiovascular disease warrant further exploration, including attempting to replicate these results in other populations with other study designs.Controlling for traditional risk factors of cardiovascular disease did not lead to substantive changes in the results with respect to statistical significance. However, this does not allow us to totally rule out chance as a potential explanation of the findings. Although there are plausible biologic mechanisms to support interactions among genes from the RA, bradykinin, and fibrinolytic systems, this study used an exploratory analysis plan searching for all possible two-way interactions between six genes. Furthermore, for each possible pair of polymorphisms, we tested nine different interactions based on different genotype coding schemes. Multiple tests increase the possibility of chance associations, especially when conducting a non-replicated study. Given that these tests are correlated in the present study, it is difficult to calculate how many interactions are expected by chance. However, by making some simplifying assumptions, and noting that a total of 105 interactions were tested (nine possible interaction effects were tested for the 15 possible pairs of polymorphisms minus 30 interactions since only dominant coding was used for the It is important to recognize that cardiovascular disease is a relatively infrequent event in this cohort due to the young age (mean 48.6 years at baseline) and gender composition . The onset of symptoms is age related and additional cardiovascular events are expected to be observed in the PREVEND cohort as more time passes. In addition, the penetrance of the alleles may also be age related. Future analyses should examine whether these potential interactions remain as additional events become available and the population grows older.Furthermore, because this study was limited to the investigation of potential two-way interactions, future studies should consider higher-order interactions using a multifactor dimensionality reduction (MDR) type analysis In summary, this study suggests possible interplay between genes from the RA, bradykinin, and fibrinolytic systems on the risk of cardiovascular disease, extending previous research that has demonstrated that interactions among genes from these systems influence plasma concentrations of both t-PA and PAI-1.Table S1Table S1a - S1f. P values for the unadjusted first-order gene-gene interactions are reported in supporting information tables S1a-S1f.(0.09 MB DOC)Click here for additional data file.


Crystal form can be crucial to the performance of a dosage form. This is especially true for compounds that have intrinsic barriers to drug delivery, such as low aqueous solubility, slow dissolution in gastrointestinal media, low permeability and first-pass metabolism. The nature of the physical form and formulation tends to exhibit the greatest effect on bioavailability parameters of water insoluble compounds that need to be given orally in high doses. An alternative approach available for the enhancement of drug solubility, dissolution and bioavailability is through the application of crystal engineering of co-crystals. The physicochemical properties of the active pharmaceutical ingredients and the bulk material properties can be modified, whilst maintaining the intrinsic activity of the drug molecule. This article covers the advantages of co-crystals over salts, solvates (hydrates), solid dispersions and polymorphs, mechanism of formation of co-crystals, methods of preparation of co-crystals and application of co-crystals to modify physicochemical characteristics of active pharmaceutical ingredients along with the case studies. The intellectual property implications of creating co-crystals are also highly relevant. Chemists and engineers in the pharmaceutical industry generally seek to deliver crystalline forms of their active compounds, mainly due to the inherent stability of crystalline materials and the well-established impact of crystallization processes on purification and isolation of chemical substances. IncreasPharmaceutical active ingredients (APIs) can exist in a variety of distinct solid forms, including polymorphs, solvates, hydrates, salts, co-crystals and amorphous solids. Each form displays unique physicochemical properties that can profoundly influence the bioavailability, manufacturability purification, stability and other performance characteristics of the drugs.Solid form discovery and design depends on the nature of the molecule of interest and type of physical property challenges faced in its development. The preferred solid form is generally the thermodynamically most stable crystalline form of the compound4. Howeve35Crystal engineering is generally considered to be the design and growth of crystalline molecular solids with the aim of impacting material properties. A principal tool is the hydrogen bond, which is responsible for the majority of directed intermolecular interactions in molecular solids. Co-crystals are multi-component crystals based on hydrogen bonding interactions without the transfer of hydrogen ions to form salts; this is an important feature, since Bronsted acid-base chemistry is not a requirement for the formation of a co-crystal. Co-crystallization is a manifestation of directed self-assembly of different components. Co-crystals have been described of various organic substances over the years8 and giv911Pharmaceutical co-crystals can be defined as crystalline materials comprised of an API and one or more unique co-crystal formers, which are solids at room temperature.Co-crystals can be constructed through several types of interaction, including hydrogen bonding, p stacking, and vander Waals forces. Solvates and hydrates of the API are not considered to be co-crystals by this definition. However, co-crystals may include one or more solvent/water molecules in the crystal lattice. Co-crysPharmaceutical co-crystallization, which has only recently gained widespread attention as a means of modifying the physicochemical properties of APIs, has two inherent advantages over the salt form. First, because co- crystal formation may potentially be employed with all APIs, including acidic, basic and non ionisable molecules and second is a large number of potential ‘counter molecules’ which may be considered to be non toxic possibly increasing the scope of the pharmaceutical co- crystallization over the salt forms.An analogy can be drawn to salt selection in which pKa arguments are used to select acid-base pairs that can be converted to salt compounds. Chemistry demonstrates that a pKa difference of at least two units (between an acid and a base) is required to form a salt that is stable in water.It is also important to remember that salt formation is generally directed at a single acidic and basic functional group. In contrast co-crystals can simultaneously address multiple functional groups in a single drug molecule. In addition space is not limited to binary combinations (acid-base pairs) since tertiary and quaternary co-crystals are realistic one18. One vThe extent of polymorphism of pharmaceutical is limited to the handful of the different crystal forms. Primary difference between solvates and co-crystals is the physical state of the individual components. If one 2426Direct answer of this question is not possible because to prove the absence of polymorphism is tantamount to the proving-the-negative. If one considers an argument that compounds have a lower degree of self-complementarity than complementarity to a rationally selected co-crystal former. Initial indications are that polymorphic substances may provide the good candidates for the co-crystals. For exaThe key benefits associated with co-crystallization approach to modify properties of pharmaceutical solids are the theoretical capability of all types of drug molecules, including weakly ionizable and non-ionizable, to form co-crystals, and the existence of numerous, potential counter-molecules, including food additives, preservatives, pharmaceutical excipients as well as other APIs, for co-crystal synthesis. Major advantage is that co-crystal synthesis may offer for the pharmaceutical industry is an opportunity to address intellectual property (IP) issues by extending the life cycles of old APIs.Co-crystals having advantages like stable crystalline form (as compared to amorphous solids), no need to make or break covalent bonds, theoretical capability of all types of API molecules (weakly ionizable/non-ionizable) to form co-crystals, the existence of numerous potential counter-molecules , the only solid form that is designable via crystal engineering patentable expanding IP portfolios and can be produced using solid-state synthesis green technologies high yield, no solvent or by-products.The crystal engineering experiment typically involves the Cambridge Structural Database (CSD) survey followed by the experimental work. Co-crystals designed on the principal of the supramolecular synthesis; it provides a powerful approach for proactive discovery of novel pharmaceutical solid phases. Co-crystals consist of multiple components in given stoichiometric ratio, where different molecular species interact by hydrogen bonding and by non-hydrogen bonding.The use of hydrogen bonding rules, synthons and graph sets may assist in the design and analysis of co-crystal systems. In general though, prediction of whether co-crystallization will occur is not yet possible and must, at present, be answered empirically. Co-crystal formation may be rationalised by consideration of the hydrogen bond donors and acceptors of the materials that are to be co-crystallized and how they might interact. Following the extensive examination of preferential packing preferences and hydrogen bond patterns in a number of organic crystals, Etter and co-workers proposed the guidelines to facilitate the deliberate design of hydrogen-bonded solids. All gooA detailed understanding of the supramolecular chemistry of the functional groups present in a given molecule is the prerequisite for designing the co-crystals because it facilitates the selection of the suitable co-crystal former. Supramoecular synthons that can occur in common functional group in order to design new co-crystals and certain functional groups such as carboxylic acids, amides and alcohols are particularly amenable to formation of supramolecular heterosynthon. The strCo-crystal formation described in the literature indicates the notoriously difficult situation these systems present with regard to preparation it has been known to take 6 months to prepare a single co-crystal of suitable quality for single X-ray diffraction analysis. This isCo-crystals can be prepared by solvent and solid based methods. The solvent-based methods involve slurry conversion solvent evaporation, cooling crystallization and precipitation. The solid based methods involve net grinding; solvent-assisted grinding and sonication (applied to either to wet or dry solid mixtures) 80° to 85°.For solution co-crystallization, the two components must have similar solubility; otherwise the least soluble component will precipitate out exclusively. However similar solubility alone will not guarantee success. It has been suggested that it may be useful to consider polymorphic compounds, which exist in more than one crystalline form as co-crystallising components. If a molecular compound exists in several polymorphic forms it has demonstrated a structural flexibility and is not locked into a single type of crystalline lattice or packing mode. Thus, the chance of bringing such a molecule into a different packing arrangement in coexistence with another molecule is increased. Clearly polymorphism alone does not guarantee the functionality of a compound to act as a co-crystallising agent, whilst the ability of a molecule to participate in intermolecular interactions obviously plays a critical role.Small-scale preparation has been described. Scale-up crystallization was performed in a 500 ml water-jacketed glass crystallization vessel. Temperature was maintained by a circulating water bath. A reflux column, digital thermometer, and overhead stirrer with a glass shaft and Teflon blade were attached to vessel ports. The drug and co-crystal former were added to a reaction vessel. The solids were dissolved in ethanol/methanol mixture and heated to 70° for 1 h under reflux. Temperature was decreased in 10° increments to induce precipitation in a stirred, unseeded system. Observe the appearance of the co-crystal. Literate to enhance solids recovery decrease the further temperature.th century report is often cited as the earliest reference to such a procedure, the recent technique of adding small mounts of solvent during the grinding process has been shown to enhance the kinetics and facilitate co-crystal formation and as lead to increased interest of solid-state grinding as a method for co-crystal preparation[When preparing co-crystals, the product obtained from grinding is generally consistent with that obtained from solution. This may indicate that hydrogen-bond connectivity patterns are not idiosyncratic or determined by non-specific and unmanageable solvent effects or crystallization conditions. Nevertheless there are exceptions. Whilst many co-crystal materials can be prepared from both solution growth and solid-state grinding, some can only be obtained by solid-state grinding. An example is that in the co-crystallisation of 2,4,6-trinitrobenzoic acid and indole-3-acetic acid, different crystal forms were obtained from solution compared with grinding. Failure to form co-crystals by grinding may be due to an inability to generate suitable co-crystal arrangements rather than due to the stability of the initial phases. When co-crystal formation has been successful from solution, but not from grinding, solvent inclusion in stabilizing the supramolecular structure may be a factor. Although co-crystal formation by solid-state grinding has been established for some time and a late 19eparation.Slurry conversion experiments were conducted in different organic solvents and water. Solvent (100 or 200 ml) was added to the co-crystal (20 mg) and the resulting suspension was stirred at room temperature for some days. After some days, the solvent was decanted and the solid material was dried under a flow of nitrogen for 5 min. The remaining solids were then characterized using PXRD.This is one of the methods for precipitation or recrystalization of the co-crystal former and active pharmaceutical ingredient. Solvents include buffers (pH) and organic solvents. For example preparation of co-crystals of aceclofenac using chitosan, in which chitosan solution was prepared by soaking chitosan in glacial acetic acid. A weighed amount of the drug was dispersed in chitosan solution by using high dispersion homogenizer. This dispersion was added to distilled water or sodium citrate solution to precipitate chitosan on drug.A crystalline particle is characterized by definite external and internal structures. Habit describes the external shape of a crystal, where as polymorph state refers to the definite arrangement if molecules inside the crystal lattice. SupersaFrom the above described methods such as solution crystallization, solvent change that create supersaturation by increasing the solute concentration and decreasing the solute solubility, respectively. There a40In case of increased saturation, rate of nuclei formation is greater than crystal growth. More growth in one direction produces fine needle shaped crystals that exhibit poor folowability, while less saturation leads to platy crystals which exhibit greater dissolution rate. Cooling a supersaturated solution of drug or pouring it into crystallizing solvent maintained at low temperature immediately decreases the drug solubility and results in rapid deposition of drug molecules on the nuclei. Rapid cooling leads to formation of platy or needle shaped crystals, slow rate of cooling forms compact, symmetric or elongated prisms. The degree of solution agitation has influence on saturation level, high speed of agitation leads to elongated crystals with small particle size distribution having good folowability and less sedimentation in suspension. Slow speed of agitation or unstirred solution forms large platy crystals.The nature of solvent has been found to have a profound effect on crystal habit of ibuprofen. Crystallization of ibuprofen from ethanol and acetone having high surface tension, dielectric constant and less specific gravity were thin, platy, and neatly circular in shape, while those obtained from propylene glycol and 2-propanol were rod shaped. When pH was decreased by addition of hydrochloric acid to sodium hydroxide solution (pH-10) resulted in formation of needle shaped crystals. However, spherical agglomerates were obtained when ibuprofen was dissolved in acetonitrile because of limited miscibility with water.Low temperature of crystallizing solvent produces irregular shaped crystals while in case of high temperature nuclei formation is delayed and fine, symmetric crystals are produced. Ions, polymeric molecules, or the other substances present in solute or solvent acts as impurities for the growing crystals and modify crystal habit. Impurity is known to modify the growing crystals into a morphology that is desirable from the viewpoint of dosage form design and performance. Grindin15The earliest example of pharmaceutical co-crystals in the context of APIs relates to a series of studies conducted in the 1950s by Higuchi and Roy. They studied complex formation between macromolecules and certain pharmaceuticals. However, these would not be classified as pharmaceutical co-crystals according to the criteria applied herein47.et al.[Perhaps the first application of crystal engineering to the generation of pharmaceutical co-crystals was a series of studies reported by Zerkowski et al. concerniet al.. Despitemax and comparable Tmax when compared with the marketed form, Tegretol®[Carbamazepine (CBZ) is an important antiepileptic drug that has been in use for over three decades. Oral administration of CBZ encounters multiple challenges, including low water solubility with high dosage required for therapeutic effect (i.e. >100 mg/day), dissolution-limited bioavailability and auto induction for metabolism. In contrast to its simple molecular structure, CBZ exhibits complexity in its crystal forms21. To da20Tegretol®.The availability and marketability of a variety of APIs as chloride salts is well recognized, and, recently, an approach to utilize such chloride salts, specifically fluoxetine hydrochloride (fluoxetine HCl), to generate co-crystals of an amine hydrochloride salt via a chloride-mediated carboxylic acid supramolecular synthon has been reported. Fluoxetine HCl is the active pharmaceutical ingredient found in the common antidepressant drug Prozac®. It is a solid under ambient conditions, only one crystalline phase is known, and it is available in the salt form. It has been demonstrated that co-crystallization of this API modifies the physical properties of fluoxetine HCl while still retaining the hydrochloride salt of the API. Fluoxetine HCl was co-crystallized with benzoic acid (1:1), succinic acid (2:1), and fumaric acid (2:1) via traditional evaporation techniques. For all three co-crystals, the carboxylic acid was found to form hydrogen bond to the chloride ion, which in turn interacted with the protonated amine, thus generating, in all three cases, amine hydrochloride salt hydrogen bonding to an additional neutral molecule. Powder dissolution experiments were carried out in water for the three novel co-crystals resulting in a spread of dissolution profiles. The fluoxetine HCl:benzoic acid cocrystal was found to have a decrease in aqueous solubility by 50%, and the fluoxetine HCl:fumaric acid cocrystal had only a slight increase in aqueous solubility. However, the fluoxetine HCl: succinic acid cocrystal exhibited an approximately twofold increase in aqueous solubility after only 5 min. The complex formed between succinic acid and fluoxetine HCl falls apart in solution to generate its pure components after about 1 h. An intriguing aspect of this study is that by simply hydrogen bonding a hydrochloride salt of an API with similar cocrystal formers, one can generate distinctively different dissolution profiles[Itraconazole is a triazole antifungal agent that is prescribed to patients with fungal infections. Itraconazole is extremely water insoluble and administered both orally and intravenously. The oral formulation of itraconazole is the amorphous form coated on the surfaces of sucrose beads, and marketed as the Sporanox® capsule. In addition, co-administration of acidified HP-β-cyclodextrin beverages with Sporanox® capsules is required to achieve the maximal absorption of the API, even though such a co-administration can cause diarrhea52. InterCrystalline phases of itraconazole can be engineered by introduction of additional molecules to match hydrogen-bond donors and acceptors50. A numSildenafil is a drug used in the treatment of pulmonary arterial hypertension, congestive heart failure, atherosclerosis, conditions of reduced blood vessel potency and peripheral vascular disease, as well as male erectile dysfunction and female sexual disorders. Sildenafil selectively inhibits cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 that is responsible for degradation of cGMP in the corpus cavernosum, leading to smooth muscle relaxation in the corpus cavernosum, and resulting in increased inflow of blood and an erection. Sildenafil citrate, with moderate water solubility, has been commercially developed and marketed by Pfizer and is available under the trademark Viagra®.It has been observed that sildenafil in a pharmaceutical cocrystal form could provide an improved solubility of the API under acidic conditions. In addition, such an improvement of solubility of sildenafil could be particularly advantageous for its orally administrable formulation. Sildenafil has been successfully co-crystallized with acetylsalicylic acid (1:1 molar ratio) by slurry or under reflux conditions.The crystal structure of the cocrystal of sildenafil and acetylsalicylic acid has been determined by single crystal X-ray diffraction, and in addition, the composition of matter was confirmed by powder X-ray diffraction and infrared spectrometry. Moreover, the differential scanning calorimetry and thermo gravimetric analyses indicate that the melting point of the cocrystal is approximately 143°, and it remains thermodynamically stable up to 165°. An intr50) of 3100 mg/kg for the male and 3900 mg/kg for the female. Also, the quantity of melamine observed in those incidents was not at levels that would normally kill. In the course of the pet food recall investigation, cyanuric acid, another relatively nontoxic compound, was also identified in the pet food as a co contaminant. Although melamine and cyanuric acid are relatively safe individually, no data could be found in the literature that could determine the potential toxicity of melamine and cyanuric acid in combination. From the crystal engineering viewpoint, melamine and cyanuric acid (1:1 molar ratio) form an extensive two-dimensional networks in the solid-state based on the robust three-point molecular recognition, and it was observed that the resulting melamine:cyanuric acid cocrystal is highly insoluble in water.In early 2007, the FDA received complaints from owners of more than 4000 pets regarding the deaths of animals after taking food that was later recalled; it was reported that majority of those deadly incidents were caused by acute renal failure. At first, melamine, which was observed in the tainted products, was the suspected contaminant, since this particular chemical could be intentionally added to raise the apparent protein content of the food. However, melamine is considered relatively nontoxic with the acute toxicity of melamine in rats was reported with oral lethal doses 50 . The possibility of crystalline hydrate formation complicates design of a consistent, reproducible for an API in the drug development process. Reversible hydrate formation is particularly problematic, as it indicates that neither the anhydrate nor the hydrate is fully stable across the range of common processing condition. Theophylline is structural analogue of caffeine. The co-crystals of the theophylline were prepared with oxalic acid, malonic acid, maleic acid, glutaric acid by solvent evaporation technique. The relative humidity stability comprised of the storage and subsequent PXRD analysis at four specific RH levels  across four different time points . Over the course of 7 week study it was found that, at 75% RH and below, theophylline anhydrate converted into theophylline monohydrate. No formation of theophylline hydrate was found in any case.The observed RH stability of theophylline co-crystal demonstrates the physical stability improvement, specifically avoidance of hydrate formation. The co-crystals formed by oxalic acid found to be more stable. This study demonstrates use of co-crystals in physical property improvement.Aceclofenac is an orally effective nonsteroidal antiinflammatory drug of phenyl acetic acid group, which possesses remarkable antiinflammatory, analgesic and antipyretic properties. Aceclofenac exhibits slight solubility in water and as a consequence it exhibits low bioavailability after oral administration. Mutalic preparedChitosan has been considered to be one of the most promising biopolymer for drug delivery purpose. Chitosan is a linear hydrophilic polysaccharide polymer of D-glucosamine. It is non-toxic natural polcationic polymer that is degraded by the microflora in the colon. It is abundant in nature and is present in the exoskeleton of crustaceans such as crabs and shrimps. Chitosan has been demonstrated to be a good vehicle for enhancing the dissolution properties and bioavailability of a number of poorly water-soluble drugs.in vitro drug release, stability and phrmacokinetic study.Chitosan was precipitated on aceclofenac crystals using sodium citrate as a salting out agent. The pure drug and prepared co-crystals with different concentrations (0.05 to 0.6%) were characterized in terms of solubility, drug content, particle size, thermal behavior , X-ray diffraction (X-RD), morphology, In vivo study revealed that the crystals provided a rapid pharmacological response in mice and rat; besides improve in pharmacokinetic parameters in rats[It was observed that particle size of co-crystals was drastically reduced during the formulation process. The DSC showed a decrease in melting enthalpy indicating disorder in crystallinity. XRD also revealed disorder in crystallinity. The dissolution study showed that marked increase in dissolution rate in comparison to pure drug. The considerable dissolution rate of aceclofenac from optimized crystal formulation was attributed to the wetting effect of chitosan, decreased drug crystallinity, altered morphology and micronization. The optimized crystals showed excellent stability on storage at accelerated conditions. s in rats.Co-crystals of 5-nitrouracil with solvent molecules, dioxane, pyridine, DMSO, formamide and ethanol as well as with piperazine, N,N'-dimethylpiperazine, 3-aminopyridine and diazabicyclooctane obtained by deliberate inclusion, have been examined by X-ray crystallography. The tape structure found in the parent centric form of nitrouracil is retained with some modifications in the co-crystals with dioxane, piperazine, diazabicyclo-octane, N,N'-dimethylpiperazine, pyridine and DMSO, with the guest molecules forming alternate tapes. In co-crystals involving formamide, ethanol and 3-aminopyridine, the molecular tapes exhibit mixed compositions. The observed bonding patterns have been classified into six schemes. Interestingly, quadruple type hydrogen bonding patterns are seen in co-crystals containing 3-aminopyridine or ethanol and water, while a network of acyclic tetrahedral pentamers of water is found in the cocrystal containing diazabicyclo-octane and water. This case study reveals that hydrogen acceptors and donors are necessary to form cocrystal.Indomethacin, a non-steroidal antiinflammatory drug (NSAID), is widely prescribed for patients with arthritis. It exists as α, γ and amorphous forms, in the solid state α-form being most stable form at room temperature. Indomethacin is practically insoluble in water  and poses severe challenges in the formulation development. Various cocrystal formers, including saccharin, were used in the screening for indomethacin co-crystals in a series of solvents.Solution evaporation method was used in the screening phase. DSC, TGA, IR, Raman and PXRD techniques characterized the potential new phases. The indomethacin saccharin co-crystal  structure was determined from single crystal X-ray diffraction data. Pharmaceutically relevant properties such as the dissolution rate and dynamic vapour sorption (DVS) of the IND-SAC co-crystal were evaluated. Solid-state and solvent-drop co-grinding methods were also applied to indomethacin and saccharin.The IND-SAC co-crystals were obtained from ethyl acetate. Physical characterization showed that the IND-SAC co-crystal is unique vis-à-vis thermal, spectroscopic and X-ray diffraction properties. The co-crystals were obtained in a 1:1 ratio with a carboxylic acid and imide dimer synthons. The dissolution rate of IND-SAC was considerably faster than that of the stable indomethacin α-form. DVS studies indicated that the co-crystals gained less than 0.05% in weight at 98% RH. IND-SAC co-crystals were also obtained by solid state and solvent-drop co grinding methods. The IND-SAC co-crystal was formed with a unique and interesting carboxylic acid and imide dimer synthons interconnected by weak N-H…O hydrogen bonds. The co-crystals were associated with significantly faster dissolution rate than indomethacin (α-form) in phosphate buffer pH 7.4 and were non-hygroscopic.“New” refers to anything under the sun that is made by the man, such as new composition of matter or any useful improvements. Thus a necessary condition to claim a new composition of matter is to describe clearly with precision the composition, a great challenge in the case of co-crystals.Aripiprazole co-crystals, the preConsequently, there is a need for soluble forms of aripiprazole that can be readily formulated for use in various modes of administration, including parenteral and oral administration. Co-crystal complexes of aripiprazole would add a powerful tool in the treatment of central nervous system disorders. The present invention provided co-crystals of aripiprazole and fumaric acid which are stable and are reproducible on an industrial scale.TDFA is the co-crystal of tenofovir disoproxil hemi-fumaric Acid. TenofovTheophylline crystallized rapidly from a hot ethylene glycol solution forms theophylline co-crystals. This teAs one aspect, novel co-crystals are provided. The novel co-crystals comprise one or more active agents, particularly of the salts of such active agents. Novel forms of salts of active pharmaceutical ingredients are provided. For example, the present invention provides novel co-crystals of fluoxetine HCl and benzoic acid; fluoxetine HCl and succinic acid; and fluoxetine HCl and fumaric acid. Novel forms or solid state phases of active pharmaceutical ingredients may be prepared for which there are no known polymorphs, solvates or hydrates, or where such polymorphs, solvates or hydrates were disfavored. Co-crys


CD209, is a receptor capable of binding and internalizing Mycobacterium tuberculosis. Previous studies have reported that the CD209 promoter single nucleotide polymorphism (SNP)-336A/G exerts an effect on CD209 expression and is associated with human susceptibility to dengue, HIV-1 and tuberculosis in humans. The present study investigates the role of the CD209 -336A/G variant in susceptibility to tuberculosis in a large sample of individuals from sub-Saharan Africa.Tuberculosis causes significant morbidity and mortality worldwide, especially in sub-Saharan Africa. DC-SIGN, encoded by CD209 -336A/G SNP (rs4804803). Significant overall protection against pulmonary tuberculosis was observed with the -336G allele when the study groups were combined . In addition, the patients with -336GG were associated with a decreased risk of cavitory tuberculosis, a severe form of tuberculosis disease . This direction of association is opposite to a previously observed result in a smaller study of susceptibility to tuberculosis in a South African Coloured population, but entirely in keeping with the previously observed protective effect of the -336G allele.A total of 2,176 individuals enrolled in tuberculosis case-control studies from four sub-Saharan Africa countries were genotyped for the CD209 -336G variant allele is associated with significant protection against tuberculosis in individuals from sub-Saharan Africa and, furthermore, cases with -336GG were significantly less likely to develop tuberculosis-induced lung cavitation. Previous in vitro work demonstrated that the promoter variant -336G allele causes down-regulation of CD209 mRNA expression. Our present work suggests that decreased levels of the DC-SIGN receptor may therefore be protective against both clinical tuberculosis in general and cavitory tuberculosis disease in particular. This is consistent with evidence that Mycobacteria can utilize DC-SIGN binding to suppress the protective pro-inflammatory immune response.This study finds that the  Mycobacterium tuberculosis infection causes significant morbidity and mortality throughout the world, particularly in resource-poor countries. M. tuberculosis infects one third of the human population and approximately two million persons die of tuberculosis every year M. tuberculosis and more than 25 million will die from the disease, despite the availability of antibiotics CD209 on chromosome 19p13.3, is a C-type lectin that is expressed on subsets of dendritic cells (DCs) and alveolar macrophages M. tuberculosis, M. leprae, Bacillus Calmette-Guérin (BCG), Heliobacter pylori, Streptococcus pneumoniae, Klebsiella pneumoniae, HIV-1, HIV-2, SIV-1, Dengue virus, Ebola Virus, Cytomegalovirus, Hepatitis C virus, Schistosoma mansoni, Leishmania pifanoi and Candida albicansDendritic Cell-Specific ICAM3-Grabbing Non-integrin (DC-SIGN), encoded by M. tuberculosis on immune cell subsets and has been shown to bind mannosylated lipoarabomannan (ManLAM) present on virulent, but not avirulent, strains of Mycobacterium spp.M. tuberculosis and infection of DCs and alveolar macrophages M. tuberculosis-induced immune suppression DC-SIGN is a major receptor for CD209 variation in human susceptibility to infectious diseases including M. tuberculosis and M. leprae, HIV-1, and Dengue M. leprae case-control study found an association of the CD209 -336A/G promoter SNP with infectious disease susceptibility or protection. Martin and colleagues demonstrated that the -336G allele was associated with susceptibility to parenteral but not mucosal HIV-1 infection, although this was not replicated in individuals of recent African descent CD209 -871 SNP was found to be the polymorphism most significantly associated with tuberculosis susceptibility in the South African Cape Coloureds, Barreiro and colleagues found that this SNP is not polymorphic in other African countries. The relatively high prevalence of the -871 SNP in the South African Cape Coloureds cases and controls is due to their inherent admixture with European and Asian populations.There has been considerable recent interest in the role of CD209 -336G allele disrupts a potential SP-1-like transcription factor binding site and that this allele is associated with lower CD209 expression in vitro than the ancestral -336A variant M. tuberculosis binding and signaling events, and in an attempt to replicate the findings of Barreiro and colleagues in a larger population, we genotyped the CD209 -336A/G SNP in five tuberculosis case-control studies from four different sub-Saharan African countries.Sakuntabhai and colleagues also presented evidence that the M. tuberculosis positivity by culture, smear, or histology. HIV-1 testing was routinely performed and the percentage of HIV-1 positive individuals among the tuberculosis index cases and controls for the various studies are as follows: The Gambia (A), cases 7.5%, controls 5.2%; Republic of Guinea, cases 9.0%, controls 5.1%; Guinea Bissau, cases 23.8%, controls 16.2%; Malawi, cases 62.2%, controls 33.5%. The Gambia (B) cohort has 330 confirmed HIV-1 negative tuberculosis cases, with an additional 17 cases which were not tested for HIV-1 status. Controls for the various studies were defined as persons without a previous history of tuberculosis and were frequency matched for age, sex and area of residence. A number of clinical correlates of tuberculosis were recorded for the The Gambia (A), Republic of Guinea and Guinea Bissau studies including haemoptysis, duration of cough, degree of sputum positivity, and the presence of cavitation and number of zones affected on chest radiograph. Cavitory tuberculosis status and number of zones affected was determined by radiological analysis by board-certified radiologists.The tuberculosis case control studies from The Gambia (A), Republic of Guinea and Guinea Bissau Informed written consent was obtained from patients or their parents or guardians. Ethical approval was provided by the joint Gambian Government/MRC Ethical Committee, Ministry of Public Health  and National Ethics Committee, Ministry of Health, Conakry, Republique de Guinee. Human experimentation guidelines of these ministries were followed. Ethical approval for the Malawi study protocol was obtained from the National Health Sciences Research Committee of Malawi and the London School of Hygiene and Tropical Medicine Ethics Committee.CD209 -871A/G (rs735239) and -336A/G (rs4804803) SNP assays were designed using the SPECTRODESIGNER software (Sequenom). The primers for the CD209 -871A/G were ACGTTGGATGCTCTGTCTGGGTCCTTTTAC, ACGTTGGATGACAGCAATGAAAAAGCAAAG, GCAAAGTACTAGTACATTTAATAAC and for CD209 -336A/G were ACGTTGGATGTGTTACACCCCCTCCACTAG, ACGTTGGATGAAAGCAGGAAAGCCAGGAGG, CCCTCCACTAGGGCAAGGGT. The assays were in 384-well plates containing 10 ng of DNA in each well were amplified by a touch-down PCR using Titanium Taq polymerase (Clontech). The touch-down PCR cycling conditions were as follows: 95°C for 15 minutes; 94°C for 20 seconds; 65°C for 30 seconds; 72°C for 30 seconds; steps 2 to 4 repeated for 5 cycles; 94°C for 20 seconds; 58°C for 30 seconds; 72°C for 30 seconds; steps 5 to 7 repeated for 5 cycles; 94°C for 20 seconds; 53°C for 30 seconds; 72°C for 30 seconds; steps 8 to 10 repeated for 38 cycles; final extension at 72°C for 3 minutes.DNA extraction from blood was performed using Nucleon II kits . DNA concentrations were determined using the PicoGreen™ kit . Genotyping was performed utilizing the Sequenom™ system  which uses mass spectrometry (MALDI-TOF) to discriminate products by their absolute mass P value was significant then further analysis was carried out using a stepwise forward binary logistic regression correction to adjust for the potential confounding factors of HIV status, age, ethnicity, and sex. Mantel-Haenszel for the combined analysis was performed using SPSS by weighting cases by frequency and stratifying by population. The Gambian (B) study utilizes the Gambian (A) study controls for this Mantel-Haenszel analysis. The Breslow-Day and Tarone's tests for homogeneity of odds ratios were found to be non-significant indicating that the odds ratio for each study did not significantly deviate from the overall, combined odds ratio. All control genotype distributions were in Hardy-Weinberg equilibrium (P>0.20).Statistical analysis of genotype associations and logistic regression was performed using the program SPSS v14.0. The genotypic and allelic tests of association utilized Pearson's chi-square test with the respective degrees of freedom. All statistics are two-tailed. If the unadjusted CD209 -871A/G and -336A/G SNPs were initially genotyped in 329 tuberculosis cases and 327 control individuals from The Gambia (study group A) CD209 -871 SNP was found to be polymorphic with controls and cases both having a 2.3% allele frequency  consistent with the Barreiro result 2 = 7.79, P = 0.020)  (The = 0.020) Table 1.P = 0.006 (P = 0.32), suggesting that there was a good consistency between the combined odds ratio with the individual odds ratios for each study.Study groups from Republic of Guinea, Guinea-Bissau, and Malawi were then used in an attempt to replicate this finding  = 0.006 Table 2.P = 0.003  and  = 0.003 Table S1CD209 -336A/G SNP was further examined in the cases for association with clinical correlates of tuberculosis, with no significant difference between sex, HIV status, age, haemoptysis, number of zones affected on chest radiograph, duration of cough or degree of sputum positivity. However, a significant association was obtained between CD209 -336GG genotype status and lung cavitation in tuberculosis patients. Patients homozygous for the CD209 -336GG allele were associated with 2.4-fold protection against cavitory tuberculosis disease  mannosylated termini which closely resemble the high mannose N-linked oligosaccharides of newly produced glycoproteins in eukaryotic cells M. tuberculosis modulate the immune response. If so, DC-SIGN is a prime target for this virulence strategy.Maturation of DCs, defined by DC upregulation of co-stimulatory molecules such as CD80/CD86, can occur on exposure to TLR ligands. This process can however be inhibited by a variety of unknown mechanisms. One possible mechanism of inhibition of DC maturation is the binding of pattern recognition receptors to molecular patterns present on host cells M. tuberculosis may use the DC-SIGN receptor to subvert the immune response.Overall, our results suggest that lower levels of DC-SIGN on immune subsets may be protective against tuberculosis in the sub-Saharan Africa setting. The findings implicate the involvement of DC-SIGN in the pathogenesis of clinical tuberculosis in general, and severe, cavitory tuberculosis in particular. This has wider implications since it provides further evidence that Table S1CD209-336A/G SNP Allelic Association in Tuberculosis Case Control Studies Excluding All HIV-1 Positive Cases and Controls. The allelic P value for HIV-1 negative individuals enrolled in tuberculosis case control studies. The Gambia (A), Republic of Guinea and Guinea-Bissau (ref. 33), The Gambia (B) (ref. 34) and Malawi (ref. 35). *The Gambia (B) study utilizes The Gambia (A) controls. Significance levels of P<0.05 are indicated in bold.(0.06 MB DOC)Click here for additional data file.


Although several attempts have been made to control enzootic bovine leukosis (EBL) at the local level, a nationwide control program has not been implemented in Japan, except for passive surveillance. Effective control of EBL requires that the transmission routes of bovine leukemia virus (BLV) infection should be identified and intercepted based on scientific evidence. In this cross-sectional study, we examined the risk factors associated with within-herd transmission of BLV on infected dairy farms in Japan. Blood samples taken from 30 randomly selected adult cows at each of 139 dairy farms were tested by enzyme-linked immunosorbent assay (ELISA). Information on herd management was collected using a structured questionnaire.Infected farms were defined as those with more than one ELISA-positive animal and accounted for 110 (79.1%) of the 139 farms in the study. Completed questionnaires obtained from 90 of these 110 farms were used for statistical analysis. Seroprevalence, which was defined as the proportions of animals that tested positive out of all animals tested on the farm, was 17.1%, 48.1%, and 68.5% for the 25th, 50th, and 75th percentiles, respectively. A mixed logistic regression analysis implicated a loose housing system, dehorning, and a large number of horseflies in summer as risk factors  and feeding of colostrum to newborn calves from their dams as a protective factor  against within-farm transmission of BLV on infected farms.Control of EBL in infected dairy farms in Japan will be improved by focusing particularly on these risk and protective factors. Bovine leukemia virus (BLV), a retrovirus of the family Retroviridae, is the causative agent of enzootic bovine leucosis (EBL). Approximately 30% of cattle infected with BLV have persistent lymphocytosis, and 1-5% develop B-cell lymphosarcoma [osarcoma  and is iosarcoma . In contosarcoma . Althougosarcoma , thus faosarcoma .In Japan, EBL is listed as a notifiable disease, but no nationwide control programs have been established. According to animal health statistics, EBL was reported in 159 cattle at 157 farms in 2000 and in 838 cattle at 677 farms in 2007. These data suggest that EBL has been gradually spreading in Japan.BLV is present in the circulating peripheral blood lymphocytes of infected cattle, and horizontal transmission of the virus occurs via infected blood often as a result of unhygienic farm practices such as the use of plastic sleeves to perform rectal palpation and the same needle for vaccination of more than one animal -11 or unThese transmission routes should be considered when designing preventive measures against EBL at the farm level. In addition, it may be important to determine which transmission routes are the most important for each farm. It is therefore essential to identify the risk factors associated with BLV transmission in the production systems targeted for disease control. However, no such epidemiological approach for BLV infection has been attempted in Japan. Therefore, the objective of this study was to clarify herd management factors associated with within-herd transmission of BLV and to facilitate a more rational and efficient approach for controlling BLV transmission on dairy farms in Japan.Results of serological tests showed that 110 (79.1%) of 139 dairy farms were infected with BLV. Statistical analyses were then performed for 90 of these farms (81.8%), for which complete data were obtained from the questionnaires.Seroprevalence at infected farms at the 25th, 50th (median), and 75th percentiles was 17.1%, 48.1%, and 68.5%, respectively  Table . A looseThis is the first epidemiological study to assess herd management factors related to the seroprevalence of BLV on dairy farms in Japan. Based on our mixed logistic model with a random herd effect, the significant factors were found to be cattle housing conditions, the presence of horseflies in summer, dehorning practices, and colostrum feeding.With regard to housing conditions, a loose housing system was found to be positively associated with seroprevalence compared with a tied housing system (p = 0.03). A possible explanation may be that direct contact between infected and uninfected cattle is facilitated in loose housing systems such as free stalls and barns. Furthermore, the chances of indirect contact between uninfected and infected cattle may be relatively high in a loose housing system because cattle randomly move about the barn or paddock and are not always handled in the same manner.Dehorning is a practice in daily herd management that can reduce the risk of injury from horn-butting in farmers and in cattle during herd conflict. This practice was found to be positively associated with seroprevalence compared with farms without it (p < 0.01). A potential explanation for this result is the unhygienic procedure of dehorning, which is a risk factor for BLV transmission. It was previously reported that calves dehorned with contaminated dehorning apparatus were more likely to be infected than those that had not been dehorned -13. FarmWith regard to the presence of horseflies in summer, a response of "very high" was positively associated with seroprevalence compared with a response of "never to seldom" (p = 0.01); however, there was no significant difference between "sometimes or often" and "never to seldom" (p = 0.49). BLV transmission by hematophagous insects has been reported -16. In tIn the present study, colostrum feeding of calves from their dams resulted in a decrease in within-farm seroprevalence . Because colostrum contains BLV and its antibodies, ingestion of colostrum from infected dams reduces the risk of BLV infection during the weaning period in calves -25. Our Because this study had a cross-sectional design, it was difficult to elucidate definite relationships between seroprevalence and each risk or protective factor. However, in spite of this constraint, our results were consistent with previous studies and confirmed the important factors that should be controlled.Controlling BLV infection at Japanese dairy farms should focus on these factors. Future research is required to compare the influences of each factor responsible for within-herd transmission and to facilitate more rational prioritization of control measures.This cross-sectional study examined dairy farms located in 7 of the 47 prefectures in Japan. Each prefectural government sought participation from approximately 20 dairy farmers, and a total of 139 agreed to enroll. At each farm, the owner was asked for the blood sampling by the sample collectors who gave the minimum possible pain to the cattle, and only those who understood the study objectives were involved in this study. Each animal was firmly restrained before sampling to prevent unnecessary apprehension and pain to the animal and unexpected accidents to the sample collectors. Samples were then collected from the jugular or caudal vein. Blood samples were taken from 30 randomly selected adult cows  at each farm between June and December, 2007. Separated sera were transferred to the National Institute of Animal Health  and stored at -20°C for serological tests. Since this study was a field survey, no ethical approval for the study was required by the animal care and ethical committee of National Institute of Animal Health.Information regarding herd management was acquired at the time of blood sampling by local veterinary officers using a questionnaire prepared in advance [Additional file Each serum sample was serologically tested at NIAH using a commercial enzyme-linked immunosorbent assay (ELISA) kit . The procedures were performed according to the manufacturer's instructions. If the ELISA results were inconclusive, confirmation was sought using an agar gel precipitation test reported previously .In the present study, an infected farm was defined as a farm with more than one serologically positive cow. To analyze the risk factors associated with within-herd transmission of BLV, the subsequent modeling process targeted the seroprevalence of each infected farm as a response variable, which was defined as the proportion of cattle testing positive among all tested cattle on the farm.To obtain a general overview of the association between seroprevalence and each herd management factor, the normality of the distribution of seroprevalence at infected farms was evaluated by visual inspection of the histogram and by the Shapiro-Wilk test, followed by univariate analysis. The Mann-Whitney U-test was used to analyze variables with two levels, and the Kruskal-Wallis test to analyze all other variables. Variables with a p value < 0.15 in these tests were used for multivariate model building. A generalized mixed linear model was used to evaluate the risk factors that influenced seroprevalence; the response variable being the logit of seroprevalence and the herd as a random effect. The model is described as follows:p = number of positive animals/number of test animals,where p represents the seroprevalence accounting for sample size, α is the model intercept, χ is fixed effects with p < 0.15 in the univariate analyses, β is its coefficient, RH is the random herd effect, and e is the binomially distributed residual term. The best model was constructed by a stepwise approach, observing the change in AIC of each model. The final model was obtained with the minimum AIC and p < 0.05 for the remaining fixed effects.All statistical analyses were performed using R version 2.6.1 , and the glmmML package  was usedSK participated in the design of the study, performed all the data handling and statistical analyses, and drafted the manuscript. TT participated in the design of the study and helped to draft the manuscript. TY and YH investigated the data analyses performed by SK. KK and MK performed serological diagnoses and the related laboratory work. KM conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Questionnaire used for the sero-epidemiological survey of the enzootic bovine leukosis in Japan, 2007. The questions inquired about cattle housing conditions, cow replacement, provision of own grazing area or free range on the farm, the presence of horseflies in summer, dehorning, the use of plastic sleeves for rectal palpation, change of needles used for herd vaccination, colostrum feeding, and general data on herd demography.Click here for file


Facial characteristics are an important basis for judgements about gender, emotion, personality, motivational states and behavioural dispositions. Based on a recent finding of a sexual dimorphism in facial metrics that is independent of body size, we conducted three studies to examine the extent to which individual differences in the facial width-to-height ratio were associated with trait dominance (using a questionnaire) and aggression during a behavioural task and in a naturalistic setting . In study 1, men had a larger facial width-to-height ratio, higher scores of trait dominance, and were more reactively aggressive compared with women. Individual differences in the facial width-to-height ratio predicted reactive aggression in men, but not in women (predicted 15% of variance). In studies 2  and 3 , individual differences in the facial width-to-height ratio were positively related to aggressive behaviour as measured by the number of penalty minutes per game obtained over a season . Together, these findings suggest that the sexually dimorphic facial width-to-height ratio may be an ‘honest signal’ of propensity for aggressive behaviour. Most studies that have investigated facial characteristics and sexual selection have focused on what is perceived as attractive to an observer . Some evIn addition to providing information as to the personality and health of a target, some recent studies suggest that characteristics of the face may also provide cues as to the behavioural tendencies of the target. For example, women's judgements of the extent to which a man was interested in infants based on his face predicted his actual interest in infants  took part for course credit and a $5 honorarium. Eighty-two per cent of the participants self-identified as Caucasian, with the remaining 18 per cent representing a diversity of ethnicities. All procedures of the study were approved by the university's ethical review committee.ImageJ (NIH open-source software) was used to measure the distance between the lip and brow (height of upper face) and the left and right zygion (bizygomatic width) of the digitized images, based on Weston et al. .In study 1, photographs were taken with a Nikon D50 digital camera. n et al. . Inter-rr=0.98, p<0.001; facial width: r=0.94, p<0.001; width-to-height ratio: r=0.90, p<0.001).For study 2, photographs were obtained from 21 undergraduate male varsity hockey players  from the university's website  and measured as in study 1. All the pictures were facing forward; however, some individuals did not have a neutral expression (i.e. some were smiling). All measurements had good inter-rater reliability . Two pictures had to be excluded because the individuals were not facing forward and the tilt of the head would compromise the measurement of the facial width-to-height ratio. Further, we excluded photographs from goalkeepers (n=12) because these individuals are typically not in a position to obtain penalties (the measure of aggression). The final sample was n=112. Facial width-to-height ratios were calculated as in studies 1 and 2. All measurements had good inter-rater reliability .For study 3, photographs were obtained for every player who played on the Canadian teams of the NHL during the 2007–2008 season  whose pictures were available on the Entertainment and Sports Programming Network (ESPN) website  on a task that required them to select among three response options to earn points that would be exchangeable for money. Pressing response option no. 1 a hundred consecutive times would cause the point counter on the screen to enlarge, flash several times with positive signs around it, and increase the point counter total by one. It was explained to participants that the point counter might flash several times with negative signs around it, resulting in a one-point decrease in the point counter total. They were told that this meant that their partner  had stolen a point, and each stolen point would be added to the partner's counter. Participants could respond by continuing to select option no. 1 (point reward) or could switch to option no. 2 or 3. Pressing option no. 2 ten times would steal a point from their partner; however, participants were instructed that they were randomly assigned to the experimental condition whereby they, unlike their partner, would not keep any points stolen. Pressing option no. 3 ten times would protect their point counter against theft of points for a brief time. Thus, the dependent variables from the PSAP measure were option nos. 1 (reward earned), 2 (aggression) and 3 (protection). Selection of option no. 2 was considered reactive aggression because the participants did not increase reward, and in fact lost opportunity to increase reward, each time option no. 2 was selected.To measure aggressive behaviour, we used a modified version of the Point Subtraction Aggression Paradigm PSAP; . This meParticipants arrived in groups of two or four and first completed a demographic and trait dominance questionnaire. Next, participants were photographed while in a seated position and maintaining a neutral facial expression. Participants were escorted to separate rooms for the PSAP procedure. The PSAP took approximately 40 min to complete, after which they completed a brief questionnaire designed to assess whether they were aware of the deception used in the experiment. Responses confirmed that participants believed that they were playing against another person.The penalty minutes that each player accrued per number of games played during the 2007–2008 season (obtained from the Ontario University Athletics website for study 2 and from ESPN's website for study 3) were used as the measure of aggression. Penalties included behaviours such as slashing, cross-checking, high-sticking, boarding, elbowing, checking from behind, fighting and so on. These behaviours meet the classic definition of aggressive behaviour as any act that is intended to harm another individual, who, in turn, is motivated to avoid the behaviour .t-tests. Multiple linear regression analysis was used to examine the relationship between predictor variables  and aggressive behaviour as measured by the PSAP. The main assumptions underlying linear regression  were examined and were all met. Also, Pearson's correlation coefficients were computed to examine the bivariate association between the facial width-to-height ratio and aggressive behaviour in varsity and professional ice hockey players. All analyses conducted were two-tailed and the level of significance was set at p<0.05.Gender differences in trait dominance and facial width-to-height ratio were examined using multivariate analysis of variance (MANOVA), with follow-up t-tests. There was a main effect of gender : men had a greater facial ratio ; scored higher on trait dominance ; and were more aggressive than women . Men and women did not differ in reward  or protection  responses . Furthermore, the face ratio by trait dominance interaction was not significant . For women, face ratio and trait dominance did not predict aggressive behaviour , nor did the interaction .Separate regression analyses for men and women were computed with trait dominance and face ratio as predictors of aggressive behaviour. For men, face ratio predicted 15 per cent of unique variance in aggressive behaviour  in NHL hockey players. Separate correlation coefficients were also computed for each individual team  in the upper face was evident in the photographs of an unselected sample of undergraduates, and this ratio predicted aggression in men assessed in a validated laboratory behavioural task and in a naturalistic setting . The data obtained here suggest that for men variation in the width-to-height ratio from neutral faces may be an honest signal of propensity for aggressive behaviour. Clearly, an angry facial expression is a direct way to communicate one's emotional state and behavioural intent. However, it remains possible that subtle cues from a neutral face may have been selected because they provide information as to an individual's behavioural dispositions. Notably, angry facial expressions consist of lowering the brow and raising the upper lip, a pattern of muscle activity that increases the facial width-to-height ratio. From an evolutionary perspective, these findings suggest that selection pressures may have shaped the perceptual system to be especially attuned to cues of threat and/or aggression. However, it will be important to examine the extent to which people are sensitive to individual differences in facial width-to-height ratio and whether this facial metric is used to guide behaviour. Another possibility is that the relationship observed between the facial metric and aggressive behaviour was partly influenced by the posture of the head in the photographs . It is also possible that stronger relationships would have been observed between the facial ratio and aggressive behaviour if direct measurements of the face were made instead of using photographs and had we been able to control facial expression in the faces of the hockey players.There was no relationship between trait dominance and aggressive behaviour in our sample, although individual differences in trait dominance were associated with self-report measures of trait aggression in both men and women in other studies . The facThere is much research literature addressing the role of the face in social interactions, and there is some literature indicating that faces can be used to gauge certain personality traits above chance e.g. . For exaThe novel finding of the present study is that individual differences in facial characteristics predict behaviour: Variability in a sexually dimorphic facial metric in men, which is independent of body size, predicted aggressive behaviour in a laboratory setting and in male hockey players. The relationship between within-sex variation in facial width-to-height and aggressive behaviour might reflect a common relationship to a third variable, such as organizational effects of testosterone as part of sexual differentiation in adolescence, which influences both the development of the physique and the nervous system (reviewed in


S-Adenosylmethionine (AdoMet) is a key biochemical co-factor whose proximate metabolites include methylated macromolecules , methylated small molecules , polyamines , ethylene, and N-acyl-homoserine lactones. Marine organisms produce numerous AdoMet metabolites whose novel structures can be regarded as lead compounds for anti-infective drug design. S-Adenosyl-l-methionine  is a biochemical intermediate which serves as precursor to a vast compendium of bioactive metabolites in all living organisms. The remarkable structural diversity that characterizes known metabolites of AdoMet is a key component of AdoMet’s ability to meet specialized needs in highly different microenvironments. Many AdoMet metabolites and AdoMet-utilizing pathways, as yet unknown, remain to be discovered and characterized. Marine environments provide an attractive and abundant reservoir to search for new classes of molecules that are structurally derived from AdoMet. This is corroborated by a recent novel finding that the chloro-substituent of salinosporamide A, a marine product now undergoing Phase I clinical trials for cancer treatment, is enzymatically derived from AdoMet n–NH3+ (n = 4–14), gives an idea of their extraordinary length . . D. pulcUM-5, UM-7); most integrate methyl substituents within unusual structural scaffolds (UM1 - UM7). From a shared chemical and biological perspective, the most extraordinary AdoMet-derived marine metabolite is, arguably, the low molecular weight boronate diester, AI-2, which acts as an interspecies bacterial signal molecule. Inclusion of salinosporamide A (H-1) in this small group of uncommon molecules reflects its novelty as the first marine metabolite known to be halogenated via an AdoMet-dependent halogenase  [i.e., spermidine) tail. Additional motuporamines whose structures differ by ring size and by the presence and positions of unsaturated bonds and/or methyl substituents, have since been isolated [UM-5 (composite structure) each contain a methyl group of unassigned position. AdoMet independently serves as polyamine precursor and methyl donor for the structural components of UM-5. MPAs have been shown to inhibit angiogenesis and tumor cell invasiveness [Motuporamines (MPAs) were first isolated from the tropical marine sponge patrick) . Their hisolated . The MPAsiveness ,159. To siveness . MPA-C isiveness –161.UM-6) was first isolated from the marine sponge Hippospongia metachromia in 1979 [S-adenosylhomocysteine hydrolase (AHH), a key enzyme in methylation pathways [S-adenosylhomocysteine, the potent product inhibitor of all AdoMet dependent methyltransferases, suggesting that AdoHcy levels are elevated in ilimaquinone-producing marine organisms. Since AdoHcy also serves as proximate precursor of the QS signal molecule, AI-2, “ilimaquinone-producers” in marine habitats may have an enhanced capability to synthesize AI-2 in response to environmental stresses.The sesquiterpene quinone, ilimaquinone  is a spermidine-dihydroxycholestane-sulfate conjugate that was initially isolated from stomach extracts of the dogfish shark Squalus acanthias [Escherichia coli, Staphylococcus aureus, Candida albicans and P. aeruginosa [The aminosterol, squalamine  were found in the marine algicolous fungus, Monodictys putredinis by Konig and colleagues who had previously isolated a group of monomeric xanthones from the same organism [UM-1 and UM-3 contain two unusual, non identical xanthone subunits and three chiral methyl substituents. UM-1 and UM-3 differ only by the site of their linkage [Monodictychromones A and B  cells and its potent antifungal activity against C. albicans [L. majuscula [C-methyl-transferase signature motif, previously identified in the biosynthetic gene clusters of curacin A and jamaicamide from other L. majuscula strains, was found to be present in the hectochlorin biosynthetic gene cluster [Hectochlorin,  leachii ,172. UM-albicans . Gerwickajuscula . During  cluster ,173–175.UM-4 was isolated from the marine ascadian, Atriolum robustum [UM-4 is derived from 3-(4-hydroxyphenyl-2-methoxyacrylic acid (HMA); the 3’-ribose ester substituent, from urocanic acid (UCA). UM-4 was consistently inferior to MTA in receptor-specific binding assays for the A1, A2A, A2B, and A3 adenosine receptors with binding constants in the micromolar range for the A1, A2A, and A3 receptors [In silico docking into homology models of the A1 and A3 receptors demonstrated that UM-4 readily docks into the adenosine binding sites of both receptors, likely due to the inherent flexibility of its long chain ester substituents [The unusual, asymmetric diester of MTA, robustum . The 2’-eceptors . In silitituents .N-acylhomoserine lactone biosyntheses. Thus, the quantities of MTA produced by marine organisms are not insignificant. MTA, like S-adenosylhomocysteine, is a proximate AdoMet metabolite that is usually recycled to methionine [UM-4 might be an MTA prodrug that is slowly hydrolyzed by marine esterases [UM-4 may also be a depot form of UCA and/or HMA. UCA’s biological properties support this possibility [trans-isomer of UCA is biosynthesized from histidine in the outer epidermal layers of marine organisms. Subsequent exposure to UV radiation converts trans-UCA to the bioactive, immunosuppressive cis-isomer. The mechanisms associated with these biological properties, although widely studied, are not well understood [UM-4 function may be forthcoming in the future. From a structural perspective alone, UM-4 is, perhaps, the most bizarre AdoMet metabolite to be extracted from the oceans’ depths.MTA is the enzymatic by product of three major AdoMet-dependent pathways: polyamine, ethylene and thionine ,177. Twothionine –181. Onesterases . Expandisibility . The traderstood . FurtherAI-2. We regard QS networks in global marine habitats as an unusually fertile source of potential, anti-infective AdoMet-derived molecules. The different types of QS-related molecules that can be used as templates for drug design include AHLs, their corresponding tetramic and tetronic acids, and halogenated furanones. Equally important drug templates will emerge from the vast array of defensive chemicals produced by marine organisms to combat the coordinated assaults of pathogenic, quorum sensing bacteria.Marine environments continue to serve as a source of newly identified, structurally novel metabolites of AdoMet. The discovery of quorum sensing activities in gram-negative marine bacteria and the ensuing studies of QS phenomena in marine environments have been instrumental to our current understanding of the complexities of this previously unrecognized bacterial signaling network. These studies provided the first evidence of two new types of AdoMet-derived metabolites, AHLs and


Assessment of coronary heart disease (CHD) risk is typically based on a weighted combination of standard risk factors. We sought to determine the extent to which a lipidomic approach based on red blood cell fatty acid (RBC-FA) profiles could discriminate acute coronary syndrome (ACS) cases from controls, and to compare RBC-FA discrimination with that based on standard risk factors.rds derivation set and validated on the remaining 1/3rd. The area under receiver operating characteristic (ROC) curves (c-statistics), misclassification rates, and model calibrations were used to evaluate the individual and combined models. The FA discriminated cases from controls better than the SRF  and the FA profile added significantly to the standard model . Hosmer-Lemeshow calibration was poor for the FA model alone (p = 0.01), but acceptable for both the SRF (p = 0.30) and combined models (p = 0.22). Misclassification rates were 23%, 29% and 20% for FA, the SRF, and the combined models, respectively.RBC-FA profiles were measured in 668 ACS cases and 680 age-, race- and gender-matched controls. Multivariable logistic regression models based on FA profiles (FA) and standard risk factors (SRF) were developed on a random 2/3RBC-FA profiles contribute significantly to the discrimination of ACS cases, especially when combined with standard risk factors. The utility of FA patterns in risk prediction warrants further investigation. Several recent risk prediction algorithms have been proposed, such as those from the Prospective Cardiovascular Munster (PROCAM) study e.g., plasma triglycerides and non-esterified FAs) are sensitive indicators of acute changes in dietary habits and in hepatic and adipocyte function. Adipose tissue FA composition is a long-term (months to years) reflection of dietary habits, whereas membrane FA composition  provides a more intermediate estimate (weeks). We Fatty acids (FAs) are powerful modulators of cell membrane receptors and affect signal transduction, gene transcription, and eicosanoid metabolism. They are present in many tissue compartments, including plasma , adipose tissue and cell membranes. Some of these compartments  excluded symptomatic ischemic heart disease or confirmed an alternative explanation for their presentation . Three physicians reviewed the charts of all patients for whom diagnostic uncertainty remained and attained consensus on the final diagnosis. With this approach, a total of 1,661 patients were included in this registry and enrolled as described in All consecutive patients admitted to two hospitals associated with the University of Missouri-Kansas City School of Medicine were prospectively screened for an ACS between March 2001and June 2004 . The subPatients having blood drawn for routine clinical testing were recruited from blood drawing centers at Saint Luke's Hospital (where 88% of the cases were derived) between March 2004 to March 2005 as outlined in ACS patients completed a baseline interview within 24 to 72 hours of admission, and detailed information on patient presentation, race, comorbidities, and treatments were obtained by chart abstraction. Standard risk factors included age, sex, total-C, HDL-C, a history of diagnosed hypertension and diabetes, and smoking status RBC-FA composition was measured as previously described 768 patients diagnosed with ACS were matched one-to-one with controls on the basis of age (5-yr windows), gender, and race (Caucasian vs. non-Caucasian). 228 were excluded due to incomplete information on HDL-C, total-C, self-reported hypertension (HTN), self-reported diabetes mellitus (DM), age, gender, or current smoking status . Two-thiStepwise unconditional multivariable logistic regression was used to develop prediction models with p = 0.01 used to enter and remain in the model. One model was developed using RBC-FAs(FA), another with the 7 standard risk factors (SRF), and another using the FAs selected in the FA model combined with the standard risk factors (SRF+FA). Natural log transformations were used for HDL-C and total-C to improve normality. Robust, non-parametric 95% confidence intervals (CI) of the parameter estimates were obtained using bootstrapping method with 10,000 replicates from the training data set for both FA models. In addition to using the stepwise selected FAs, four pre-specified FA metrics were also tested for their ability to add to the SRF model: the omega-3 index (eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA)) iβ, i = 0 to p) multiplied by each subject's FA levels  or by the standard risk factors  as follows:For each MLR model a single continuous variable, a risk score, was calculated (equation 1) as the linear combination of the parameter estimates  was determined for each model and the difference compared to the SRF alone. The standard error (SE) for the c-statistic was computed as described by Hanley and McNeil Due to matching on age, sex and race, there were no differences in these attributes . As expeOdds ratios for the 7 standard risk factors alone, FAs alone and the combination are presented in Using the standard risk factors, and the parameter estimates for blood cell FAs, the ability of MLR models to discriminate cases from controls were compared, both alone and in combination . The FA i.e., p>0.05) were those that included the SFR, either alone or when combined with FAs  trans-oleic (or elaidic) acid and eicosadienoic acid. Associations of increased intakes and/or in vivo levels of industrially-produced trans FAs with CHD risk are well established The relations between risk for CHD and RBC levels of the individual FAs included in the model generally fit well with previous observations: inverse associations with omega-3 and omega-6 FAs and direct associations with trans FAs. Of the ten FAs included in the model, increasing levels of eight were inversely associated with odds for ACS case status. These included the FAs of both the omega-3 and omega-6 series, the monounsaturated FA palmitoleic acid, and the saturated FA, stearic acid. These levels reflect a mixture of diet and metabolism since the essential FAs (omega-3 and omega-6) are strongly affected by diet whereas palmitic and stearic, which can be synthesized de novo, reflect metabolic processes. Direct associations were found only with A potential weakness of case control studies is that the exposure of interest could be altered by the clinical event it is intended to predict. The use of RBC-FAs is attractive in this regard as levels remain stable for at least 4–6 weeks and are not appreciably affected by CHD events in primate models e.g. discrimination, positive likelihood ratios, misclassification rates, etc.), FA profiles performed well and show promise as a new risk marker for CHD. Other proposed criteria such as the potential to reveal novel disease mechanisms are also satisfied since FAs affect a variety of metabolic and regulatory pathways linked to CHD . These may be in part mediated by alterations in the activity of membrane-associated receptorse.g., inflammatory) Based on those criteria set forth by Vasan Strengths of this study include a large sample size, a rigorously-defined ACS population, detailed FA analysis, the use of stable biomarker of tissue FA status, use of separate derivation and validation data sets, and a comprehensive examination of several metrics of model utility. Potential limitations should also be considered. It is possible that the outpatients who agreed to participate were not truly representative of the case population. Nevertheless, the fact that the standard risk factors predicted case-control status very comparably to other prospective studies suggests that control selection bias was unlikely to have materially affected our results. Hypertension and diabetes were self-reported in controls. Since these conditions are often under-diagnosed some controls may have incorrectly reported normal blood pressure or glycemia. Under-reporting by controls would help the SRF, but not the FA, model discrimination. In addition, FA profiles have been reported to predict the presence of vascular disease independently of hypertension and diabetes In conclusion, an RBC-FA lipidomic approach added substantially to standard risk factors for prediction of ACS. These findings suggest that substantial, previously unrecognized biological information may reside in membrane FA patterns. A deeper appreciation of the mechanisms by which FAs modulate cellular metabolism could lead to a new understanding of causes and pathways of CHD as well as to improved clinical risk prediction and treatment strategies.