In this article, we review the contributions of genetic polymorphisms to major individual variations in drug pharmacotherapy, focusing specifically on the pharmacogenomics of phase-I drug metabolizing enzymes and transporters. Substantial frequency differences in key variants of drug metabolizing enzymes and transporters, as well as their possible functional consequences, have also been discussed across geographic regions. The current effort illustrates the common presence of variability in drug responses among individuals and across all geographic regions. This information will aid health-care professionals in prescribing the most appropriate treatment aimed at achieving the best possible beneficial outcomes while avoiding unwanted effects for a particular patient.The interindividual genetic variations in drug metabolizing enzymes and transporters influence the efficacy and toxicity of numerous drugs. As a fundamental element in  Pharmacogenomics is the understanding of how individuals differ in their response to drug therapy and the mechanisms underlying variable drug response by utilizing genomics, proteomics, transcriptomics, and metabolomics based knowledge. Every individual has a different genetic makeup, which influences the risk of developing diseases as well as responses to drugs and environmental factors Drug response of individual patients is primarily determined by the pharmacokinetic and pharmacodynamic properties of prescribed drugs, which is directly or indirectly affected by polymorphisms in drug metabolizing enzymes and transporters. Different populations have varied allele frequencies in genes of both drug metabolizing enzymes and transporters. For precision medicine, the molecular and clinical information is integrated in order to understand the biological basis of disease and develop medications with better outcomes for patients It is well known that individuals vary significantly in their clinical responses to administered drugs and the outcomes, which can be inherited or acquired, are always patient-specific Different patients can respond differently to the same drug and dose. Sometimes, the effective drug dose for a particular patient may prove lethal to or result in therapeutic failure in others , leading to serious adverse effects or no effects at all. Continuous drug monitoring is recommended when prescribing drugs with known serious side effects and narrow therapeutic indexes to avoid unexpected and undesirable outcomes The recommended daily maximum dose of simvastatin for the management of blood cholesterol levels is 40 mg. In a cohort study of 156 patients, 95% of them showed reduced levels of low-density lipoprotein (LDL) cholesterol, whereas the remaining 5% exhibited no reduction was observed for the remaining 5% of the patients, even at doses as high as 160 mg/day of simvastatin Individual-specific response to medication can be attributed to many multifold and complex factors including the unique genetic makeup . These genetic factors, as well as physiological conditions ; environmental influences ; and pathological factors  can work alone or in combination to influence drug responses Disease conditions of individuals used to be diagnosed based on signs and symptoms, which may be indicative of several different diseases or somewhat related to the family history. In the past, clinicians could only attempt to cure or treat disease upon its onset Genetic polymorphisms may influence a drug’s effect by altering its pharmacokinetics, pharmacodynamics, or both , which aMutations in the gene coding regions could cause alterations in gene expression or protein structure, leading to variations in protein quantity and quality. In the case of enzymes, such mutations affect both the protein function and the rate and kinetic constants. Changes in drug-receptor or drug–enzyme interactions due to structural alterations of enzymes or receptors could also result in variations in drug responses Twin studies have provided evidence supporting the contribution of genetic factors to individuals’ varied drug responses. For instance, in the late 1950s, it was found that dizygotic twins exhibited more metabolic variability than did monozygotic twins for isoniazid metabolism CYP2D6. The highly polymorphic CYP2D6 gene is located on the chromosome 22q13.1, consisting of nine exons and eight introns (GenBank accession No. NM 000106.5) CYP2D6 genetic variants have been described  to date, resulting from point mutations, duplication, insertions or deletions of single or multiple nucleotides, and even whole-gene deletion. Individuals carrying different CYP2D6 allelic variants have been classified as poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs) according to the metabolic nature of the drugs and degree of involvement in drug metabolism of these variants Cytochrome P450 (CYP), which represents a large and diverse group of heme-containing enzyme superfamily, is involved in oxidative metabolism of structurally-diverse molecules like drugs, chemical, and fatty acids. The genetic polymorphism in the genes encoding CYP members was firstly reported for CYP2D6 gene significantly affects phenotypic drug responses. Up to a 10-fold difference in the required dose was observed in order to achieve the same plasma concentration in different individuals in vivo CYP2D6 phenotyping. According to the probe substrate metabolic capabilities among the sampled individuals in a population, patients can be categorized into the following four phenotypic groups: poor, intermediate, extensive, and ultra-rapid metabolizers , respectively CYP2D6 allelic variants . These allelic variants lead to inactive CYP2D6 enzymes CYP2D6*3 (3.3% in Sardinians), CYP2D6*4 (23%–33% in Polish and Faroese populations), CYP2D6*5 (5.9%–6.2% in Spaniards and African Americans), and CYP2D6*6  were also calculated in diverse populations  in the Faroese population CYP2D6*4/*4 genotype than in patients with one or no CYP2D6*4 alleles (20%) CYP2D6, codeine is not recommended as an analgesic because of the minimal enzymatic conversion from codeine to morphine. Conversely, a higher risk of morphine toxicity may occur in patients with the UM phenotype owing to the rapid conversion of codeine to morphine. The situation would be more devastating in UMs who are lactating mothers because the normal codeine dose can translate into fatal morphine concentrations into the breast milk CYP2D6 allelic variants *10, *17, and *41 exhibit normal catalytic activity but are sometimes associated with intermediate to low metabolic activities CYP2D6*10 allele has been found more common than other alleles  and it causes a greatly decreased (but not deficient) enzyme activity The prodrug tamoxifen is a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer patients CYP2C9 gene . Among them, CYP2C9*2 (R144C) and CYP2C9*3 (I359L) are the most common variants associated with highly-reduced CYP2C9 enzymatic activities in comparison with the wild-type allele (CYP2C9*1) CYP2C9 is another important member of the CYP superfamily. The gene coding for CYP2C9 is located on chromosome 10q24.2, and spans more than 55 kb in length. CYP2C9 constitutes approximately 18% of the total CYP protein in the human liver microsomes CYP2C9*2 variant results in a markedly decreased enzyme activity due to higher km value and lower intrinsic clearance of drugs like S-warfarin CYP2C9*2 allelic variant has been reported with up to 25% allelic frequencies in the Iranian population CYP2C9*1/*2, homozygous CYP2C9*2 or CYP2C9*3 carriers were lower (0.1%–1%) in the Chinese and Japanese populations compared with those in Caucasians and Iranians. Caucasians have approximately 1% CYP2C9*2 and 0.4% CYP2C9*3 homozygotes, respectively *1*2 or the *1*3 genotype, while more than 2% have the *2*2, *2*3, and *3*3 genotypes CYP2C9*2 and CYP2C9*3 is greater than that in the other studied populations CYP2C9*1 allelic frequency variant of >90%, whereas allelic CYP2C9*2 variant was not detected in the Korean, Chinese, and Vietnamese populations but occurs 1% in the Japanese. Furthermore, no individuals from the South African and Zimbabwean populations have been reported to carry the CYP2C9*2 allele -5-phenylhydantoin (phenytoin metabolite) than those with the CYP2C9*1/*1 genotype. Multiple studies have also shown that the CYP2C9*3/*3 genotype is associated with reduced metabolisms and altered pharmacokinetic properties of substrates such as phenytoin, warfarin, losartan, and tolbutamide Both CYP2C9 and CYP2C19 are involved in microsomal hydroxylation of phenytoin to its R and S enantiomers CYP2C19, which is located on the chromosome 10q24 encodes another CYP family member. CYP2C19 can metabolize numerous routinely-administered drugs such as anxiolytics (diazepam), proton pump inhibitors (omeprazole), anticonvulsants (S-mephenytoin), and antimalarial biguanides CYP2C19 variants and approximately 2000 SNPs have been identified , with continuous increase in SNP numbers reported. Among them, CYP2C19*2 and CYP2C19*3 are the most common variants that have been studied extensively. Both of them are null variants and patients carrying these variants are therefore categorized as PMs. CYP2C19*2 is the most common allelic variant caused by a single nucleotide alteration in exon 5 (G > A), resulting in an abnormal splicing site and conferring reduced enzymatic activities of CYP2C19 The polymorphic CYP2C19*2 variant is found at a high allelic frequency (30%) in South Indians, but occurs with the lowest frequency (2.9%) in the Faroeses. In contrast, CYP2C19*3 is found at higher allelic frequencies in the Japanese (approximately 13%) but lower (0) among the Italians, South Africans, Greeks, European-Americans, and other populations  and most of these variants are responsible for varied enzyme activities ranging from modest to highly reduced catalytic efficiencies among the affected individuals CYP3A4 gene (CYP3A4*2 and CYP3A4*3) were observed in Caucasian whereas high frequencies of allelic variant CYP3A4**18 were observed in Chinese people  is associated with impaired ability to accept electrons from NADPH POR*13 (Q153R) variant leads to severely-impaired steroid biosynthesis in Antley–Bixler skeletal malformation syndrome (ABS) CYPOR genes have been described .CYP oxidoreductase (CYPOR) is the catalytic partner and compulsory element to all CYP-mediated metabolisms. The interaction between the CYP and CYPOR is essential for the metabolic activities of CYPs A drug could produce a beneficial or toxic effect in a particular patient. The nature and extent of the resulting effect is largely dependent on the absorption, distribution, and excretion rates of the drug. Drug transporters primarily control the movement of all drugs and their active or inactive metabolites into or out of cells. Therefore, polymorphisms of drug transporter genes can modify the absorption, distribution, and excretion rates, and ultimately safety and efficacy of the administered drugs. The ABC and solute-carrier (SLC) transporters are two superfamilies of transport proteins are ubiquitous membrane-bound transport proteins that are involved in the absorption, distribution, and elimination of drugs ABCA to ABCG (http://nutrigene.4t.com/humanabc.htm). The impact of some important polymorphisms on the drug transport activities of various ABC transporters is summarized in http://www.bioparadigms.org/slc/menu.asp). Among them, members of the organic anion transporter (OAT), organic anion transporting polypeptides (OATP), and organic cation transporter (OCT) subfamilies are of particular significance in drug disposition ABC transporters often transport drugs and other substances against the concentration gradient using ATP as an energy source ABCB1 gene, also known as the multidrug resistance 1 (MDR1), encodes a P-glycoprotein (Pgp), which is involved in the cellular efflux of numerous chemotherapeutic agents, physiological metabolites, and carcinogens ABCB1 is highly polymorphic with allelic variants found in varied frequencies in different populations  in Asians but low (34%–42%) in Caucasians. The substrate-dependent effects of Pgp on pharmacokinetic and pharmacodynamics properties remain obscure due to controversial studies on digoxin disposition. For example, for patients with a mutant allele (3435C > T) that were administered a single oral dose of digoxin, Sakaeda et al. ABCC2 and G671V variant with 28% allelic frequency in Caucasians have been reported ABCC2  that has been extensively studied for its role in drug resistance development in cancer and human immunodeficiency virus type 1 (HIV-1)-infected patients As the important ABC members, both ABCC1 and ABCC2 are involved in the transport and excretion of several chemotherapeutic agents, toxicants, and organic anion molecules ABCG2 gene have been identified Similar to ABCC1, ABCG2 was first discovered in multidrug-resistant cell lines ABC transporters would be essential to enhance the understanding of the genetic impact on pharmacotherapy.The C421A is widely distributed in many ethnicities with frequencies of 27%–35% in Asians, 9%–14% in Caucasians, and 1%–5% in Africans . GefitinOATPC*5 and OATPC*9 allelic variants are associated with a reduced uptake of OATPC substrates such as estrone sulfate and estradiol-17-β-D-glucuronide OATPC*5 allele OATPs are a large family of membrane-bound influx transporters that are responsible for the cellular uptake of a wide range of endogenous and exogenous substances including bile salts, hormones, and clinically administered drugs such as antibiotics, cardiac glycosides, and anticancer agents SLCO1B1 and is essential for the hepatic uptake of the simvastatin active metabolite, simvastatin acid SLCO1B1 gene with their allelic frequencies and functional consequences in Asian, African and Caucasian have been discussed in SLCO1B1 is associated with reduced OATP1B1 activity, which is responsible for the higher blood concentrations of simvastatin acid, as well as the consequently increased toxicity and reduced efficacy of simvastatin OATP1B1*15 was associated with increased plasma concentrations of pravastatin and 7-ethyl-10-hydroxycamptothecin (irinotecan active metabolite), whereas OATP1B1*17 variant is linked with an increased cholesterol synthesis mediated by pravastatin On the other hand, OATP1B1, OATP2B1, and OATP1B3 are mainly expressed on the hepatocyte sinusoidal membrane, which can facilitate the hepatic drug uptake SLC22A family and in humans, which are present in the basolateral cell membrane of the renal proximal tubule OCT1, OCT2, and OCT3, have been identified in humans OCT2 is highly expressed in the kidneys. OCTs mediate the cellular uptake of a wide range of structurally-different organic cations including clinically-administered drugs such as metformin and procainamide OCT2 270S variant has been associated with low activity while the 270A variant induces high activity of OCT2 OCTs are proteins encoded by the in vivo by CYP1A2, CYP2B6 and CYP2C19 for its anti-platelet activity CYP2C19, CYP1A2, 2B6*6, and CYP3A5*3 were found to be associated with the varied degree of drug–drug interactions for clopidogrel, due to its highly-complex pharmacokinetics and variable drug response as compare to other anti-platelet drugs Effects of one drug are modified by other concomitantly administered drugs due to drug–drug interactions, which may be attributed to the altered pharmacokinetic or pharmacodynamic properties of one drug induced by the coadministered drug. The polymorphisms in drug metabolizing and transporter genes are an important risk factor of drug–drug interactions and varied interindividual drug responses Mutations in the drug transporter genes also contribute to drug–drug interactions and adverse drug reactions. HMGCR inhibitors such as atorvastatin, rosuvastatin, and pravastatin are actively transported by OATP1B1 and ABCG2 The genetic variations of CYPs and transporters have been described in diverse populations. In this review, we review the different allelic variants that are responsible for altered drug activities in diverse geographic regions. Some populations exhibited extremely high frequencies of allele variants that are associated with several significant clinical consequences. Taking advantage of pharmacogenomics, researchers have assessed some specific genetic variants responsible for the particular drug responses of individuals.Whole genome SNP profiling, haplotyping, multigene analysis, and gene expression studies by biochip or microarrays are all in place to study drug responses of individuals, which would aid in drug discovery, development, and individualized treatments. Given the common variability in drug responses among patients, the optimization of dosage regimen at the individual level is not an easy task. Comprehensive appreciation of the contributing factors associated with interindividual and interethnic differences in medication responses is a must for the development of precision medicine, and help health-care professionals in recommending the proper treatment with the best possible beneficial outcomes while preventing unwanted drug effects in the particular patients. The development of clinical practice strategies based on accurate genotype testing will facilitate the enhanced understanding of altered drug responses and drug–drug interactions. Furthermore, the development of more reliable biomarkers based on polymorphisms in genes responsible for the adverse events will hopefully create strategies for administering drugs based on the genotype and phenotype of patients, to minimize unwanted drug reactions.The authors have declared no competing interests.


Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection. As exemplified by the highly successful, yellow fever virus (YFV) 17D vaccine developed in the early 1930’s and more recently Japanese encephalitis virus (JEV), vaccination is a feasible strategy for preventing and controlling mosquito-borne flavivirus infections4. In other flavivirus infections where neutralizing antibody titers >10 protect6, similar titers in DENV infection are complicated by the existence of four heterotypic serotypes and heterotypic cross neutralization. While the presence of neutralizing antibodies has been long considered a correlate of protection for flaviviruses, recent data from dengue vaccine trials prove that the presence of antibodies that neutralize DENVs in cell culture do not necessarily confer protection7. New assays and reagents are needed to characterize human antibody responses to dengue virus infections and vaccination and to identify requirements for protection beyond mere neutralizing antibodies.The four serotypes of dengue viruses (DENV1-4) are estimated to cause around 100 million cases of dengue fever or dengue hemorrhagic fever each year8. Additionally, people experiencing a secondary DENV infection with a new serotype face a greater risk of progression to severe DHF (Dengue hemorrhagic fever) and DSS (Dengue shock syndrome). Severe disease is a result of immunopathology, likely mediated by aberrant T cells9 and non-neutralizing antibodies induced by the first infection. Moreover, pre-existing antibodies may increase viral load in secondary infections through the process of antibody-dependent enhancement (ADE) of infection of Fc receptor bearing cells10. As such, a successful DENV vaccine should ideally elicit robust anti-DENV protective immunity against all 4 serotypes to prevent subsequent dengue illness, especially severe illness that can result from ADE infection. To date this has been a difficult challenge to overcome, especially in those seronegative at the time of vaccination.A major challenge to DENV vaccine development is the existence of 4 serotypes and the need for vaccines to confer protection against all 4 serotypes. With an approximate 60% amino acid divergence between the E proteins of the 4 serotypes, immunity to one serotype usually does not confer long-lasting cross-protective immunity to the other serotypes11. Structural studies with human monoclonal antibodies (hmAbs) isolated from dengue patients have provided high resolution maps of epitopes on the viral surface. These studies have also led to the development of new tools and reagents to identify correlates and mechanisms of protective immunity following natural infection or vaccination16.It has been known since the early 1980s through passive transfer experiments that antibodies targeting the E glycoprotein can protect from lethal flavivirus challenge14. Our group and others have characterized DENV-specific antibodies in people exposed to natural and experimental infections or live attenuated vaccines18. Both serotype-specific and cross-reactive strongly neutralizing mAbs have been isolated from the memory B cells of donors with a history of primary and secondary DENV infections20. The location of mAb epitopes on the envelope glycoprotein often, but not always, differs between serotypes and commonly the paratope recognizes a complex quaternary epitope that is present only on fully assembled and intact virions. While the structural footprints of several human neutralizing mAbs on the viral envelope have been determined through the use of cryo-electron microscopy, the resolution of these structural studies can only predict the relative contribution of different fractions of the quaternary epitope to monoclonal and/or polyclonal antibody neutralization phenotypes. Recently, DENV3-specific potently neutralizing antibody, 5J717, was identified as using a complex quaternary epitope to neutralize DENV3 in a manner previously hypothesized for West Nile virus (WNV)22. Importantly, the 5J7 monoclonal antibody footprint interacts with distinct residues encoded in 3 adjacent monomers, A, B’ and B  system for a clinical isolate of DENV4 isolated in Sri Lanka in 1992 and belonging to genogroup I26. To circumvent instability and toxicity during bacterial plasmid amplification in E coli, the DENV4 genome was subcloned into 4 distinct fragments nts Fig. . Naturalnts Fig. . DENV4 r17. Using cryo-electron microscopy, the foot print of hmAb 5J7 was recently mapped to a quaternary epitope that spans 3 adjacent E protein molecules on the viral envelope15  hinge region was superimposed upon the DENV3 E protein crystal structure to approximate the footprint of an antibody epitope binding region. Following amino acid (AA) and nucleotide alignments between DENV3 and DENV4, interserotypic variant AA were identified between DENV3 and DENV4  5J7 is a strongly neutralizing serotype specific antibody isolated from a patient who recovered from a DENV3 infectione15 Fig. . We recee15 Fig.  into DEN B’ Fig.  for hmAbNV4 Fig. . Three dNV4 Fig. . NucleotNV4 Fig. . DENV4/3All three of the rDENV4/3 viruses  were successfully recovered following electroporation and subsequent passage on C6/36 cells. Using sequence analyses and RT-PCR RFLP analyses, all viral stocks were pure and contained only the rDENV of interest  Fig. . In factTo eliminate the possibility that the panel of rDENV4/3 viruses had been modified in such a way as to increase their sensitivity to neutralization by DENV immune sera in a non-specific manner, neutralization assays were performed with immune sera collected at late convalescence (>6 months) from subjects exposed to a primary DENV2 (DT100) or DENV1 (310 and 147) infection and secondary DENV infections (DT 000). These immune sera cross-neutralized DENV3, DENV4 and DENV4/3 M14 and M16 in U937 + DC-DIGN cells to similar degrees and were not significantly different by a 2-way Wilcoxon matched-pairs rank test. Fig.  indicati20. Some of these quaternary structure-dependent epitopes are present on the E dimer but some are only present on the intact virion and may span across dimers to bind to neighboring monomers/dimers, as is seen with the 5J7 epitope. It is becoming apparent that knowing the epitope specificity of an antibody response to a vaccine may be essential for understanding the correlates of DENV protective immunity. Large-scale clinical trials of new vaccine candidates have demonstrated that in vitro gold standard neutralization results do not always predict vaccine efficacy, current data indicate that the standard Vero neutralization assay without regard to epitope specificity does not always correlate with protective immunity30. This is not the first time that failure of in vitro characteristics, previously small-plaque and temperature-sensitive phenotypes failed to be predictive of vaccine efficacy33. Measuring epitope specificity of polyclonal sera is a daunting task, although recent advances in elucidating the contributions of individual hmAb to the overall serological repertoire have demonstrated that only a few neutralizing antibodies may dominate a protective response against influenza virus35. While this approach provides rich molecular information on an individual basis, new reagents are needed for population based studies. Single and multiple mutations in the E protein have been effectively used to measure type specific antibodies in human polyclonal sera by loss of neutralization36. Using epitope-transplanted chimeric DENV viruses, such as the set described herein, gain of neutralization can be measured to easily and accurately measure epitope specific antibody binding and neutralization responses in complex polyclonal sera. Such chimeric viruses may be combined with the 4 serotypes of DENV or they may be used in combination with antigen depletion of sera to measure epitope specificity of polyclonal sera.Human mAbs that bind quaternary epitopes on flaviviruses were first described for WNV followed by the 4 DENV serotypes37.Here, we show incremental gain of binding of the 5J7 monoclonal with the increasing size of the transplanted epitope in both binding and neutralization assays. It is also abundantly clear that additional residues in adjoining monomers significantly enhanced 5J7 binding to DENV4/3 M16 virus, clearly demonstrating that residues on A, B’ and B monomers are critically important for 5J7 recognition, maximal neutralization and quaternary epitope function. More importantly, we show that the larger epitope transplants that include residues of 3 different E proteins can be used to measure the amount of antibody that targets this antigenic site in DENV 3 primary immune polyclonal sera. Because individual monoclonal antibodies like 5J7 can only infer the location of the primary neutralizing antigenic site that is recognized by the polyclonal response, epitope transplant provides a means of demonstrating the importance of the A, B’ and B monomers in the global polyclonal neutralization response against DENV3. We do not always capture the entire DENV3 neutralization titer present in the polyclonal sera, suggesting that additional neutralizing sites and/or genetic variability within the epitope must exist in DENV3, supporting the critical need for identifying and charactering more type specific hmAb for each DENV serotype. Importantly, our data argue that our rDENV 4/3 panel is a powerful tool for measuring DENV3 epitope-specific antibodies in polyclonal sera. Indeed, the DENV4/3 M14 and M16 have very recently been successfully used to track development of antibodies to the 5J7 epitope in a large scale DENV3 cohort study with similar results41. To date, none have presented complex quaternary epitopes that span multiple monomers. We have previously demonstrated that residues in the A monomer of the 5J7 epitope can be transplanted into DENV125. The transplant of the DENV3 5J7 epitope into DENV 4 is unique from previous transplants in that this is the first transplanted epitope in any virus in which residues of adjoining monomers and dimers were also modified to capture an entire complex quaternary epitope. We detected a clear gain of epitope specific responses to 5J7 in the DENV4/3 recombinant viruses as the size of the transplanted region was increased to capture residues adjacent in B and B’ E molecules. With the DENV4/3 panel we have enlarged the scope and complexity of epitopes that can be successfully transplanted, effectively reconstructing a functional epitope that spans multiple monomers in the E glycoprotein raft15. In the DENV4/3 M16 recombinant virus, it is interesting that a single nucleotide change emerged in cell culture and that some minor differences were noted in the peak binding levels of broadly cross reactive fusion loop antibodies. DENV4/2 M16 displayed higher peak binding to 1M7 and 1N5 fusion loop antibodies, perhaps reflecting different maturation states and prM levels on virions. Future studies will focus on unraveling the role of this mutation and its phenotypes in epitope exchange viruses.A variety of approaches are being investigated to present complex epitopes in virus-like particles and on scaffolds, both for diagnostic and vaccine applications42. New developments in reverse genetics can be utilized to generate infectious clones for a number of viruses, including flaviviruses50. Importantly, quaternary epitopes and complex conformational neutralizing epitopes exist in many highly evolving and variable viruses, thus the methods described herein should be applicable to broad, based immunogen design for multiple pathogens. Live virus vaccines for yellow fever virus and more recently Dengue viruses provide new avenues for improving vaccine immunogenicity and safety, using reverse genetic approaches. In particular, the DENV tetravalent vaccine usually elicits immune responses against all four serotypes, regardless of manufacturer54. Nonetheless, the scope of the response varies based on vaccine serotype and the pre-immune status of the host. A detailed understanding of the location and functional activity of conformational and quaternary epitopes in vaccine performance offers novel opportunities for developing epitope specific correlates of protective immunity, engineered serotypes for improved vaccine immunogenicity and performance, and simplified bivalent vaccine formulations. (reviewed in55).The advent of modern molecular techniques and genomics has allowed for a new, highly-rational approach to be taken in the development of tools for epitope analysis and of new vaccine candidates13 The rDENV3 clone, based on a Sri Lankan 1989 strain, was initially described in detail23 and the DENV4 clone was based on the Sri Lankan 1992a sequence26 and has only briefly been described previously13.Vero cells (ATCC# CCL-81), C6/36 cells (ATCC CRL-1660) and DC-SIGN-expressing U937 cells (ATCC CRL-1593.2 transfected with a retrovirus coding human DC-SIGN) and their growth conditions have been previously described57.Convalescent DENV immune blood samples were collected from travelers visiting countries where dengue is endemic. Our protocol for collecting dengue immune blood samples was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol 08-0895). Written informed consent was obtained from all subjects before their participation in the study. Studies at UNC and NIH were performed in accordance with the relevant guidelines and regulationsDENV serum samples used in this study were obtained from the existing dengue traveler collection as mentioned above. The samples were not from acute clinical patients and hence were not PCR confirmed. All samples were coded and analyzed anonymously.24, all changes were isolated to the A fragment of the DENV4 clone genome. The 3 increasing sizes of the 5J7 epitope transplant were synthesized  and incorporated into 3 different DENV4 fully assembled DNA genomes, transcribed, and then the genome length RNAs electroporated into Vero-81 cells to generate the rDENV4/3 panel of viruses, all 3 of which were viable. The first attempt at DENV4/3 M16 acquired a tissue culture adaptation, Env K323Q. The DENV4/3 M16 described in this paper was rederived with this additional Env K323Q residue and is otherwise isogenic with DENV4. Recombinant viruses were subjected to full length sequencing to demonstrate the presence of appropriate subsets of mutations as previously described13.To generate the 3 different size rDENV4/3 viruses , increasing numbers of amino acid residue sequences were transplanted into DENV4 from DENV3. As a result of our quadripartite infectious clone designFor viral titrations, viral stocks were diluted 10-fold serially in dilution medium  supplemented with 2% heat-inactivated fetal bovine serum  and 1x antibiotic/antimycotic. Following a 1hr infection, cells and inoculum were overlaid with overlay medium: OptiMem  containing 5% carboxymethylcellulose, 2% HI-FBS (Hyclone defined), and 1x antibiotic/antibiotic . Following a 4–6 day incubation (to achieve countable foci of infection), cell monolayers were extensively washed with 1xPBS followed by fixation in a solution of 80% methanol:20% phosphate-buffered saline (PBS) (v:v). Fixed cell monolayers were blocked in a solution of PBS containing 5% non-fat milk, and incubated with a primary antibody cocktail of 4G2 and 2H2 murine mAbs. Following thorough PBS washes, infectious foci were visualized using HRP-goat-anti-mouse secondary antibody , followed by TrueBlue substrate and foci were enumerated and used to calculate infectious titer.DENV3, DENV4, and the rDENV4/3 panel were subjected to multi-step (MOI = 0.1) growth curve analyses on Vero or C6/36 cells. Briefly, Vero or C6/36 cell monolayers in 12-well plates were inoculated with indicated virus at an MOI = 0.1. At 24hr intervals, all medium was removed and an equivalent amount of fresh medium was added. Supernatants were clarified by centrifugation and stored at −80 °C until viral titrations were performed. Virus grown on Vero cells was titrated on Vero cells, and C6/63-derived virus was titrated on C6/36 cells, enumerated, and plotted over time to generate growth curves.13. Briefly, monolayers of Vero-81 cells in 24 well plated were infected with a virus:antibody/serum cocktail that had previously incubated for 1hr at 37 °C to allow for Ab:virion binding. Following a 1hr incubation at 37 °C for infection, cells and inoculum were overlaid with overlay medium (see above). Following a 4–6 day incubation at 37 °C, monolayers were fixed, stained, and geometric mean neutralization titers enumerated using wells containing no antibody as a control for infection.Neutralization on Vero-81 cells has previously been described4 cells were added and the infection was allowed to proceed for 2 hours at 37 °C. Unbound virus was washed with infection media and the volume of media in each well was increased to 200 μl, and the cells were returned to 37 °C for a total of 24 hours. After 24 hours, the cells were washed with PBS, fixed in paraformaldehyde, permeabilized with saponin, blocked with normal mouse serum in permeabilization buffer, and stained with AlexaFluor 488-conjugated  2H2 antibody. Unbound antibody was washed off, and cells were resuspended in Hank’s Buffered Salt Solution  supplemented with 2% fetal bovine serum. Assays were performed twice and in duplicate. Samples were read on a Guava easyCyte 5HT flow cytometer (Millipore) as previously described by our group27. All serum neutralization curves are shown in Supplementary Figures Human sera or monoclonal antibodies were serially diluted 3-fold and mixed with sufficient virus to cause 15% infection in U937 + DC-SIGN cells. Dilution media contained reduced 2%) fetal bovine serum. The virus:antibody mixtures were incubated for 45 minutes in a 96-well plate at 37 °C. Following this incubation, 5 × 10% fetal b13. Briefly, RNA isolated from viral stocks, DNase treated , was reverse transcribed and the resulting cDNA amplified  using primers specific for each serotype. Positive controls from WT DENV RNA were run for each reaction as was a no template control. Presence of amplicons for rDENV4 and no other rDENV indicates absence of any species of DENV1-3.In order to confirm our recombinant virus stocks were pure and did not contain any WT DENV, highly sensitive RT-PCR and RFLP were performed as previously describedFor RFLP, RNA isolated from viral stocks was converted to cDNA and the portion of the E protein gene containing the transplanted epitope was amplified by PCR. The resulting amplicon was gel purified on a 1% agarose gel, and subjected to specific restriction endonuclease (RE) digests designed to cut the WT amplicon, but not the rDENV amplicon (due to ablation of the RE site by epitope transplantation). While the approximately 1000 bp wild-type amplicon was cut by BsiWI into an 800 bp and 200 bp fragments, the rDENV were not digested, indicating the absence of WT DENV4 in the recombinant viral stocks.Geometric mean neutralization titers were determined by 4-way non-parametric curve fitting with upper and lower bounds of 100 and 0. Nonparametric column statistic  were used to calculate p-values. All statistics were calculated using Graphpad Prism .Supplementary Information


Bpifa1 have been associated with OM susceptibility, its role in the ME is not well characterized. We investigated the role of BPIFA1 in protection of the ME and the development of OM using murine models. Loss of Bpifa1 did not lead to OM development. However, deletion of Bpifa1 in Evi1Jbo/+ mice, a model of chronic OM, caused significant exacerbation of OM severity, thickening of the ME mucosa and increased collagen deposition, without a significant increase in pro-inflammatory gene expression. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions and its loss in the presence of inflammation, exacerbates epithelial remodelling leading to more severe OM.Otitis Media (OM) is characterized by epithelial abnormalities and defects in innate immunity in the middle ear (ME). Although, BPIFA1, a member of the BPI fold containing family of putative innate defence proteins is abundantly expressed by the ME epithelium and SNPs in  Acute OM is primarily caused by the normally commensal bacteria non-typeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis2 while chronic forms of OM show a genetic component of 45–75%3. The large numbers of OM susceptibility genes that have been identified emphasize the importance of innate immunity in protection against OM development7. More than 20 mouse genetic models of chronic OM have been characterised in recent years and these have proven to be a powerful tool in understanding the pathobiology of OM  One such model is the N-ethyl-N-nitrosourea (ENU) mutant mouse, Junbo, that spontaneously develops chronic suppurative Otitis media (CSOM) under specific pathogen free (SPF) conditions, characterised by development of cellular fluid and hypoxia in the ME and inflammatory thickening of the mucoperiosteum. The causative mutation is a SNP in the gene encoding the transcription factor EVI1, also known as MECOM9. EVI1 is an inducible negative regulator of NFkB10 and a repressor of the TGFβ signalling pathway11. The heterozygous mutant Evi1Jbo/+ mouse has increased responsiveness to microorganisms and inflammatory stimuli10.Otitis Media (OM), or middle ear (ME) inflammation, is the most prevalent paediatric disease and the leading cause of paediatric surgery, antibiotic prescription and conductive hearing impairment13, where it localises to a non-goblet, non-ciliated population of secretory cells14, minor glands and mucous cells of the sub-mucosal glands15. Secreted BPIFA1 is found in nasal lavage, saliva, sputum and bronchoalveolar lavage (BAL)13 and in the apical surface lining (ASL) fluid from air liquid interface cultures of differentiated tracheobronchial epithelial cells17.The ME is an anatomical extension of the upper respiratory tract (URT) and demonstrates similar physiological and immune mechanisms. The epithelium of the URT and its secretions play a key role in protection against environmental insults and infections. BPIFA1, a member of the bacterial permeability-increasing (BPI) fold containing family of putative innate defence proteins, is one of the most abundant secretory proteins in the URT. Its expression is restricted to the nasal passages, oropharynx, trachea and bronchi18 and chronic rhinosinusitis19. Despite the lack of compelling mechanistic evidence to identify the precise biological function of BPIFA1, an increasing number of studies suggest that it plays a multifunctional host defence role in the conductive airways16. In line with the structural similarity to BPI20, BPIFA1 exhibits antimicrobial activity against a variety of respiratory pathogens and deletion of Bpifa1 in mice is associated with increased susceptibility to pulmonary infections25. Other proposed functions of BPIFA1, include a surfactant-like activity, dispersal of bacterial biofilms, and regulation of ASL volume by modulation of epithelial sodium channel (ENaC) function16.Alterations of BPIFA1 levels are associated with multiple URT diseases such as cystic fibrosisBpifa1 expression in vivo using siRNA in chinchillas led to impaired mucociliary clearance23. BPIFA1 protein was present in the effusions of patients with chronic OM27 and BPIFA1 was also identified as one of the top hits in a Genome Wide Association Study of genes associated with OM susceptibility28. We recently showed production of BPIFA1 in the murine ME, in vivo and in vitro29 and it has been reported that Bpifa1−/− mice greater than 10 months-of-age show increased susceptibility to spontaneous development of chronic OM30. Together this data suggests that BPIFA1 may be a determinant for predisposition to OM.Recently, evidence of BPIFA1 expression in the ME and its involvement in OM pathogenesis has started to emerge. Knocking down Junbo model of chronic OM. Our data suggests that BPIFA1 is involved in maintaining homeostasis within the ME under steady state conditions. This is the first study to report the use of compound Bpifa1 mutants and it underlines the importance of investigating the additive effect of multiple genetic defects in order to better understand the aetiology of multifactorial diseases such as OM.In this study, we have studied the role that BPIFA1 plays in protection of the ME and its association with OM development. Our data shows that although the loss of BPIFA1 did not lead to the development of spontaneous OM in mice up to 6 months-of age, it significantly exacerbates the inflammatory phenotype in the 31. In order to evaluate the onset of BPIFA1 production during the post-natal development of the bulla, immunohistochemistry (IHC) was performed from the day of birth (P0) to P30 at intervals of 5 days. BPIFA1 was detected in the ME throughout this period. As described previously, between P0 to P10, the ME space is occupied by mesenchyme32. BPIFA1 was detected in the non-ciliated epithelium enclosing this mesenchyme mass, in the region where it retracted from the opposing epithelial surface  or 12 weeks of age .Having established that BPIFA1 is present in the ME epithelium during the process of bulla cavitation, we went on to determine whether loss of the protein caused OM. Hearing thresholds, measured using broad-spectrum Auditory Brainstem Response (ABR) (which is indicative of conductive hearing loss), did not differ significantly in age Fig. . Histoloeks Fig.  or 6 moneks Fig.   and Bpifa1/−+ (2/8) bullae contained fluid and 2/12 Bpifa1−/− ears and 1/8 Bpifa1/−+ ears were NTHi culture positive. WT bullae (n = 16 ears) did not have fluids and all were NTHi culture negative. Although this data was suggestive of an effect of loss of Bpifa1 on infection these differences did not achieve statistical significance. A similar outcome was achieved when we challenged WT, Bpifa1/−+ and Bpifa1−/− mice with NTHi using baroinoculation34 and sampled bulla fluids at day 3 post-inoculation (data not shown).As the development of OM is often associated with bacterial infection, we investigated whether the loss of BPIFA1 modulated the ability of mice to respond to the intranasally (IN) inoculated otopathogen NTBpifa1 does not lead to spontaneous development of OM and does not lead to an increased ME infection or overt induction of OM following NTHi inoculation.Taken together, the data suggested that deletion of Evi1Jbo/+ mouse. OM initiates spontaneously in Evi1Jbo/+ mice from P21 onwards and is characterised by significant remodelling of the middle ear epithelium9. At P28, IHC staining for BPIFA1 appeared to be reduced in the ME epithelium of Evi1Jbo/+ mice compared to their age matched WT littermate controls  from WT and Evi1Jbo/+ mice using RT-qPCR. This analysis showed that there was a significant down-regulation of Bpifa1 mRNA in Evi1Jbo/+ mMMCs when compared to WTs  compared to Evi1Jbo/+ mice  from 7 to 10 week old Bpifa1−/−Evi1Jbo/+, Evi1Jbo/+, Bpifa1−/− and WT mice. Although mMMCs of Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ mice had significantly elevated levels of the pro-inflammatory cytokines Cxcl2, Il1β, Tnfα and the profibrotic mediator, Tgfβ, compared to WT mMMCs, there was no significant difference in the expression levels for any of these genes between WT and Bpifa1−/− mMMCs or between Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ mMMCs  and fibroblasts isolated from the bullae of Bpifa1−/−Evi1Jbo/+, Evi1Jbo/+, Bpifa1−/− and WT mice. No significant differences in doubling times were seen in either mMECs  by 10–18 months of age30. This apparent difference could be due to strain differences (as we have studied mice on C57BL/6 J and C3H/HeH backgrounds) and could be further examined by studying our Bpifa1−/− model at a comparable age. However, OM is typically studied in young mice as this better represents the timing of the paediatric disease in humans28 and the relevance of any late development of the disease is unclear. As mice over expressing BPIFA1 showed reduced levels of lung fibrosis when exposed to a sterile irritant37, it is possible that extended exposure to respiratory irritants or previous episodes of bacterial infection may be contributory to the low penetrance OM in aged Bpifa1−/− mice30.However, our data shows that loss of bpifa1 has previously been shown to predispose mice more severe respiratory infections following an experimental challenge and yet we found that IN challenge of Bpifa1−/− mice with the major otopathogen NTHi, did not result in significant ME infection, although our data did appear to suggest that Bpifa1−/− mice infection prevalence was marginally higher. NTHi is a significant causative pathogen isolated from a majority of AOM and COME patients38 and the potential role that BPIFA1 plays in defence against this organism requires further studies.The loss of Bpifa1−/− mice, we investigated the contribution of the protein to OM disease progression by studying compound mutant Bpifa1−/−Evi1Jbo/+ mice. Evi1Jbo/+ mice represent an established model of OM39 and develop hallmarks of the disease soon after middle ear cavitation occurs. Our analysis suggests that BPIFA1 protein staining was reduced in the remodelled epithelium in Evi1Jbo/+ mice compared to WT. It seems possible that this loss of staining may be due to an alteration of cell populations in the mucosa, with a reduction in the number of BPIFA1 producing cells being present. However we could still readily detect the protein in the ME exudates from Evi1Jbo/+ mice. This is consistent with proteomic data from human ME exudates27 and our own data from the apical secretions of differentiated mMECs29.Given the high levels of BPIFA1 seen in the ME, but the absence of spontaneous OM or lack of significantly increased susceptibility to ME infection by NTHi in our Bpifa1−/−Evi1Jbo/+ bullae demonstrated an exacerbation of OM severity compared to Evi1Jbo/+ mice. This suggests that removal of BPIFA1 from the middle ear environment allows for a more significant remodelling response. This further suggests a shifting of the time required to produce the OM phenotype. Indeed analysis of compound mutant mice at day P21 also revealed an exacerbation of OM severity (results not shown). It seems likely that the loss of BPIFA1 from the middle ear mucosa during the process of cavitation directly exacerbates the development of OM. Previous studies of Evi1Jbo/+ bulla exudates demonstrated elevation of inflammatory gene networks and hypoxia signalling genes compared to venous blood as a baseline control39. We have shown up-regulation of the pro-inflammatory mediators Cxcl2, Tnfα, Il1β and Tgfβ in the ME mucosa of Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ mice. Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ bullae also appeared to demonstrate epithelial cellular alterations characterised by down regulation of the ciliated cell marker, Foxj1, and the secretory cell marker, Muc5ac, and Evi1Jbo/+ mice demonstrated reduced levels of Bpifa1 expression. However, despite the exacerbated OM phenotype in Bpifa1−/−Evi1Jbo/+ mice, there was no significant additive effect of the loss of Bpifa1 on the expression of these inflammatory and epithelial markers. This may be a consequence of comparison based on the already inflamed Evi1Jbo/+ epithelium that has high basal expression levels of these inflammatory genes. At this time we are unable to identify exactly which cell type(s) express these pro-inflammatory markers but our mucosal cell isolation procedure generates a population of structural  cells without a significant inflammatory cell component.The most striking observation from these studies is that Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ mice do not appear to be explained by proliferation rates of the epithelial cells and fibroblasts measured ex-vivo. It seems likely that inflammatory changes such as connective tissue oedema, dilation of lymphatics and blood vessels, infiltration of inflammatory cells and fibrosis are responsible for the mucosal thickening seen in the ME. An interesting feature observed in the inflamed Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ MEs was fibrosis of the sub-epithelial connective tissue, indicated by collagen deposition and accumulation of myofibroblasts. These features are characteristic of fibrotic pulmonary diseases such as asthma and idiopathic pulmonary fibrosis40 and indicative of epithelial remodelling due to an over-active wound repair response following inflammation. In turn this is suggestive of over-activation of the TGFβ signalling pathway (a target for EVI1)41. The enhanced thickness of the Bpifa1−/−Evi1Jbo/+ mucosa compared to that of Evi1Jbo/+ mice suggests greater fibrosis due to loss of BPIFA1. BPIFA1 has been previously associated with an allergen-induced asthma-like phenotype in mice42 and overexpression of BPIFA1 has been shown to protect against pulmonary fibrosis37. A recent report suggesting a role for BPIFA1 as an epithelial derived smooth muscle relaxing factor43 is supportive of a potential role for this epithelial protein in mediating crosstalk with the underlying connective tissue.The differences in the mucosal thickening of Bpifa1 in the inflamed microenvironment of the Evi1Jbo/+ ears. This reduction in BPIFA1 may represent transcriptional down regulation of Bpifa1 or perhaps more likely is a consequence of phenotypic changes in the epithelial cell populations. Previous work with BPIFA1 has largely focused on evaluation of a role for the protein in bacterial and viral pathogen challenge studies or in the presence of pre-existing chronic allergic and inflammatory pulmonary disease (for review see16). Importantly, we did not observe any phenotypic abnormalities or signs of organ inflammation in the non-challenged Bpifa1−/− mice. Thus, the presence of an infectious or inflammatory stimulus seems to be a pre-requisite to uncover functional roles of BPIFA1. This may also explain why Bpifa1 deletion alone does not lead to the spontaneous development of OM in Bpifa1−/− mice. Consistent with this hypothesis we have recently shown that Bpifa1−/− mice are more susceptible to influenza A infection through a mechanism that allows Bpifa1 deficient cells to be more readily infected by the virus44.An important observation in this study was the downregulation of epithelial 46. Further studies are required to address this point and whether the mechanism(s) are via alteration in surfactant function, epithelial permeability, and/or impaired antimicrobial action.This work provides new insights into the function of BPIFA1 in the ME. From birth onwards BPIFA1 is secreted at high levels into a film that overlies the epithelium lining the ME, but its absence does not predispose to spontaneous OM or increase the susceptibility to bacterial infection. This indicates that, within normal limits, BPIFA1 is a functionally redundant innate immune protein that plays a homeostatic role in mucosal protection in a manner described for multiple respiratory proteinsWe have explored the role of BPIFA1 in mice bearing an OM predisposing Evi1 mutation, and shown an upregulation of ME epithelial cytokine expression and down-regulation of epithelial markers, with dispersal of BPIFA1 from this surface layer into bulla fluid. Deficiency of BPIFA1 on an Evi1 mutant background does not markedly alter epithelial cytokine and cell marker expression probably via perturbation of epithelial cell populations, and consequently exacerbates inflammatory thickening of the submucosal connective tissues.Bpifa1 on an OM prone background results in the earlier initiation of OM and whether, once initiated, the progression of disease is significantly altered. It is also important to study if loss of BPIFA1 exacerbates the OM phenotype in other OM mutant mouse lines. Given that features such as mucosal fibrosis have been noted in chronic OM in Tlr4 and Evi1 mutant mice47, early fibrosis, and mucosal thickening may suggest accelerated disease in the absence of BPIFA1 rather than substantially altered pathogenesis.Together this suggests that BPIFA1 aids homeostasis in the ME by contributing to the epithelial barrier function, and its role is revealed only under significant epithelial insult. Future time course studies are needed to define whether the deletion of Mice were bred and maintained by the Mary Lyon Centre, MRC Harwell and were housed in specific-pathogen free conditions. All animal experimentation was approved by the Animal Welfare and Ethical Review Body at MRC Harwell. The humane care and use of mice in this study was under the authority of the appropriate UK Home Office Project License.Bpifa1−/− mice on a C57BL/6 background were obtained from Professor Ralph Shohet at the University of Hawaii. The generation of these mice in which exons 2 and 3 of the Bpifa1 gene have been deleted have been described in detail elsewhere44. C57BL/6 Bpifa1−/− mice were re-derived at MRC Harwell Institute, UK and backcrossed 5 times to C3H/HeH mice to produce a congenic background. Junbo heterozygote (Evi1Jbo/+) mice were maintained on the C3H/HeH background9. Compound Bpifa1−/−Evi1Jbo/+ mutants and their littermate controls were generated by intercrossing Bpifa1−/− and Evi1Jbo/+ mice and mice were examined at P28 and at 6 months of age. P0-P30 WT C3H/HeH mice, used for IHC of BPIFA1, were obtained from MRC Harwell Institute. For comparative purposes Bpifa1−/− mice on the original C57BL/6 background and a mixed C3H/HeH-C57BL/6 background were used in some infection studies. Husbandry of SPF mice and microbiological surveillance is described elsewhere9.9.The hearing thresholds of 8 and 12 week-old mice were assessed using ABR measurements as previously describedMice were euthanized by intraperitoneal overdose of sodium pentobarbital . The mouse was decapitated and head skin removed. The tympanic membrane was examined under 10× binocular magnification to establish whether there was evidence of opacity which is indicative of macroscopic bulla fluid accumulation.Bpifa1−/− mice at 6 months of age were analysed for morphological abnormalities.Tissue samples were fixed in 10% formaldehyde (Surgipath Europe) for 48 hours at room temperature and the skulls decalcified in Kristenson D.F.B agent (Pioneer Scientific) for 72 hours before wax embedding. Four µm H&E stained sections of the bullae and nasal passages were scanned using the NanoZoomer Digital Pathology system and morphometric measurements made with NanoZoomer software (Hamamatsu). Thickness of the ME mucosa was measured in a defined 1 mm area in the promontory region opposite the tympanic membrane. Average mucosal thickness was calculated using 5 measurements taken at a distance of 250 μm within this region. Both male and female mice were analysed and no sex-related differences were seen in the development of OM. H&E slides for sections of a total of 42 target tissues from 31 or rabbit anti-αSMA (1:200) (Abcam 5694) antibodies overnight in a humified chamber at 4 °C then washed twice in PBS, incubated in 0.5% biotinylated polyclonal goat anti-rabbit secondary antibody  for 30 minutes at room temperature, followed by incubation for 30 minutes with the ABC reagent for signal amplification before colour detection using a NovaRed system . Images were captured with an Olympus BX61 light microscope and Olympus colour view digital camera.Four µm wax sections were used for IHC. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide in methanol . Non-specific binding was blocked by incubating the sections in 100% goat serum for 30 minutes at room temperature. Sections were washed and incubated in rabbit anti-Bpifa1 (1:750)SR), was performed as previously described48.Intranasal bacterial challenge experiments, using genomically sequenced, streptomycin resistant strain NTHi #375 . The pressure was raised to approximately 1.4 pounds per square inch (Psi) by compressed air through an inlet port. Thereafter, the pressure was increased by approximately 0.7 Psi every 15 secs to achieve a maximum pressure of 5.8 Psi, then after 2 minutes was decreased by venting air via an outlet port at 30 secs intervals to return the chamber to atmospheric pressure.The baroinoculation protocol was adapted from previously published studies in mice29. Fibroblasts were isolated using a differential adherence method described in the same paper. Adherent fibroblasts were cultured by adding 5 ml of mMEC basic 10% FBS media to the tissue culture dish29 and incubating at 37 °C in a 5% CO2 incubator till confluent. Upon confluence, cells were washed with warm HBSS and sub-cultured twice to obtain a pure fibroblast population. For the third passage, 3mls of Trypsin-EDTA solution (Sigma) was added to the dish and incubated as above for a minute to dislodge the cells. Trypsin was neutralised by addition of 3 ml neutralising medium (mMEC basic plus 10% FBS) and cells were centrifuged at 500 × g for 5 minutes. Cell pellets were resuspended in 5 mls of fresh mMEC basic 10% FBS and split into five T25 flasks for expansion of the fibroblast cultures. 4 ml of media was added to each flask and cells were cultured at 37 °C in 5% CO2 till confluence. When calculating population doubling time (PD), the purified fibroblasts were seeded at 5 × 104 cells/well in mMEC basic 10% FBS media in 6 well tissue culture plates . Similarly, fresh mMECs were seeded at 5 × 104 cells/well in mMEC- Plus media29 in submerged culture. Media was changed every 48 hours. Fibroblasts were harvested every 24 hours from day1 to day 3 whereas mMECs were harvested every 48 hours by trypsinisation and quantified using a haemocytometer from day 2 to day 10.The isolation of mMECs was performed as previously described10, C1 = initial cell count, C2 = final cell count.Population doubling time was calculated using the formula.48, these were then mixed with an equal volume of 2XSDS lysis buffer. Samples were denatured at 95 °C for 5 minutes and resolved on a 12% polyacrylamide gel, transferred to a PVDF membrane using a semi dry blotting system (Biorad Trans-blot turbo) then probed with rabbit anti-Bpifa1 primary antibody (1:20031), overnight at 4 °C. The primary antibody was detected using a polyclonal goat anti-rabbit secondary antibody (Dako P0448) conjugated with HRP (1:2000) and bound antibody was visualised using the EZ-Chemiluminescence detection system  and manual development. Images were scanned as a whole with no modification.Bulla fluids were sampled by pipetting as previously describedBpifa1−/−, Evi1Jbo/+ and Bpifa1−/−Evi1Jbo/+ MEs, mouse mucosal epithelial cells (mMMCs), i.e. ME epithelial cells before undergoing the differential fibroblast separation, were used. Total RNA was extracted, cleaned from residual genomic DNA contamination, quantified and reverse transcribed into cDNA as previously described29. RT-qPCR was performed using Applied Biosystems TaqMan gene expression assays for Cxcl2 (Mm00436450_m1), TNFα (Mm99999068_m1), TGFβ (Mm01178820_m1), Bpifa1 (Mm00465064_m1), FoxJ1 (Mm01267279_m1) and Muc5ac (Mm01276718_m1) on a 7500 Fast Real Time PCR system (Applied Biosystems), with 2× Taqman Fast Universal Master Mix . 10 ng cDNA was added to each reaction and three technical replicates were performed for each assay in each batch. Target gene expression levels were normalized to three endogenous controls: Atp5b, Cyc1 and Canx (Primerdesign geNormTM Reference Gene Selection Kit) and analysed using ABI 7500 software v2.0.1 using the 2∆∆Ct− method. Data is presented as mean Relative quantification (RQ) and error bars represent standard error of the mean.For comparison of gene expression between WT, t-test was used to compare two groups of data. One way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to compare more than two groups, for parametric data. Fisher’s exact test was used for analysing frequency data such as presence or absence of bulla fluids or genotype ratios . Significance threshold was set at p < 0.05. Data are presented as the mean +/− standard error of the mean (SEM) unless otherwise stated. Significance levels represented on graphs are as follows: ns = not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.The normality of experimental data was determined by performing Levene’s test prior to the statistical analysis. GraphPad Prism (version 6.0) was used to perform all statistical tests. 2 tailed students Supplementary Dataset 1


PGD2 is released from guinea pig bladder urothelium/suburothelium and can inhibit detrusor contractile responses. We presently wished to investigate PGD2 actions in guinea pig out‐flow region and the distribution of DP1/DP2 receptors. The effects of PGD2 on urothelium‐intact trigone and proximal urethra contractility were studied in organ bath experiments. Expression of DP1/DP2 receptor proteins was analysed by western blot. Immunohistochemistry was used to identify distribution of DP1/DP2 receptors. PGD2 in a dose‐dependent manner inhibited trigone contractions induced by electrical field stimulation (EFS) and inhibited spontaneous contractions of the proximal urethra. PGD2 was equally (trigone) or slightly less potent (urethra) compared with PGE2. Expression of DP1 and DP2 receptors was found in male guinea pig bladder trigone, neck and proximal urethra. In the trigone and proximal urethra, DP1 receptors were found on the membrane of smooth muscle cells and weak immunoreactivty was observed in the urothelium. DP2 receptors were distributed more widespread, weakly and evenly in the urothelium and smooth muscles. Inhibitory effects by PGD2 on motor activity of guinea pig trigone and proximal urethra are consistent with finding DP1 and DP2 receptors located in the urothelium and smooth muscle cells of the trigone and proximal urethra, and PGD2 may therefore be a modulator of the bladder out‐flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome.The proximal urethra and urinary bladder trigone play important roles in continence. We have previously shown that  The lower urinary tract (LUT) consists of the urinary bladder and the urethra with the functions of urine storage and periodic urination. Continence and micturition involve a balance between detrusor activity and urethral sphincter closure. Bladder filling and voiding are controlled by a complex pattern of afferent and efferent signalling in parasympathetic, sympathetic and somatic pathways In the LUT, the bladder trigone is a smooth region in the base of urinary bladder within the two ureteral orifices and the internal urethral orifice which has different embryological origins from the rest part of the bladder. The majority of vessels and nerves of the bladder enter and concentrate in the trigone making it very sensitive to expansion 2 is an important lipid mediator that exerts its biological functions via the G protein‐coupled receptors prostaglandin D receptor type 1(DP1) and prostaglandin D receptor type 2 (DP2), the later also known as chemoattractant homologous receptor expressed on Th2 cells (CRTH2). We have previously shown that PGD2 and PGE2 were released from guinea pig urinary bladder and that PGD2 inhibited induced bladder detrusor contractions 2 on the trigone and proximal urethra has been studied for decades. Andersson and colleagues showed that PGE1 and PGE2 relaxed pre‐contracted human circularly cut urethral rings 2 was shown to relax the pre‐contracted trigone and longitudinally cut human and pig urethra i.e. PGE2 relaxed the pre‐contraction of circularly cut hamster and longitudinally cut dog urethra 2 contracted the longitudinal urethra strips but relaxed the circular urethra muscle 2 enhanced the tone and increased spontaneous activities 2 is involved in the regulation of trigone and urethra motility remains unknown. In the human, expression of functional DP receptors was found in corpus cavernosum smooth muscle 1 receptors was found in the smooth muscle and urothelium with a dominant localization to smooth muscle membranes, DP2 was also found on the bladder wall 1 and DP2 receptors in the proximal urethra and trigone regions have not been reported.Prostaglandin D2 and PGE2 on male guinea pig trigone and proximal urethra and report on the efficacy of PGD2 and PGE2 in these tissues. We describe the expression and distribution of DP1 and DP2 receptors in trigone and proximal urethra with respect to their distribution in both urothelium and muscle components.In the present study, we examined the effects of PGDen bloc. Seminal vesicles, deferent ducts, coagulating gland ducts and ejaculatory ducts were removed at duct openings. The trigone was dissected by locating the urethra and ureter openings. A trigone strip about 7 × 2 mm was made from each guinea pig and with the urothelium intact. The IUS ring from the level of bladder neck to above duct openings of the proximal urethra was dissected with intact urothelium was opened and cut into 1–2 strips for organ bath experiments. All tissue strips were tied at both ends with thin cotton threads and equilibrated in 5.5 ml organs bath containing Tyrode's solution  and aerated with 5% CO2 in O2 at 37°C.All experiments were approved by the local animal ethics committee (Dnr N178/11). Male albino guinea pigs weighing 500–750 g were anaesthetized with midazolam 1 mg/kg + sodium pentobarbital 120 mg/kg and exsanguinated. The urinary bladder and proximal urethra were removed −6 M was given to the trigone tissues to inhibit the production of endogenous prostaglandins. After 10 min. incubation with diclofenac, the tissues were washed and diclofenac 10−6 M was reapplied to trigone strips throughout the experiment.After 30 min. equilibration, one end of the tissue was connected to an isometric transducer and the other end to a hook at the bottom of the bath. Tissues were carefully washed with Tyrode's three times. The initial resting tension of the trigone and urethra strips was adjusted to 5 mN. Proximal urethra strips were left unstimulated to record the spontaneous contractions. When stable tension developed, trigone strips were electrically stimulated by means of two platinum electrodes on the walls of the organ baths . The evoked contractions were recorded with a computerized acquisition system . When stable contractions were recorded, diclofenac 102 and PGE2 were added to the tissues cumulatively in log increments from 10−9 to 10−6 M. Each dose was applied for 10 min. Control contraction amplitudes were measured before application of PGD2 and PGE2. Contractile response at 10 min. at every dose of PGD2 and PGE2 were measured and compared with control amplitude. Log concentration‐response curves were constructed.PGD2 and PGE2 were given to the urethra strips cumulatively in half‐log or log increments from 10−9 to 10−6 M at 8–10 min. intervals in the absence of diclofenac since cyclo‐oxygenase inhibitors were reported to inhibit spontaneous contractions in the urinary tract 2 and PGE2 and expressed as area per min. Log concentration‐response curves were constructed.After regular spontaneous contractions were developed, PGD2 and PGD2 was applied cumulatively in log increments to trigone and urethral tissues without any compound. The final concentration of ethanol in each organ bath was less or equal to 0.1%.The corresponding amount of ethanol used to dissolve PGE1 receptor C‐terminal antibody  or rabbit anti‐human DP2 (CRTH2) receptor antibody  diluted in PBS‐Tween 20 with 5% skim milk. HRP‐conjugated goat anti‐rabbit secondary antibodies  and Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific) were used to detect protein signal on autoradiographs .Male guinea pigs were anesthetized as above and the abdominal aorta was flushed distally with 30–40 ml warm saline to achieve blood‐free tissues. The bladder trigone, neck and proximal urethra were dissected and isolated apart. For protein extraction, each mg wet tissue was subjected to 20 μl of lysis buffer (pH 7.6) containing 20 mM Hepes, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 25 mM KF, 1 mM sodium orthovanadate, 0.5% Triton X‐100, 20% glycerol and 1% protease inhibitor cocktail . Tissues were homogenized using an Ultra‐Turrax for 2 min. and then homogenized for 4 min. in a Dounce glass homogenizer. Lysates were centrifuged at 13,000 × g gravity for 20 min. at +4°C. Protein content of the supernatant was determined with the Bradford protein assay . 7 μg of protein was loaded onto 8–16% SDS Pierce Protein Gel  and separated by electrophoresis. Proteins were transferred onto PVDF membranes using dry blot/iBLOT according to the manufacturer's instructions . Membranes were blocked for one hour with 5% skim milk dissolved in PBS‐T . Membranes were probed overnight with rabbit anti‐human DPen bloc and cleaned from connective tissues. The bladder was flushed of urine before fixation which was by immersion in ice‐cold 4% paraformaldehyde 0.1 M phosphate buffer fixative solution for 4 hrs at 4°C. After fixation, tissues were cryoprotected by incubation in 0.1 M phosphate buffer with 30% sucrose solution for 16–20 hrs at 4°C. The trigone and urethra were then dissected from the bladder. The proximal urethra was cut transversely into several ring segments. Small pieces of tissue were covered with Neg‐50™ (Thermo Scientific) and quickly frozen in liquid nitrogen cooled isopentane and stored at −80°C for later cryostat sectioning. 10 μm cryostat sections of vertical trigone and transverse urethra were mounted on gelatin coated slides.Male guinea pigs were anaesthetized and perfused as above. The urinary bladder with short ureter remains and proximal urethra were taken 1 receptor antibody  or labelled with the rabbit anti‐human DP2 receptor antibody . All the antibodies were diluted in blocking buffer. The sequential control sections were treated with blocking buffer. Cryostat sections labelled with antibodies were incubated overnight at 4°C. Before secondary antibody treatment, the sections were rinsed three times for 5 min. in PBS. Then, sections were incubated for 1 hr with secondary antibodies at room temperature. Counterstaining of smooth muscle actin, F‐actin and nuclei was made by monoclonal anti‐α‐smooth muscle actin antibody , phalloidin  and Hoechst33258 , respectively. The secondary antibodies used in this study were: donkey anti‐rabbit antibody labelled with Alexa Fluor 568  and donkey anti‐rabbit antibody labelled with Alexa Fluor 488 . After 1 hr incubation, sections were rinsed three times in PBS for 5 min. in the dark. The sections were then mounted in S3023 medium with anti‐fading agent  and covered with coverslips.Cryostat sections were blocked in blocking buffer with PBS (pH 7.2) containing 0.5% Triton X‐100 and 5% normal bovine serum for 20 min. at room temperature. Sections were either labelled with the rabbit anti‐human DPAll immunolabelled sections were observed under an Axioplan 2 imaging fluorescence microscope  equipped with FITC (Chroma 41001), TRITC (Chroma 41002a) and DAPI (Chroma #31000) filters. Sections were photographed with a Nikon D7000 digital camera using NKRemote software  for camera control with 12‐bit image acquisition followed by subsequent background subtraction and contrast enhancement in ImageJ (NIH open source).2 and PGD2 were generous gifts from Professor Ernst H Oliw . Diclofenac, atropine, tetrodotoxin and D‐tubocurarine were from Sigma‐Aldrich. All the data are presented as mean ± S.E.M. PGE2 and PGD2 log dose–response curves were fitted by a 4‐parameter sigmoidal curve model using Prism 5  to estimate the IC50 of PGE2 and PGD2.PGEIn vitro, unstimulated urothelium intact guinea pig urinary bladder trigone strips showed irregular, fast and fast‐relaxing (3–4 sec.) spontaneous contractions with a frequency of 4.71 ± 0.74 contractions per min (n = 7). When the trigone strips were electrically stimulated , monophasic and reproducible post‐stimulation contractile responses were observed. These were nerve‐mediated as indicated by their sensitivity to tetrodotoxin. Muscle strips from the level of bladder neck had no spontaneous activity and did not respond to nerve stimulation. Strips of proximal urethra taken below the bladder neck and above the duct openings, representing the major region of the IUS, showed regular, slow and long‐lasting (30–40 sec.) spontaneous contractions , PGE2 or PGD2 was applied cumulatively from 10−9 to 10−6 M. PGE2 and PGD2 inhibited the EFS‐induced contractions in a dose dependent manner. In some experiments, low concentrations of PGE2 (10−9 to 10−8 M) elicited a weak enhancement of contractile responses, whereas higher concentrations (above 10−8 M) always elicited inhibition. Dose–response curves  and an estimated pIC50 value for PGD2 of 7.08 ± 0.27 (n = 6). The corresponding amounts of ethanol used to dissolve PGE2 and PGD2 had no effect on EFS contractions when applied to the trigone strips.Reproducible repeated contractile responses of isolated trigone were induced by EFS as above. After treatment with diclofenac  for PGE2 and 7.09 ± 0.22 (n = 14) for PGD2. Ethanol at the corresponding concentrations as used to dissolve PGE2 and PGD2 did not modify the IUS spontaneous contractions.Urethral strips from below the neck and above the duct openings, which is the IUS region exhibited regular spontaneous contractions that were unaffected by atropine 5 × 101 and DP2 (CRTH2) receptor proteins in the guinea pig trigone, bladder neck and proximal urethra was examined by Western blot. Tissue extracts, containing the whole layers of both muscle and urothelium from these regions were exposed to DP1 and DP2 antibodies and which were the same antibodies used in the immunohistochemistry. As shown in Figure 1 and DP2 proteins. In Figure 1 panel, two groups of protein bands at, respectively, the predicted molecular weight for DP1 (40 kD) and at around 95 kD were seen. This result fits the data provided by the manufacturer in tests with the antibody on different cell lines. In Figure 2 panel, only one group of DP2 protein bands was observed at around 75 kD. The predicted band location for DP2 is 40 kD, the reason for observing an increased size of the protein bands is likely a reported post‐translational modification, e.g. phosphorylation, glycosylation etc. The expression of DP for DP1 0 kD and 1 and DP2 receptors was seen throughout the trigone urothelium and smooth muscle. DP1 receptors were more prominent in the smooth muscle layer while DP2 receptors were more evenly distributed in the urothelium and smooth muscle layers but not as strong and localized as DP1 receptors in the muscle cells. Figure 1 receptor distribution in the urothelium of the trigone. The border between urothelium and suburothelium was heavily stained for DP1 receptor with localization to the cells immediately below the membrane but only faint fluorescence was seen in the urothelium layer, with a predominant localization to urothelium cell membranes. Figure 1 receptor distribution in the smooth muscle bundles. As shown in Figure 1 receptor antibody was found surrounding the red fluorescence for muscle actin, indicating localization of DP1 receptor mainly in the membranes of smooth muscle cells. The blue fluorescence in Figure i.e. not exposed to primary antibody, only secondary antibodies) showed no staining for DP1 or DP2 receptors.Fluorescence immunohistochemistry results from sequential vertical sections of the trigone with transitional urothelium (uro) and part of the smooth muscle (sm) layers are shown in Figure The morphology of the male guinea pig urethra differs depending on the position at which sections are taken. In this study, we focused on the proximal urethra with the IUS. Figure 1 receptor was seen distributed in the proximal urethra urothelium  and anti‐α‐smooth muscle actin (red) shows strong yellow fluorescence indicating co‐localization of DP1 receptor on the smooth muscle bundles  and striated muscle (st) in male guinea pig proximal urethra is shown in Figure les Fig. C2. The dcle Fig. C1.2 can exert inhibitory influence on smooth muscle contractile responses induce by EFS and on spontaneous contractions. DP1 and DP2 receptors are found expressed in the trigone and proximal urethra. In the bladder trigone, DP1 receptors are markedly located on the suburothelium layer and smooth muscle cells membranes similar as in the proximal urethra. DP2 receptors are found weakly and evenly on the urothelium/suburothelium layers and also in the smooth muscle.The major novel findings in the present study are that, in male guinea pig trigone and proximal urethra PGDin vitro study using human bladder strips found no effect of PGD2 up to 3 × 10−7 M on resting tension and was contractile at higher concentrations 2 was produced in guinea pig urinary bladder in a urothelium‐dependent fashion and exerted its inhibitory effects on EFS induced contractions via DP1 receptors localized on the detrusor membrane 2 together with other relaxing mediators such as PGE2 is produced by the urothelium/suburothelium to relax the underlying smooth muscle. When the bladder reaches a threshold volume, an emptying process is triggered where PGD2 together with other mediators might reduce the amplitude of initial detrusor muscle contractions, but also by relaxation of the internal sphincter might facilitate entry of urine into the proximal urethra. Passage of urine into the proximal urethra is a signal in the initiation of micturition A previous We observed distinct regional inherent activity and responses to EFS along the proximal urethra. An earlier study measuring the urethral pressure profile of the male feline urethra using a silicone rubber catheter with pressure transducers showed increased urethra pressure from proximal to distal with several peaks at the region of prostatic urethra, bulbourethra and penile urethra 1 and DP2 receptor distribution is supported by our Western blot data and by results with similar antibody in a study by Zhang et al. who showed that DP1 and DP2 receptors are present in guinea pig oesophageal nodose ganglia by immunostaining and Western blot with similar antibody, and by RT‐PCR 1 and DP2 receptors located in the urothelium and smooth muscle and that DP1 receptors were prominent on the membranes of smooth muscle cells, in agreement with our previous study on bladder detrusor 2 might therefore be suggested to directly bind to the receptors located on the smooth muscle, tentatively regulating the contractility by modulating muscle cAMP level since this is a known mechanism in the PGD2 inhibitory action on smooth muscle The validity of our histochemical results on DPin vitro design using male guinea pig trigone and proximal urethra strips. In vivo experiments dealing with the whole LUT with intact neural system will be necessary to determine the exact functional implications of our results. Studies on female urethra should also be carried out in the future.Some limitations of this study include the fact that it was an 2 on the guinea pig trigone and proximal urethra and is consistent with the expression and distribution of DP1 and DP2 receptors in these regions. PGD2 may therefore be a modulator of the bladder out‐flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome. The information is of value for our further understanding of the LUT physiology and provides a foundation for future studies on the human out‐flow region.The present study suggests an inhibitory influence of PGDNG conceived and designed the study and performed all the organ bath experiments after discussion with LG. NG and PdV performed the Western blots and NG and KS performed the immunohistochemistry experiments which were evaluated together with LG and PW. NG compiled the data and made all the draft figures which were finalized together with LG. NG drafted the first manuscript which was reviewed and revised by all authors.The authors confirm that there are no conflicts of interest.


Fibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.We used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification  was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression . Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients.We successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong RNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.The online version of this article (doi:10.1007/s10120-017-0758-x) contains supplementary material, which is available to authorized users. Gastric cancer is the third most frequent cause of death from cancer worldwide . WorldwiOverexpression and gene amplification of fibroblast growth factor receptors 2 (FGFR2) has been associated with poor outcome in gastric cancer , 13. DifFGFR2 gene copy number . For analysis of both overall survival (OS) and recurrence-free survival (RFS), univariate Cox proportional hazards models and Kaplan–Meier estimates were computed, complemented by a multivariate Cox model analysis adjusting for clinical covariates. The latter models were moreover subjected to Akaike information criterion (AIC)-based backward variable selection to ensure robust and informative statistical modeling.All analyses were done in R v3.2.3  ; FGFR2 gene amplification and patient outcome data. Univariate Cox PH and Kaplan–Meier analysis suggested that patients with tumors that had FGFR2 gene amplification had shorter RFS than patients with nonamplified tumors   Fig. a. After  OS Fig. b.Fig. 7PFGFR2 gene amplification with different methods are currently ongoing; however, no FGFR2-targeted agent along with a biomarker assay has been approved yet. FGFR2 mRNA expression rather than FGFR2 gene amplification has been discussed as a biomarker to select patients for FGFR2-targeted therapies in gastric cancer [FGFR2 gene amplification and mRNA expression as biomarkers in gastric cancer. The gold standard to detect FGFR2 gene amplification is FISH. However, the method is expensive and requires fluorescence microscopy, limiting its use in clinical practice. Polymerase chain reaction (PCR)-based methods are cheaper but lack spatial resolution; the latter might be problematic considering that gastric cancer has been shown to have a very high rate of intratumor heterogeneity [FGFR2 gene amplification determined with another in situ technique, dual-color in situ hybridization (DISH), which does not require fluorescence microscopy and is more convenient to use compared to FISH. Very recently, Han et al. performed a study exploring FGFR2 mRNA expression by in situ techniques and FGFR2 protein expression by IHC in 362 surgically resected gastric cancer tissues and 135 matched metastatic lymph nodes from Korea. FGFR2 gene amplification was determined by FISH in 188 of the samples [FGFR2 has been proposed as a target for the treatment of gastric cancer . Severalc cancer . We wantogeneity . We therFGFR2 gene amplification in 578 patients. We were able to generate both FGFR2 gene amplification and mRNA expression for 468 patients. We detected a high level of FGFR2 gene amplification (FGFR2:CEN10 >10) in 2% (12/578) of the samples, and high FGFR2 mRNA expression score 4 was detected in 4% (29/718) of samples. Moderate FGFR2 gene amplification  was detected in 8% (47/578). Previous studies have reported FGFR2 gene amplification in 3.6–9% of gastric cancer cases [FGFR2 gene amplification and 5.8% FGFR2 mRNA overexpression, which is very similar to the 2% of cases with high FGFR2 gene amplification and 4% mRNA score 4 patients we detected in a large cohort of Japanese gastric cancer patients [Not all the available samples were analyzed by DISH; 1036 samples were analyzed by RNAscope and only 967 of those were also analyzed by DISH. Some samples could not be analyzed because the core in the TMA was missing or the sample did not have sufficient tumor content. The percentage of successful analysis of samples was acceptable, with 69% for RNAscope and 60% for DISH (Supplementary Table 3). For 27% of the tested samples the RNAScope assay failed; 36% of the assayed DISH samples did not give a result. The samples analyzed in this study were fixed in unbuffered formalin and stored for more than 10 years, explaining the rather high failure rate. Both in situ techniques detect nucleic acids, and factors preserving or degenerating nucleic acids will affect both methods in a similar way. In line with that expectation, 68% of the samples tested either failed both assays or were successfully analyzed with both methods (Supplementary Table 3). There was no major difference in failure rate between the two methods. The success rate is slightly higher for RNAscope. We cannot exclude the possibility that degradation of nucleic acids in some samples biased the analysis. FGFR2 mRNA was successfully determined in 718 patients and er cases , 37–43, FGFR2 gene amplification also showed high levels of FGFR2 mRNA expression. All samples with dense clusters of RNAscope signal, visible under a 1× objective, showed a high level of FGFR2 gene amplification with a FGFR2:CEN10 ration >10. Surprisingly, these samples also showed a more homogeneous FGFR2 expression within the tumor sample, suggesting that FGFR2 gene amplification might have been an early event in the development of these gastric cancer cases. The scoring algorithm described in the RNAscope kit instructions does not allow separating this potentially biologically distinct subgroup from regular score 4 cases. We therefore suggest adding a score 5 category for samples with dense clusters of RNAscope signal visible under a 1× objective. There were only 12 score 5 samples with high FGFR2 gene amplification in our study. Further studies may help to explore if this subgroup is indeed biologically distinct from other gastric cancer patients.We assessed intratumor heterogeneity using TMAs. The small size of TMA cores as compared to whole-tissue sections is limiting this analysis. As recommended by previous reports, we sought to overcome this limitation by obtaining two cores from different areas to evaluate the tissue heterogeneity, with one sample collected from the intramural area and one from the invasive front area. The statistical power given by the large sample size is thought to compensate for the small size of the TMA cores , 45. In FGFR2 gene amplification with histological subtypes [FGFR2 gene amplification in this study Supplementary material 2 (XLSX 12 kb)Supplementary material 3 (XLSX 11 kb)Supplementary material 4 (XLSX 12 kb)Below is the link to the electronic supplementary material.


Tardive dyskinesia (TD) is a motor side effect that may arise after long-term treatment of antipsychotic drugs. Its etiology is not well understood, but a number of risk factors have been associated with TD. TD occurrence appears to be familial, thus suggesting a genetic component. We previously reported on an association between the SLC18A2 gene that codes for the vesicular monoamine transporter 2 (VMAT2) that packages monoamines including dopamine from the cytoplasm into synaptic vesicles . In the present study, we examined the dopamine transporter gene SLC6A3 by itself and in conjunction with SLC18A2 for possible association with TD.We genotyped and analyzed the variable-number tandem repeat (VNTR) polymorphism in the 3’ untranslated region of the SLC6A3 gene in our European sample of 187 schizophrenia/schizoaffective disorder patients assessed for TD occurrence based on the Abnormal Involuntary Movement Scale (AIMS). We also explored the interaction between the VNTR and the TD-associated SLC18A2 marker rs363224.Our preliminary analysis did not show the SLC6A3 VNTR to be associated with TD occurrence or severity. There also appeared to be no significant interaction between SLC6A3 VNTR and SLC18A2 rs363224 in TD occurrence or severity (p>0.05).Our findings did not support a major role of the dopamine transporter gene in TD risk or severity, but we will examine additional putative functional markers in this gene.


The diagnostic performance of cardiac magnetic resonance (CMR) imaging in suspected myocarditis is still limited. Recently, CMR T1 and T2 mapping has been suggested to yield excellent diagnostic accuracies in patients with suspected myocarditis as compared to healthy controls. However, the true diagnostic performance of CMR mapping when compared to endomyocardial biopsy (EMB) and the impact of CMR field strength in patients with a variety of pathologies is still unknown and was therefore assessed in this study.Within the final analyses, 129 consecutive patients with suspected acute or chronic myocarditis were included. Patients had to fulfill indications for CMR imaging according to the JACC White consensus paper.All patients underwent biventricular EMB, cardiac catheterization for exclusion of coronary artery disease, and CMR on a 1.5  and 3.0  Tesla tomography.The CMR protocol included standard Lake-Louise (LL) parameters as well as native and post contrast T1 and T2 mapping. Relative Enhancement was assessed with a T1 weighted TSE sequence, edema ratio with a T2 weighted STIR sequence. T1 Mapping was measured with a MOLLI-Sequence pre and post contast  and T2 Mapping with a Multi Echo Spin Echo sequence on the 1.5T scanner and a T2 prepared SSFP sequence on the 3T scanner. Patients were divided into 2 groups according to duration of symptoms: patients with acute symptoms (≤14 days) and those with chronic symptoms (>14 days).The diagnostic performance of LL criteria, native T1 and extracellular volume fraction (ECV) as well as T2 mapping is summarized in the table. On 1.5 Tesla, in patients with acute symptoms, native T1 yielded best diagnostic performance (81%) followed by ECV (75%) and T2 (72%). In chronic patients, T2 was the only technique achieving an acceptable accuracy (72%). Accuracies of imaging techniques on 3.0 Tesla CMR were slightly lower as compared to 1.5 Tesla. However, T2 mapping on 3.0 Tesla appeared to be unsatisfactory for the diagnosis of myocarditis.In patients with acute symptoms and suspected myocarditis, mapping techniques provides a useful tool for the confirming or rejecting the diagnosis of myocarditis and are superior to LL criteria. In contrast, only T2 mapping results in an acceptable diagnostic accuracy in patients with chronic symptoms. The impact of field strength on diagnostic accuracies requires further exploration.


Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4C's membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a ‘relay’ mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.The chromosomal passenger complex (CPC)—composed of Aurora B kinase, Borealin, Survivin and INCENP—surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes  In addition, these filaments appeared to promote the closure of membrane gaps over time . This behaviour is similar to that observed previously for the C. elegans CHMP4B homologue  Methionine-labelled CHMP4C and MKLP1 polypeptides were prepared from corresponding PCR products amplified, using primers harbouring a T7 promoter and then transcribed and translated in vitro using the TnT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [35S] methionine (Perkin Elmer). The binding reaction contained 150 mM NaCl and subsequent washes varied from 150 mM to 1 M NaCl. GST pull down assays were carried out as described previously [DNA fragments coding for CHMP4C and MKLP1 fragments were generated by PCR and cloned into pDEST15 (Thermo), using Gateway technology to express N-terminal GST-tagged polypeptides in eviously .


Complete genome sequences of 13 human respiratory syncytial virus strains were determined from samples obtained from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection. We identified amino acid polymorphisms between the NA1 and ON1 genotypes in the P, G, F, and L proteins. Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infection in infants and young children . CurrentWe obtained nasopharyngeal swab samples from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection and isolated HRSV strains after culturing the swab samples in HEp-2 cells . Thirtee7–Among the 13 sequenced isolates, 6 were identified as genotype NA1 and 7 as genotype ON1, both of which belong to HRSV subgroup A. The complete genome sequences of the NA1 and ON1 strains were 15,204 and 15,277 nucleotides long, respectively. As previously reported, the ON1 strains had a 72-nucleotide duplication in the second hypervariable region of the G gene (The reason the ON1 strain has replaced the previously circulating NA1 strain and has become a globally predominant genotype remains unclear . The comKY654506 to KY654518.The complete genome sequences of the 13 HRSV strains in this study have been deposited at GenBank under the accession numbers