Pathways leading to socioeconomic inequalities in young children's body mass index (BMI) are not well understood. This study examined whether parenting related to the physical and social context of children's food consumption helped to explain associations between maternal educational level and child BMI trajectory.The study used data from 2957 families in a nationally representative birth cohort study surveyed from 2004 to 2011, with child BMI z-score measured 3 times . Latent growth curve models examined associations between maternal education and BMI z-score trajectory, exploring mediating effects of parenting  and unhealthy child diet.After adjusting for maternal BMI, maternal education predicted increased inequality in child BMI z-score trajectory slope over the study period. The slope index of inequality coefficient for maternal education, that is, the change in z-score associated with the lowest relative to the highest maternal education level, was 0.17, p<0.001. Indirect effects of lower maternal education on steeper BMI trajectory via parenting and/or unhealthy diet represented 89% of the total effect. Pathways via parenting and then unhealthy diet accounted for 68% of significant indirect pathways, with the remainder via unhealthy diet only. Bedroom TV was the most important parenting pathway, followed by informal meal setting.Pathways via parenting helped to explain the emergence of inequalities in young children's BMI related to maternal education. Interventions targeting parental provision of child bedroom TV and informal meal setting might reduce these inequalities. Parental socioeconomic position (SEP) is inversely associated with childhood obesity in high-income countries. A review found that parental education is the SEP indicator most strongly associated with childhood obesity, even after controlling for maternal body mass index (BMI).Family mealtimes have been a primary focus of research interest in relation to children's diet and weight gain. Eating together is a less-established practice among lower SEP families,Other contextual aspects of family food consumption have been less extensively researched. There are indications that an informal mealtime setting—not being seated at a table—has associations with children's unhealthy dietLower maternal education will be associated with three aspects of parenting: less positive mealtime interaction, an informal mealtime setting  and bedroom TV;All aspects of parenting will have positive associations with unhealthy child diet;Unhealthy child diet will be associated with a steeper BMI trajectory, as evidenced in recent longitudinal research.19This study uses a nationally representative cohort sample to examine associations between maternal education and children's BMI trajectory over a 4-year period, from 46 to 94 months: a critical developmental period when SEP inequalities are likely to emerge and increase.Data were from the first birth cohort of the Growing Up in Scotland study, a nationally representative cohort of families with children born between June 2004 and May 2005. Details of the sampling framework are provided elsewhere.2) was calculated, and standardised BMI z-scores were derived from 1990 British growth reference charts.Height and weight measurements were obtained by trained researchers when the child was 46, 70 and 94 months old. BMI (weight (kg)/height (m)i These were: (1) degree-level qualifications; (2) advanced vocational qualifications ; (3) upper level secondary school qualifications, leading to university entry ; (4) lower level secondary school qualifications ; (5) no qualifications. A slope index of inequalityhighest ranking SEP and one the lowest possible SEP rank.This was based on maternal reports and classified into five groups, using the Scottish Credit and Qualifications Framework.Unhealthy diet was measured at 58 months using six indicators: (1) skipping breakfast on a typical day; number of different types of (2) vegetable and (3) fruit consumed the previous day; and how often the child consumed (4) sweets and chocolates; (5) crisps; and (6) soft drinks, excluding diet or sugar-free drinks. Response options were: skipping breakfast, no (0), yes (1); vegetable and fruit consumption open-ended response (range 0–8); consumption of sweets, crisps and soft drinks, eight-point scale from (1) never to (8) more than once a day. Two indicators of positive mealtime interaction represented mothers' views at 58 months on whether ‘mealtimes are enjoyable for everyone’ and ‘mealtimes give us time to talk to each other’; with responses from (1) never to (4) mostly. Indicators of informal mealtime setting included eating a main meal watching TV (58 months); and the room where the child typically ate a main meal (22 and 58 months). Eating while watching TV was coded from (1) never to (4) often. For room information, seven response options were grouped into (0) rooms with a dining or food preparation area  or (1) those without a dining area . Child bedroom TV was measured using two indicators at 46 and 58 months, coded as (0) ‘no’ or (1) ‘yes’.A latent variable approach was used to model mediators, offering advantages in terms of accurate estimation of indirect effects and allowance for non-normality.These included child gender, exact age in months at first time point of data collection (mean age 46 months), maternal ethnic group (White or Minority) and maternal BMI at child aged 70 months.In total, 3196 families were interviewed at all relevant time points from 10 to 94 months. The analysis was restricted to 2957 cases where the child was a singleton birth, the natural mother was interviewed on all occasions and child BMI information was available at one or more measurement points.Initial visualisation of the data suggested a linear decline in BMI z-scores over the study period. A linear latent growth curve model of mean child BMI was constructed using Mplus V.7.3,A path model was constructed to examine mediation from maternal education to child BMI trajectory via parenting and then child unhealthy diet, as well as via unhealthy diet only. We also included alternative pathways via parenting directly to child BMI , as well as allowing for a direct effect of maternal education on child BMI . Mediation was assessed using the Mplus Model Indirect function, which decomposes the total effect of maternal education into direct and indirect effects. Bias-corrected bootstrapped estimates were produced following recommended practice.Information on child obesity levels and risk factors is provided in ii although mean BMI z-score fell from 0.45 (95% CI 0.40 to 0.49) to 0.36 . The z-score decrease is consistent with declining raw BMI scores across this age range in other UK studies,Obesity levels in the sample  increased slightly from 11% at 46 months to 13% at 94 months,−0.06 . The SSI for lower maternal education was not associated with child BMI intercept at 46 months  and a negative slope of 6 months , but it 6 months  S1.10.1136/jech-2015-206616.supp1Supplementary dataA measurement model was constructed to assess the four latent constructs  used as potential mediators of associations between maternal education and child BMI trajectory. Two indicators of unhealthy diet (soft drink and fruit consumption) with loadings <0.4 were dropped. Further details of this model are provided in an online 10.1136/jech-2015-206616.supp2Supplementary dataEstimates of indirect, direct and total effects of lower maternal education on child BMI slope are shown in The indirect effects of lower maternal education via all mediators (0.16) comprised 89% of the total effect of maternal education on BMI slope. Pathways to BMI slope from maternal education through parenting and then unhealthy diet accounted for 68% of the statistically significant indirect pathways. The main indirect pathway involving parenting was via the effect of bedroom TV, with smaller effects of informal meal setting and less positive mealtime interaction.A sensitivity analysis to allow for the complex survey design was conducted, using mediator factor scores to replace latent variables  require investigation. Our study has several other limitations. We only had child BMI information for three time points, restricting us to a linear latent growth curve model; and modelling maternal education as a slope index assumed a linear relationship with BMI. With the exception of height and weight measurements for child and mother, we relied on maternal reports. The Growing Up in Scotland study is a multipurpose study: thus, many measures are indicative, rather than comprehensive. Maternal BMI was not measured until the child was aged 70 months: future studies should incorporate earlier measures of maternal and child BMI over a longer period. Strengths of the study include use of a large, longitudinal sample that was nationally representative at baseline, incorporating weights to compensate for differential attrition, and use of a latent variable mediator approach.23Inequalities in body mass index (BMI) relating to maternal educational level start to emerge when children are about 4 years old.Pathways underlying the development of these inequalities are not well understood.The study modelled pathways from maternal education to young children's BMI trajectory via parenting and unhealthy diet. It focused on three aspects of parenting more commonly reported by mothers with lower, rather than higher, education levels: provision of bedroom TV for the child; informal main meal setting and less positive mealtime social interaction.A substantial part of the inequalities in child BMI trajectory associated with maternal education could be interpreted in terms of pathways from lower maternal education via these three aspects of parenting to unhealthy child diet, and in turn to child BMI trajectory.Interventions targeting these three aspects of family life might reduce inequalities in young children's BMI trajectories associated with maternal education.In conjunction with the widespread phenomenon of high rates of childhood obesity over recent decades, there is evidence for growing socioeconomic inequalities.


De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody–antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp–Tyr–Lys–Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics.  They have conventionally been developed through experimental approaches, such as animal immunization followed by hybridoma generation and, more recently, screening of synthetic libraries. These approaches are limited for targets that harbor poorly accessible epitopes or require precise molecular engagement to achieve the desired biological effects. Moreover, since sequence diversity expands at a rate of 20n, where n is the number of randomized amino acids, synthetic library sizes rapidly approach the limit of what can be reasonably screened using display technologies. Computational approaches have the potential to dramatically reduce the resources required for antibody discovery while increasing success rates for challenging targets. The growing utility of de novo protein design is demonstrated by a number of recent successes with therapeutic potential, including the design of anti-HIV2 and antimicrobial peptides3, epitope mimics for vaccination4 and influenza inhibitors5.Antibodies are one of the most important protein classes, widely used in commercial diagnostics and therapeutics that garnered global sales of $75 billion in 20136, they are rare. Soon after the first antibody structures were solved, it was recognized that CDR backbones cluster into distinct groups of canonical structures7 with unique amino acid sequence preferences. This observation has facilitated the development of several methods for predicting antibody tertiary structures from their amino acid sequences8. However, despite successes in de novo protein design in general, computational antibody engineering to date has typically focused on improving characteristics of existing antibodies rather than designing novel specificities. Examples include identifying charged mutations that confer thermo-resistance9, guiding affinity maturation10, improving association rates11 and identifying aggregation prone regions12. A recent exception to this trend was the development of a method to rationally design antibodies to bind epitopes in disordered portions of the antigen13. While effective, this method uses an atypical binding mode and is limited to epitopes in disordered regions of protein structure.Antibody-antigen interactions are dominated by the complementarity determining regions (CDRs), three on each of the heavy and light variable domains. Typically, CDRs bind antigens by forming a shape-complementary pocket with favorable interactions distributed throughout the CDRs. Although there are exceptions to this typical binding mode, such as the VRC01 class of broadly neutralizing anti-HIV antibodiesde novo design of antibody binding interfaces complementary to specific three-dimensional epitopes was recently reported14. This approach is unique in that it first selects canonical CDR backbone loop structures, then decorates them with specific amino acid side chains, using energy minimization to refine the structure and maximize predicted interactions with the target epitope. Here, we used this approach to design CDRs binding the minimal FLAG peptide (sequence: DYKD)15. The FLAG peptide is widely used for protein detection and affinity purification in conjunction with commercially available antibodies binding the extended form of the peptide. Additionally, the FLAG peptide has been incorporated into external protein loops without altering core structure16, allowing for its use in combination with an anti-FLAG crystallization chaperone to guide high-throughput structural biology efforts17. As a target sequence, its short length restricts the range of possible conformations while its residues are capable of forming nonpolar, hydrogen-bonding and cation-π interactions. Moreover, the FLAG peptide has been observed to form specific structures18 and thus is a candidate to assess our ability to design antibodies binding small, conformational epitopes versus more flexible linear epitopes.To address these limitations, a computational method, Optimal CDR (OptCDR) for 50] ranging from 4–50 nM) and are extremely specific, as even conservative substitutions in the peptide sequence result in complete loss of binding. As Fabs, they expressed well, were predominantly monomeric and retained binding after several months of storage. These results support de novo CDR design to target specific epitopes as a viable engineering approach.Two libraries were created by full design of all six CDRs (EEf library) or just the three heavy chain CDRs (EEh library). Ten EEf and forty EEh designs were synthesized as single chain variable fragments (scFv) and screened by phage ELISA for binding to the FLAG peptide when presented by a carrier protein. Four antibodies, two from each library, exhibited FLAG peptide binding which was maintained after conversion to a soluble antigen binding fragment (Fab) format. These antibodies bind the minimal four residue FLAG peptide with high sensitivity  optimization formulation then selects the combination of canonical structures with the best score. Generating thousands of random antigen positions leads to the identification of high-scoring antigen position/canonical structure combinations. Because the canonical structures only contain backbone atoms, the rotamer library and rotamer-selection MILP from the IPRO Suite19 are used to fill in the amino acid side chains of the canonical structures while obeying both CDR and canonical structure specific sequence rules. Finally, several thousand iterations of the IPRO algorithm are used to computationally affinity mature the CDRs through the identification of favorable mutations.The OptCDR method 20. The carrier protein used in binding assays was previously crystallized with an n-terminal DYKD tag 18. Only one of the four structures (3ESV) has three of the four tag residues resolved, although the D in peptide position P4 is resolved in all structures. In 3ESV, the kinked conformation of DYKD is stabilized by a hydrogen bond between this D in peptide position P1 and a Q on the carrier protein. Together, these observations suggest that the structure of DYKD in this system is moderately mobile with some stabilization provided by the carrier protein. Since a full structure of the target was not known, a model of the FLAG peptide was built as described in the methods. The minimal DYKD sequence was specified as the epitope in the design calculations. While an intact antibody has six CDRs, the three heavy chain CDRs can be sufficient to confer novel specificity21. Therefore, two libraries were designed: EEf, in which all six CDRs were designed to fully test the modeling process, and EEh, in which only the three heavy chain CDRs were designed and the light chain was not modified.As the framework for the designed CDRs, an scFv with known structure was selected that we previously engineered to bind an EE peptide in silico.For each library, 30 preliminary designs were generated. The top 15 designs, composed of three EEf library Table  and twelFor each selected antibody, several derivative designs were also chosen for experimental characterization. These designs correspond to intermediate CDR amino acid sequences identified during the computational affinity maturation, but prior to the final design being selected. Designs were selected such that they collectively distributed differences throughout as many CDRs as possible. Antibodies are labeled according to whether all six or just the three heavy chain CDRs were designed , followed by a preliminary design number (1–30) and then a derivative design number (0.0–0.6), in which increasing values correspond to increasing numbers of residue changes relative to the preliminary design suggested by computational affinity maturation. Despite better computational predictions, ultimately more EEh than EEf antibodies were selected for experimental characterization based on the hypothesis that grafting fewer CDRs would be less likely to disrupt the framework scFv and thus more likely to yield well-behaved proteins. In total, 10 EEf and 40 EEh designs were synthesized as scFv genes.L-linker-VH orientation were constructed using automated protein fabrication. The synthesis products for each individual library were pooled, cloned en masse into a phagemid display vector and transformed into Escherichia coli. Cloning errors were determined to be less than 5% based on colony PCR, while gene synthesis errors were found to be present in nearly 60% of clones by DNA sequencing, primarily localized to the repetitive GlySer linker region. Accounting for synthesis and cloning errors, sufficient colonies were screened to sample the library size at about three times coverage (72 clones for EEf and 368 for EEh). Phage from individual colonies were propagated and tested by ELISA for binding to three forms of the FLAG peptide: the minimal DYKD tag at the n-terminus and the full-length DYKDDDDK peptide at the n- or c-terminus of the same carrier protein  ranged from 4–50 nM  are on the edges of the binding pockets and are not expected to contribute significantly to binding Figs  and 5B. 23. For comparison, a recent survey of antibody-peptide complexes found these to have average shape complementarity values of 0.75 ± 0.06, while antibody protein complexes were 0.7 ± 0.0624. In EEf15.4, the shape complementarity derives from the peptide P2 tyrosine and P3 lysine residues penetrating deeply into the cavity at the antibody heavy-light chain interface, with the P1 and P4 aspartic acid residues sitting near the antibody surface  in CDR L2. Another key difference is that M2 can bind the full FLAG peptide at any location within a protein, but not the n-terminal core peptide DYKD recognized by the OptCDR designs.These Finally, each of the successful designs interacts with the peptide in a manner that does not disrupt the DYKD peptide structure observed in PDB 3ESV. In particular, the stabilizing hydrogen bond between the peptide P4 aspartic acid and the carrier protein is maintained and the antibodies interact with FLAG from a direction that is not expected to cause irreconcilable steric clashes with the carrier protein. As discussed in the methods, these features were not constraints used during the design process. Moreover, while all four Fabs bind strongly in ELISA, none bind denatured n-DYKD-carrier protein on a Western blot. Conversely, the commercial M1 antibody binds the same n-DYKD ligand in Western blot but not in ELISA, suggesting the peptide is not fully accessible when fused to the carrier protein (data not shown). These data are consistent with the designed antibodies binding a specific three-dimensional conformation of DYKD, as opposed to the extended linear peptide.de novo design. Using two different library approaches, we recovered four unique antibodies with different CDR sequences that each bind the same DYKD peptide conformation, with exquisite specificity. While novel antibody specificities have been rationally designed before26, these earlier examples introduced specificity by designing only the H3 CDR via introduction of hydrophobic or β-strand features that target linear epitopes. Although effective, this mode of binding is rare for natural antibodies. Additionally, the only antibodies designed with such methods have been single-domain antibodies (i.e. a VH domain without a VL domain). In contrast, we designed all or several CDRs to bind structural or linear epitopes by forming a pocket that complements the antigen shape with favorable interactions distributed throughout the CDRs and typical CDR sequences. Antibodies binding a dodecapeptide mimic of mannose containing carbohydrates were designed by a similar approach but required several rounds of molecular dynamics optimization and extensive protein refolding efforts27, whereas the DYKD-peptide binding antibodies reported here relied solely on the initial computational designs and resulted in high yields of folded protein.We have performed one of the first successful demonstrations of epitope-specific antibody binding based on de novo protein design successes is increasing, it is still common for tens or hundreds of designs to be screened before one shows the desired activity28. At a structural level, it is surprising that three of fifteen unique canonical structure combinations resulted in antigen-binding variants. For antibodies to be viable experimental or therapeutic agents, they must possess good affinities and high specificities, and these antibodies meet those criteria. The four successful designs described here exhibit sensitive binding, which was readily detectable at concentrations of 4–50 nM in ELISA using purified Fab proteins and phage-displayed scFv antibody formats. This result contrasts with common results from de novo designed binding interfaces, which usually exhibit micromolar binding affinities5 and require experimental affinity maturation to achieve nanomolar binding affinities29. Beyond sensitive binding, these four designs are also highly specific for the cognate DYKD peptide, as even conservative changes to this sequence abrogated binding . Since shape complementarity correlated with affinity better than any other biophysical parameter in a previous analysis of lysozyme binding Fabs31. Notably, PIGS26 and RosettaAntibody32 predicted main chain breaks in select OptCDR designs (data not shown), suggesting the structures may be poorly matched to the underlying framework. This interpretation is supported by the reduced thermal stability of Fab EEF15.4 as compared to the parent αEE Fab  ligand was created using the build function in PyMOL and docked to the Fab structure of a partially refined commercial FLAG peptide-binding antibody20. The framework residues were included in the OptCDR calculations but not permitted to change. Separate approaches were used to create two unique libraries: a full design in which all six CDRs were optimized (termed EEf) and a heavy chain-only design (EEh) that constrained light chain CDR sequences as wild-type. OptCDR design was performed as previously described14 to (i) select CDR canonical structure backbones most likely to allow favorable interactions with the antigen, (ii) individually initialize amino acid side chains onto the canonical structure using a rotamer library, energy functions, a mixed-integer linear programming optimization formulation and sequence-based constraints; and (iii) several thousand iterations of the Iterative Protein Redesign & Optimization (IPRO) procedure19, which simultaneously refines the CDR backbone structures and amino acid sequences.An scFv we previously engineered to bind an EE peptide served as a framework upon which the CDRs were built, as this framework is predisposed to support peptide binding 38. Briefly, amino acid sequences for VL and VH were reverse-translated using an E. coli class II codon table and combined in the scFv format in the orientation VL-(GGGGS)4-VH flanked by bidirectional SfiI cloning sites. The antibody sequences were clustered based on amino acid sequence similarity prior to reverse-translation of the sequences to enable efficient gene synthesis. Sequence sub-groups were then sequentially assembled to make each of the individual antibody genes. The synthesized products for each library were pooled and cloned into the phage display vector pMopac24, which expresses an scFv fused to a truncated C-terminal pIII protein and co-expresses the protein chaperone Skp to enhance scFv folding. Colony PCR confirmed >95% cloning efficiency, and ten colonies for each library were sequenced to identify the proportion of correctly assembled genes. All oligonucleotides were purchased from Integrated DNA Technologies and all DNA sequencing was performed at the University of Texas at Austin Core Facility.Genes for all 50 designs were synthesized by protein fabrication automation as described previously39. Mutagenic primers were used to alter or eliminate the n-terminal DYKD and to introduce the full FLAG peptide and the n-and c-terminal locations. Briefly, the parental plasmid was methylated using CpG Methyltransferase  for 1.5 h at 37 °C and then used as template in PCR-based site-directed mutagenesis. The PCR reaction followed the commercial instructions, after which DpnI was added to digest methylated template plasmid. The products were transformed into E. coli strain XL1-Blue and sequenced at the University of Texas at Austin Core Facility. The previously described MBP-KEE40 ligand was used to detect binding by the parental α-EE antibody. Production and purification of all peptide ligands was performed as previously described40.All peptide variants were constructed by site-directed mutagenesis of the pAK400/14B7 plasmid that contains a minimal DYKD epitope at the protein n-terminus and employs the irrelevant scFv 14B7 as a carrier protein20 to assess scFv expression level and ability to bind the FLAG peptide and variants. High-binding 96-well plates (Costar) were coated with carrier protein presenting various peptide ligands or control protein lacking peptide (4 μg/ml) or anti-c-myc antibody . Purified phage were added to blocked wells and detected with anti-M13-HRP (1:3000 in 2.5% non-fat milk in PBST). Specific peptide binding activity was calculated the ratio of signal on peptide-coated wells to anti-c-myc coated wells = A450 (n-DYKD)/A450 (anti-c-myc). Secondary screens were performed with phage purified by double precipitation with NaCl-PEG and quantified by plaque formation, as described41. ELISAs were repeated with phage serially diluted at known phage concentrations (measured as phage forming units) to assess binding to the panel of peptide variants.Individual colonies were screened using monoclonal phage ELISA as described previouslyH or VL domain was amplified and sub-cloned into pFabF vector using conventional cloning or the Gibson assembly method, followed by DNA sequencing. These were expressed and purified as described41, with the exception that 10% glycerol was added to all buffers and a 125 mM imidazole wash step was added to selectively elute free light chain.For expression as soluble Fabs, the VCircular dichroism experiments were conducted using a Jasco J-815 CD Spectrometer equipped with Brinkman RC20 water bath and Jasco MPTC-490S/15 temperature control cell. Two spectra from independent purifications of EEF15.4 were measured in a 0.1 cm cuvette with 20 μM EEf-15.4 in TBS , one sample with and the other without 10% glycerol. For thermal melts, spectra were acquired from 200 to 300 nm between 4 to 90 °C with a ramp rate of 1 °C/minute, data pitch of 2 °C and 1 nm bandwidth. Ten spectra were measured at each temperature with a 200 nm/min scan rate. Origin Pro 8.5 was used to plot spectra analyze melt data (CD minimum as function of temperature) using the Boltzmann sigmoid equation.TM) were coated with the naked control carrier protein, n-DYKD and peptide variants (10 μg/mL). After blocking, purified Fabs were serially diluted and detected with goat-anti-human kappa-HRP . Commercial monoclonal anti-Flag M2-HRP  was used as control antibody. GraphPad Prism 6 was used for representation of all ELISA data and all ELISAs were repeated at least twice with separate ligand, Fab and phage preparations.To measure Fab activity and specificity, ELISA was performed with high-binding 96-well plates (Costarhttp://www.maranasgroup.com/submission/OptCDR_2.htm.The data that support the findings of this study are available from the corresponding author upon reasonable request. OptCDR computer codes are freely available with registration on-line at Supplementary informationSupplementary Datasets 1, 2 & 3


Streptococcus strains. These strains otherwise shared a common genome backbone with serotype V ST1 strains. However, the genomes of ST1 serotype V strains and those of serotypes VI, VII, and VIII strains differed substantially.We report several cases of recombination events leading to capsular switching among sequence type (ST) 1 group B  Streptococcus (GBS) is a major cause of invasive infections in neonates. GBS also causes invasive disease in adults, with incidence rates that have been increasing in North America, particularly among the elderly . Because multiple recombination events can endow the receiving GBS strain with an enhanced virulence arsenal, obtaining information about additional genome changes is important.Homologous recombination occurs frequently in GBS and can involve vast areas of the genome in some lineages  that includes all hospitals (n = 28) providing care to and all laboratories (n = 25) processing sterile site cultures from residents of the population area. MLST and serotyping were performed as previously described . These 8cps loci of the 8 strains. In all cases, blastn (http://blast.ncbi.nlm.nih.gov) comparisons identified the cps locus expected for the serotype identified by latex agglutination. To characterize recombination leading to capsular switching, we first used the variant ascertainment algorithm VAAL (https://www.r-project.org), we created plots of polymorphism distribution relative to the genome of the reference strain with a sliding window of 5 kbp. We observed a nonrandom polymorphism distribution in all 8 strains . Based on the available data, it is difficult to ascertain if those strains were originally ST1 strains that acquired a Our data suggest that extensive recombination might be a key contributor to clonal diversification and emergence of GBS serotypes VI, VII, and VIII, which are less often identified in North America but have substantial regional dominance in other geographic areas (Streptococcus isolates collected by the Toronto Invasive Bacterial Diseases Network, 2009–2015, relative to the genome of serotype V sequence type 1 reference strain SS1.Results of a sliding-window analysis of the distribution of single-nucleotide polymorphisms identified in the genomes of 8 non–serotype V sequence type 1 group B


Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis.  The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase,Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection.Methods:In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry.In vivo consequences of infection with PPHD deficientP. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge.Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatantsin vitro.  In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers ofP. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain.  This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain.Conclusions: Our data show thatPseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactionsin vitro andin vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest. Pseudomonas aeruginosa is an opportunistic pathogen, which colonizes the airways of patients with chronic inflammatory lung diseases including cystic fibrosis (CF) and bronchiectasis2, and is an important pathogen in the setting of acute ventilation-associated pneumonia3. In the 2004 US CF patient registry, 57% of patients were found to be colonized withP. aeruginosa4, whilst children with CF who have sputum positive forP. aeruginosa experience more frequent hospitalisation and higher mortality5. These patients have chronic sputum production, with areas of mucus ‘plugging’, resulting in local hypoxia, a condition in which bacteria thrive6. Despite high levels of neutrophilic inflammation,P. aeruginosa continues to survive in these patients, evidence that the bacteria employ a number of effective immune-evasion strategies.P. aeruginosa impairs host neutrophil function is by generation of phenazine metabolites, particularly pyocyanin, which contributes to the characteristic blue-green colour of infected sputum7. Pyocyanin has previously been shown to accelerate neutrophil apoptosis through activation of the lysosomal death pathway, a process dependent upon the generation of reactive oxygen intermediaries within the neutrophil10 and thus on the availability of molecular oxygen11. More recently, the possibilityPseudomonas itself can directly sense and respond to changes in local oxygen availability was raised by the observation thatPseudomonas spp contain a 2-oxoglutarate (2OG)-dependentPseudomonas prolyl hydroxylase (PPHD), which acts on the abundant translation elongation factor Tu (EF-Tu) and is homologous to the oxygen sensing hypoxia inducible transcription factor (HIF) prolyl hydroxylase (PHD) enzymes described in eukaryotes12.A key mechanism by whichP. aeruginosa lacking PPHD manifests increased production of pyocyanin under normoxic (room oxygen) standardized broth culture conditions. Moreover, growth ofP. aeruginosa under conditions of hypoxia has been observed to reduce the pathogenicity ofP. aeruginosa through repression of production of the siderophores pyoverdine and pyochelin and the secreted virulence factor Exotoxin A14. Thus, the outcomes of host-pathogen interactions may be in part defined by adaptation of both the host and the pathogen to local oxygen availability. In this context, we hypothesised that (1) a PPHD-deficientP. aeruginosa sp might demonstrate a survival advantagein vivo as a consequence of increased pyocyanin production, leading to accelerated neutrophil apoptosis and impaired neutrophil mediated bacterial killing, and (2) that these effects would be influenced by oxygen availability.Importantly, an insertional mutant strain of15.All participants gave written informed consent in accordance with the Declaration of Helsinki principles, with AMREC approval for the study of healthy human volunteers through the MRC/University of Edinburgh Centre for Inflammation Research blood resource (15-HV-013). Human peripheral blood neutrophils were isolated from whole blood using dextran sedimentation and discontinuous Percoll gradientsPA0310 insertional knockout mutant strain (PPHD knockout) pseudomonas and then incubated overnight at 37°C. The following day, ten colonies were taken from the plate using a sterile inoculating loop and used to inoculate 15 ml of sterile Luria-Bertani (LB) broth  in a 50ml Falcon tube. The tube was then incubated at 37°C on a shaking platform with the lid loosened. Optical density at 595 nm was measured regularly until plateau.A Columbia blood agar culture plate  was inoculated with a single bead from a thawed master stock vial of either wild-type (PA01) orad librium. All efforts were made to ameliorate any suffering of the animals. Mice were closely monitored over the course of the experiments and humanely culled once threshold of severity was reached.All animal experiments were conducted under an Home Office approved project license in accordance with the Home Office Animals (Scientific Procedures) Act 1986 and University of Edinburgh guidelines in line with the NC3Rs. Six to eight week male C57Bl6J mice were group-housed under standard 12hr light/dark cycles with access to food and water7 cfu of either PA01 (wild-type) or PPHD knockout out (mutant) pseudomonas instilled in 50μl PBS via the endotracheal. Twenty minutes after anaesthesia, the mice were given atipamezole , an anaesthetic reversal agent, and recovered for six hours. At indicated time points  mice were assessed and tissues harvested. For the Kaplan-Meier plots, mice were culled once the threshold of sickness was reached.Mice were anaesthetised with ketamine  and medetomidine  intraperitoneally. Once adequately anaesthetised, the animals were suspended from a frame by the upper incisors and a blunt needle was passed into the trachea via the orotracheal route. Each mouse then had 1×10Bronchoalveolar lavage (BAL) was obtained by cannulation of the trachea. Total cell counts were calculated using haemocytometer counts and differential cell counts assessed on cytocentrifugation slides. IgM levels were quantified using commercially available kits .For histological analysis, lungs were fixed with 10% buffered formalin and embedded into paraffin blocks. Tissues slices were fixed and stained with haematoxylin and eosin.6 cells were centrifuged at 300g for 5 minutes at 4°C. Cell pellets were resuspended in 50uL of FC block  and 1:10 mouse serum in FACS buffer (PBS with 0.5% BSA and 0.02mM EDTA) and incubated on ice for 15 minutes. Subsequently, cells were stained with 50ul anti-Ly6G Ab  at 1:200 final concentration and incubated on ice for 30 minutes in the dark. Following a wash with FACS buffer and centrifugation at 300g for 5 minutes at 4°C, cells pellets were resuspended in Annexin-binding buffer and Annexin-V PE stain (Becton Dickinson) for 15 minutes at room temperature in the dark. Prior to flow cytometry acquisition, cells were stained with Topro3 APC (Molecular Probes). Neutrophils were gated based on Ly6G expression and Annexin-V and Topro3 expression was quantified.BAL cells were counted and 1×10Cells were acquired using a BD Calibur machine and analysed using FlowJo version 10 software (Tree Star).10-fold serial dilutions were performed on whole blood aliquots and lungs homogenized in sterile tubes following collection of BAL fluid. Three 10μl drops from each of 6 dilutions were then plated onto blood agar plates and cultured overnight in 37°C to calculate viable bacterial counts, which were normalized to count per ml of blood or per pair of lungs.Ten colonies were taken from the blood agar culture plate and used to inoculate plates containing 20ml of pseudomonas isolation agar (Difco). These plates were incubated overnight at 37°C and then placed in direct sunlight for 48h to allow pigment to develop. Each plate was then flooded with 6ml RPMI media  and left at room temperature for 2 hours. The RPMI was removed, spun at 4000g for 15 min, twice, and filter sterilised through a 0.22μm filter to remove any bacteria. To ensure sterility, 100μl of each supernatant was used to inoculate a blood agar plate and cultured for 48h at 37°C. Supernatants were stored at -80°C.2) and hypoxia (3% O2) and supernatants produced as detailed above. 4.5ml of chloroform was added to 7.5ml of sterile bacterial supernatant and vortexed. Samples were centrifuged at 2000g for 10 minutes. 3ml of the chloroform layer was transferred to a clean tube and 1.5ml 0.2M hydrochloric acid was added. Tubes were vortexed and spun at 2000g for 2 minutes. 1ml of the top layer was removed, absorbance at 520nm measured and pyocyanin concentrations determined16.PPHD mutant and wildtype colonies were inoculated into 10 ml of LB broth and incubated overnight at 37°C in a shaking incubator. 1ml of the overnight cultures were then inoculated into 9ml of LB broth and incubated for 2 hours at 37°C in a shaking incubator. 100 μl of each strain was then pipetted onto Pseudomonas Isolation agar (Difco) plates and incubated overnight under conditions of normoxia (21% O6/ml. 75μl of this suspension was cultured with 75μl of either wild-type (PA01) or mutant (PPHD knockout) pseudomonas supernatant for five hours in either normoxia (room air) or hypoxia . After 5 hours, cells were removed from the culture plate and pelleted at 400g for 5 minutes. The pellets were resuspended in 95μl annexin binding buffer and 5μl annexin V/PE (Becton-Dickinson) and incubated on ice for 20 minutes. 100μl of Topro3/APC (Molecular Probes) and 5×104 Countbright™ absolute counting beads  were added to each sample, and samples run using a BD FACSCalibur .Human peripheral blood neutrophils were isolated from whole blood using dextran sedimentation and discontinuous Percoll gradients. Neutrophils were resuspended in RPMI with 20% fetal calf serum  at 10x10Data were analysed using Prism 7.0 software . Unpaired t-tests were used for comparisons between wild-type and knockout sample means. Two-way ANOVA with Bonferroni’s post-test comparisons was performed if multiple time points were used. For comparison of viable bacterial counts, Mann-Whitney test was performed. Survival was analysed using log-rank test. Statistical significance was accepted when p<0.05.P. aeruginosa would affect rates of neutrophil apoptosis, freshly isolated human peripheral blood neutrophils were cultured for 5h with sterile supernatants harvested from wild type PA01 and PPHD-deficient bacterial culturesin vitro, a time-point at which pyocyanin markedly accelerates neutrophil apoptosis8. Total neutrophil numbers and neutrophil viability were assessed by flow cytometry , the PPHD-deficient strain produced significantly higher levels of pyocyanin than the wild-type strain (2) cell culture (2), whilst impairing bacterial growth, had no differential growth effects on PAO1 compared with the PPHD-deficient strain.To address whether the observed differences in neutrophil loss reflected altered pyocyanin production, we measured pyocyanin production over a 48 hour inoculation of blood agar following recovery into RPMI media. Under conditions of normoxia  or PPHD-deficient bacteria. 50% of animals receiving PPHD-deficientP. aeruginosa reached sickness thresholds requiring the animals to be culled by day 5 (4CFU/lung± 5.99×104) compared to wildtype infected mice  was similar between wildtype andPPHD-deficientP. aeruginosa infected mice , suggesting that iron regulation may also be of critical importance in defining the activity of PPHD enzyme activity in a physiological setting12. This is of particularly relevance toPseudomonas spp where enhanced iron redox states enable competitive outgrowth from other bacterial species34.Suppression of PHD activity is described in eukaryotic systems in the context of hypoxia – indeed is central to regulation of the hypoxic responsein vivo data, describing the clinical outcomes when mice are challenged with acuteP. aeruginosa infection in the context of normal lung architecture and therefore relatively preserved local tissue oxygenation. In this setting we observe increased mortality with PPHD-deficient strains as a consequence of insufficient neutrophil host defense and failure to control bacterial replication. Thus, we can now extend the concept that immune cell loss of PHD2 in the context of preserved tissue oxygenation promotes a detrimental immune response to also include detrimental consequences of prokaryotic loss of PPHD expression. This has potentially important ramifications in light of the current development of relatively non-selective PHD inhibitors (which may well inhibit PPHD), as well as the use of iron chelators in the clinical arena, and how they may impact more widely on the host pathogen response with consequence both for the host and the pathogen.In this work, we provideThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Dickinson RS et al.https://doi.org/10.6084/m9.figshare.5484178.v135Raw data counts available via Figshare: In this manuscript the authors clearly describe the fatal effects of PPHD knockout for the clinical outcome of mice challenged with this bacterial strain. The data are convincing and point to the problems possibly occurring with the use of small molecule inhibitors of PHDs. I have a question regarding the signalling pathway(s) underlying the increased Pyocyanin production in PPHD knockout bacteria. If the classical inhibition of PHD activity under hypoxic conditions is involved why is the production of Pyocyanin not increased in the wt strain under these conditions?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this manuscript, Dickinson and colleagues reveal the impact of P. aeruginosa PPHD on the bacteria virulence using in vitro and in vivo models. In particular, the PPHD mutation-dependent  increased concentration of pyocyanin in bacterial supernatants was characterized and associated with increased neutrophil apoptosis. The manuscript is clear, well written and the presented data support the conclusions.The authors studied the impact of pyocyanin on neutrophil survival using bacterial supernatants form WT and PPHD-deficient mutant. In order to evaluate the contribution of pyocyanin on P. aeruginosa-dependent neutrophil apoptosis induction, the authors should additionally infect neutrophils with WT and PPHD-deficient mutant strains and assess neutrophil viability (w/wo oxygen). The comparison with neutrophil apoptosis levels induced with bacterial supernatant should be then discussed.It would be informative to assess the ability of neutrophils to kill WT and MT strains (w/wo oxygen) and the results should be discussed to better interpret in vivo results.In the discussion, the authors say that "the possibility that oxygen may also regulate the behavior of bacterial pathogens has not been considered”. This statement is not true, additional references should be included regarding the O2-modulation of bacteria virulence, adhesion, secretion, etc. In order to better appreciate the contribution of pyocyanin on P. aeruginosa mediated neutrophil apoptosis, the following points should be addressed:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In vitro co-culture of neutrophils directly with pseudomonas species rapidly results in neutrophil loss. This is why we firstly explored the consequence of neutrophil culture with bacterial supernatants before undertaking in vivo experiments in which we were able to directly address the consequence of infection with the different strains of P. aeruginosa on neutrophil survival in a biological setting. Due to the ability of pseudomonas to induce neutrophil apoptosis, this is a difficult question to directly address.  We would however argue that it is the ability of Pseudomonas to evade the host response that is critical in defining the outcome of the infection challenge, a concept supported by the published literature  and our in vivo observations.We are sorry for any confusion caused, in the discussion we do actually state that “until recently, the possibility that oxygen may also regulate the bacterial pathogens has not been considered” and provide a number of references specific to Pseudomonas throughout the text that reference that capacity of hypoxia to alter pathogenicity .We thank Dr Marteyn for the review of our manuscript. The authors very elegantly describe a key role for Pseudomonas prolyl hydroxylase (PPH) in the sensing of oxygen tension and the release of the phenazine exotoxin pyocyanin, this enhances the rate of neutrophil apoptosis and compromises the host innate immune response. The authors competently used two model systems, human neutrophil incubated in the presence of supernatants from wt Pseudomonas or mutant lacking PPH cultured in either normoxia or hypoxia, and a murine pneumonia model. Both systems clearly show that infection with Pseudomonas deficient in PPH results with increased neutrophil cell death and compromised host response to the infection resulting in increased mortality in the murine model. I have one minor question regarding the murine model used. Fig 2E shows a significant drop in BAL neutrophil number at 24hrs, however in Fig 2I the authors analysed BAL from the 12hr time point showing a small but significant shift in neutrophil cell death. The description in the results refers to BAL 24hr data set. If the data is available from 24hr BAL I believe this would enhance Fig 2 and benefit the readers understanding. This manuscript further highlights the importance of understanding how the new generation of small molecular inhibitors that interact with the oxygen sensing pathway need to be comprehensively investigated not only at the cellular/tissues level but also at the point of host pathogen interaction.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank Dr Cowburn for highlighting the importance of the observations detailed in the manuscript.  With respect to the time points studied, given we were already detecting a marked difference in BAL neutrophil counts at 24 hours, and that apoptotic cells are rapidly cleared in the in vivo setting, we chose to study a time point preceding one associated with significant cell loss (12 hours), to enable us to measure changes in surface apoptosis markers. in vivo evidence that prolyl hydroxylase-decientP. aeruginosa infection results in increased levels of neutrophil apoptosis, impaired control of infection, and consequently increased mortality in mice. The authors conclude that the expression of the oxygen sensing prolyl hydroxylase homologue (PPHD) in Pseudomonas regulates neutrophil host responsesin vivo. The importance of this finding is in light of current development of relatively non-selective PHD inhibitors that most likely inhibit both, the host and Pseudomonas prolyl hydroxylase and thus impact both, host and pathogen.The authors provide convincingin vivo experiments and analyses presented in this work appear robust and of high quality. Thein vitro studies in Fig 1B: I only have a minor comment regarding It seems that under normoxia, there was no significant change in neutrophil numbers cultured with WT or PPHD mutant supernatant . Since pyocyanin was significantly increased in the MT under normoxia , why was that not reflected in significant decrease of neutrophil counts under normoxic conditions in MT versus WT (FIG 1B)? Please provide comments. What is the p value: WT versus MT under normoxia in Fig 1B? Is the mild reduction of neutrophil counts significant under normoxia (WT vs MT)?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank Dr Schlisio for her careful review of our manuscript and comments. Although the mean number of neutrophils following co-culture with mutant supernatants is lower than wildtype (1610±426 MT vs 2591±749) this does not reach statistical significance (P=0.89 by two way ANOVA).  We attribute this to significant cell loss with both wildtype and mutant supernatants under normoxic culture conditions given both strains of P. aeruginosa produce toxic levels of pyocyanin in normoxia.


Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches. Esophageal cancer is the sixth leading cause of cancer death worldwide  and esop4EAC is frequently associated with both lymphatic and distant metastases yet current staging modalities including computed tomography (CT), positron emission tomography (PET) and endoscopic ultrasound (EUS) are limited in both sensitivity and specificity . SurgicaThe normal squamous epithelium-lined esophagus is vulnerable to toxic insult from the esophageal lumen. Indeed, chronic reflux of gastric acid and bile is thought to underlie the development of columnar metaplasia, “Barrett's esophagus”, the precursor lesion of EAC . AlthougIntriguingly many of the genetic mutations present in EAC have also been demonstrated in nondysplastic Barrett's epithelium raising the possibility that a transcriptional change such as splicing or RNA-editing, or a post-translational modification is responsible for transformation . If suchCharacterizing the proteomic changes associated with EAC may also allow novel therapies to be designed. Tumor-specific proteins have been exploited as immunotherapeutic targets in other cancer types by engendering a host response to the cancer , in somede novo, expression must be measured using untargeted proteomic methods.To date, no specific markers of EAC have been identified. To identify candidate proteins Quantitative proteomic methods have now been applied across many cancer tissues. Most previous proteomic studies in EAC, however, have only identified a small number of dysregulated proteins limiting the comparisons that can be made between studies or with other cancers  (R&D ID 2006/W/PA/01). All patients gave informed consent and participants and their donated samples were de-identified at the time of recruitment. Patients were selected for relative clinical homogeneity with respect to known prognostic variables including lymphatic metastasis and tumor differentiation . The cliAt the commencement of this study, no shotgun proteomic data were available for esophageal adenocarcinoma tissue to inform a power calculation for sample size determination. The sample number was therefore based on previous esophageal discovery-phase proteomic studies or studies in similar tissue types –22, and supplemental Table S1).Because of the risk of false-positives because of the small sample size, proposed tumor-specific proteins identified by mass spectrometry were additionally verified by immunohistochemistry (IHC) using cores from archival tumors and matched normal and metastatic tissues from an independent cohort of 115 patients with esophageal or EGJ adenocarcinoma  method was adapted for protein extraction and tryptic digestion from esophagogastric tissue .−1. Homogenates were dissolved in FASP lysis buffer , mixed for 20 min at RT and sonicated at maximum amplitude, for 30 s, on ice, using a needle sonicator . Sonicated lysates were heated for 5 min at 95 °C and clarified by centrifugation at 14,000g for 5 min at 20pC before buffer-exchange as per the published FASP protocol (Biopsies between 30 mg and 60 mg in weight were maintained on dry ice until rapid disruption at room temperature (RT) in low-binding micro-centrifuge tubes containing 1 mm ceramic beads  by rapid shaking in a bench-top homogenizer  for 40 s at 6msprotocol . TrypsinTryptic peptides from each tissue sample were independently labeled with one of the 6 Tandem Mass Tag (TMT) reagents  in technical duplicate per manufacturer's instructions . Labeled2O and ∼0.5 μg loaded onto a homemade, 100 μm internal diameter, 20 mm long trapping column packed with 200 Å, 5 μm Magic C18 AQ . Trapped peptides were eluted into a 75 μm internal diameter, 150 mm long analytical column packed with 100 Å, 3 μm Magic C18 AQ (Microcom).Dried, desalted peptide fractions were reconstituted in 5% (v/v) acetonitrile, 0.1% (v/v) Formic Acid (FA) in dH2O (solvent A) and 0.1% (v/v) FA in acetonitrile (solvent B). The gradient was run as follows: 0–1 mins, 95% (A) and 5% (B), 1–56 mins, 65% (A) and 35% (B), 66–76 mins, 20% (A) and 80% (B) using a flow rate of 220 nL/min.For ultraperformance liquid chromatography (UPLC), peptides were separated using a variable solvent gradient created by a combination of 0.1% (v/v) FA in dH5 with an m/z window from 400 to 2000. A maximum of three precursor ions with the greatest peak intensities were selected for both collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD) in the LTQ with analysis in the OT. For fragment ion analysis in the LTQ, the ion population was set to 7 × 103 (isolation width of 2 m/z) whereas for detection in the OT, the ion population was set to 2 × 105 (isolation width of 2.5 m/z), with resolution of 7500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD.Peptides were analyzed in positive ion mode after electrospray ionisation on an LTQ-Orbitrap Velos mass spectrometer . For MS survey scans, the Orbitrap (OT) resolution was set to 60,000 and the ion population was set to 5 × 10Protein identifications were made using the Easyprot platform  . Data maThermo RAW files were converted to peak lists using ReAdW  and CID and HCD spectra were merged for simultaneous identification and quantification as previously described . Peaklism/z, corresponding proteins, protein coverage and raw data files have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository (28) with the dataset identifier PXD004962.All data sets were searched separately, once using a forward and once using a reversed protein database. The peptide false discovery rate was set to 1%. A single unique peptide was accepted for protein identification. Identified peptide sequences, scores, precursor For relative protein quantitation, the TMT reporter ion intensities were extracted for each peptide. An isotopic purity correction was performed within Easyprot for each reporter based on the isotopic distribution of the sixplex-TMT reporters provided by the manufacturer.The ratios of peptide expression between EAC and normal esophagus (TvE) and EAC and normal gastric epithelium (TvG) were calculated for each peptide as the ratio of 126/128 reporter ion intensities and 126/130 reporter intensities respectively. This was repeated for 127/129 (TvO) and 127/131 (TvG) reporter ions as a technical replicate. The geometric mean peptide TvE and TvG ratios were calculated for each protein to derive an estimate of the relative protein expression between patient-matched tissue types, and limiting the skew introduced by outlier ratios (supplementary Methods). The unweighted arithmetic mean and variance of the ratios across experiments was calculated for proteins identified by a single unique peptide in multiple replicates. The log2-transformed protein expression ratios (TvE and TvG) were median normalized to account for systematic errors during TMT labeling such as minor variations in protein loading.A mean expression value was derived across pooled technical and biological replicates by a meta-analysis approach using a fixed-effect model . The invt test  .The intertissue ratios (TvE and TvG) were compared with technical replicates  using Welch's modified t test  as the vt test ; FlignerMethods) . All p v testing . Signifisupplemental Table S2.For verification of relative protein expression, 4 μm sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks derived from the same resection specimens used to collect the fresh tissue for this study and representing normal esophagus, normal stomach and EAC. Sections were subjected to immunohistochemical staining using standard techniques as previously described . Staininsupplemental Table S1). Representative cores (0.6 mm) were transferred to a separate paraffin block as a tissue microarray (TMA). TMA sections (4 μm) were stained as previously and scored using a modified Allred method , FFPE blocks comprising normal esophagus, normal stomach, EAC, normal lymph nodes and, if present, lymph node metastasis were identified for an independent cohort of 115 patients undergoing esophageal resection for EAC between 1994 and 2005  as previously described . WesternFor siRNA experiments, OE33 cells were transfected with vector, nontargeting scrambled sequence siRNA  or siRNA to ARHGDIB  as previously described . Cells wA total of 6349 proteins were identified and quantified across all samples corresponding to 4772 unique Entrez GeneIDs with 744 proteins quantified in both replicates from all seven samples. The protein identifications per patient are shown in A, 2B, median Pearson correlation coefficient (PCC) = 0.9811, p < 0.001). There was also a reassuringly good correlation between different patients  when TvE ratios were considered  demonstrating concordance of the protein expression from histologically similar tissues within a relatively clinically homogeneous patient cohort. As expected, there was no significant correlation between TvE and TvG ratios across patients  underscoring the diversity in protein expression of the tissues studied.The reproducibility of quantification of protein expression was assessed by comparing technical replicates. Expression levels were highly correlated between technical replicates from the same patient's tissues . Those 14 proteins identified by a single peptide and only in a single replicate were considered low confidence identifications and were excluded from further analysis. The expression ratios for the 3082 proteins significantly dysregulated between tissues (FDR-corrected p < 0.05 for either TvG or TvE ratios) were used to produce a two-dimensional protein expression map with vectors of TvG expression on the x axis and TvE on the y axis  and Mucin 5AC are known to be specifically highly expressed in gastric epithelium , 39. Reasupplemental Table S3) were selected for verification by immunohistochemistry (IHC).To test this, a further two proteins predicted to be upregulated in squamous tissue  member 1; HSPB1 and transglutaminase 3; TGM3), two proteins upregulated in tumor but also generally expressed , two proteins upregulated in both tumor and gastric tissue  member 5; HSPA5) and one protein predicted to be specifically highly expressed in tumor  .Sections from the FFPE tissue blocks from the original resection specimens used to derive the fresh tissue samples for proteomic analysis were subjected to IHC . All fivAlthough false positive homogeneous cytoplasmic staining was observed in all or scattered basal crypt epithelial cells in most gastric tissue sections , both HSSAMHD1 showed more widespread nuclear expression across tissues but was mildly upregulated in EAC cells, as predicted. In contrast, EPCAM was very highly expressed in tumor cells with only moderate staining in basal epithelial squamous cells and gastric epithelial cells. Although overall expression of ARHGDIB was indeed higher in EAC compared with normal squamous and gastric tissue, this staining was observed in stromal cells, most likely lymphocytes, rather than epithelial-derived tumor cells.These findings support the accuracy of the quantitative proteomic approach for each of the seven candidates selected. As expected from the proteomic data, EPCAM demonstrated the greatest specificity for tumor cells.To determine the specificity of EPCAM for EAC cells compared with surrounding normal tissues, protein expression was determined by IHC using a tissue microarray consisting; normal gastric tissue, normal squamous tissue, uninvolved lymph nodes, involved lymph nodes and primary tumor samples from resection specimens from 115 patients whom had undergone surgical resection for EAC . EPCAM wThis proteomic strategy has therefore demonstrated the ability to detect relatively specific markers of EAC. Those proteins predicted to be highly expressed in EAC compared with surrounding normal tissues, including several novel candidate therapeutic targets, are presented in more detail in There is a need to identify selective markers of esophageal adenocarcinoma for early diagnosis, to enhance clinical staging and direct novel therapies. This shotgun proteomic study compared protein expression from matched esophageal adenocarcinoma, normal esophagus and normal gastric samples from seven patients and provides quantitative data on protein expression for over 6000 proteins across these tissues. A comparative analysis approach was employed to select tumor-specific proteins and this method was verified to be accurate by IHC. Multiple novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases.Incomplete proteome coverage remains a significant limitation of all proteomic studies, however, and only 744 proteins (12%) were detected in all technical replicates across the seven patients' tissues. Despite this intrinsic limitation of proteomic studies, the technical reproducibility of protein quantitation was very high and a strategy was developed to identify significantly dysregulated proteins.supplemental Table S3). This provides confidence in the predicted expression for proteins with an unknown pattern.The combination of TvE and TvG ratios for each quantified protein allowed the generation of a 2D expression map. The relative quantitative accuracy of this approach was confirmed first by evaluation of proteins with a well validated expression profile  and then subsequently by IHC for 7 further proteins with varied expression profiles . In each case the observed staining pattern mirrored the expected expression from the proteomic data . The variable expression of ARHGDIB noted in the panel of esophageal cell lines may reflect context-dependent regulation. Staining across a larger number of esophageal tumors could establish greater confidence over the cell-specific expression profile.Divergent roles for ARHGDIB have subsequently been proposed in the literature with some evidence for a role in the suppression of metastasis in bladder cancer  and in cThe cancer antigen EPCAM was predicted to be highly expressed in EAC cells compared with surrounding normal tissues and this was indeed observed. This has previously been demonstrated for several cancer types, including esophageal , 56. EPCThe expression of EPCAM was examined in detail by IHC in an independent cohort of 115 esophageal adenocarcinomas with matched normal and metastatic tissues. EPCAM was highly expressed in the clear majority of EACs with higher EPCAM histoscores observed in 85/87 EACs than the median gastric or esophageal scores. EPCAM was also highly specifically expressed in lymph node metastases compared with surrounding normal lymph nodes raising the possibility that it could exploited to enhance clinical staging using novel techniques .in vitro and the authors proposed that EPCAM expression reduced during the process of invasion as cells adopted a more mesenchymal phenotype. This may have clinical implications if anti-EPCAM therapies are to be considered.These data agree with previous work demonstrating overexpression of EPCAM in EAC compared with surrounding normal tissues . A furthn = 97) and women (n = 18) in the TMA.Samples were obtained from six men and one woman  reflectiEAC exhibits a high frequency of DNA mutation and mutant proteins are highly likely to be tumor specific . A limitPotentially because of this limitation, no entirely tumor-specific proteins could be identified in this study. Importantly, however, a group of proteins highly expressed in tumors relative to surrounding normal tissues was proposed . ImmunotThese data have been presented, in part, at the Human Proteome Organisation (HUPO) World Congress in 2012 and the Association of Upper-GI Surgeons of Great Britain and Ireland (AUGIS) in 2013 and published in abstract form :55).http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (28) with the dataset identifier PXD004962. Relative quantitation and significance are provided for all identified proteins .Corresponding proteins, protein coverage and raw data files have been deposited to the ProteomeXchange Consortium (


Bos taurus and Sus scrofa and identified reproductive driver genes based on Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm. Our strategy resulted in 11007 and 10445 unique genes consisting of 9 and 11 reproductive modules in Bos taurus and Sus scrofa, respectively. The consensus module calculation yields an overall 167 overlapped genes which were mapped to 846 DEGs in Bos taurus to finally get a list of 67 dual feature genes. We develop gene co-expression network of selected 67 genes that consists of 58 nodes (27 down-regulated and 31 up-regulated genes) enriched to 66 GO biological process (BP) including 6 GO annotations related to reproduction and two KEGG pathways. Moreover, we searched significantly related TF  and miRNAs  which targeted the genes in co-expression network. In addition we performed genetic analysis including phylogenetic, functional domain identification, epigenetic modifications, mutation analysis of the most important reproductive driver genes PRM1, PPP2R2B and PAFAH1B1 and finally performed a protein docking analysis to visualize their therapeutic and gene expression regulation ability.The biology of sperm, its capability of fertilizing an egg and its role in sex ratio are the major biological questions in reproductive biology. To answer these question we integrated X and Y chromosome transcriptome across different species:  The sex chromosomes XY or ZW in case of mammals or birds varies both phylogenetically and structurally among different taxonomical classes with many similar features and is under selective pressure that shapes their evolution , 2, 3. TBos taurus and Sus scrofa to identify reproductive driver genes based on Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm. The schematic diagram for a multi-step strategy to identify reproductive driver genes is shown in Figure Reproduction is the fundamental biological process by which animals produce their offspring. In mammals it is done by sexual reproduction where a haploid sperm from male fertilizes a haploid oocyte to restore the chromosome number. To explore the reproductive driver genes that can improve the reproductive health and fertility is at the core of reproductive biology. The recent advances in sequencing technologies and its analysis have a strong potential to find out genes that can improve the overall reproductive performance of farm animals including cow and pig. To this end, we first integrated X and Y chromosome transcriptome across different species: C. elegans have been shown important phosphatases for sperm motility [The paternal genetic material across different species is transferred to oocyte by sperm after several dramatic morphological and physical changes . A globamotility . The promotility .The genes involved in sexual development, spermatogenesis, spermatid development and differentiation and pregnancy are of vital importance in reproductive biology. Spermatogenesis is a complex process that is involved in the development of highly differentiated haploid sperms from diploid spermatogonial stem cells that is further divided into three phases of almost equal length, the spermatogoniogenesis , meiosisBos taurus and Sus scrofa and identified important reproductive driver genes including PRM1, PPP2R2B and PAFAH1B1. Interestingly various immune related genes are also expressed together with genes involved in reproduction suggesting its probable role in reproduction specifically in preferential selection of X and Y chromosome bearing spermatozoa before fertilization. The preferential selection of X or Y chromosome bearing spermatozoa by female can have a profound importance for farm animals where farmers can select animals gender without the risk of losing subsequent fertilization ability of sperm in female oviduct that are exposed to sorted sperm [The present study explores the integrated X and Y chromosome trancriptome across ed sperm . FurtherBos taurus and Sus scrofa, respectively, where we normalized expression data with Limma package. Expression data, before and after normalization can be found in Bos taurus and Sus scrofa expression data shared 555 homologous genes which were listed in In present study, we got 11007 and 10445 unique genes in In order to satisfy the preconditions of scale-free network distribution, we need to explore the value of the adjacency matrix weight parameter: power. The power plot was shown in Figure Bos taurus gene expression data as training dataset, constructed network and mined gene modules based on 555 shared genes in Bos taurus. Firstly, the coefficients of dissimilarity between genes were calculated, and the system clustering tree was obtained. Then cut the clustering tree according to standard for hybrid dynamic shear trees. We set minimum number of genes per module to 30 genes and the pruning height to cutHeight = 0.9. We finally got 9 different modules in Bos taurus gene expression data, shown in Figure We regarded Sus scrofa by using same method and parameters as in Bos taurus. We obtained 11 gene modules in Sus scrofa, 2 more modules than that in Bos taurus, as shown in Figure Bos taurus and Sus scrofa. The consistency of modules was displayed in Figure Bos taurus and Sus scrofa can be found in We also screened gene modules in We collected significantly overlapped genes in consensus modules for further analysis, that is to say, a total of 167 genes in column 6 of Table Bos taurus by using Limma package in R language, statistic test volcano plot and heatmap were shown as Figure A gene set with 846 gene which met cutoff (p<0.05 and |log2FC|>0.585) were screened as DEGs in We calculated expression correlation coefficient of 67 interested genes and ticked out gene pairs whose correlation coefficient below 0.8  annotation and 2 KEGG pathways with cutoff of p<0.05. We extracted 6 GO annotations which were related to reproduction to display, as shown in Figure We searched significantly related TF using The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 and got 4 related TFs: ISRE, AP1FJ, RP58, CREL, see Table It can be observed that RP58, ISRE and APIFJ are significantly related to 30, 27 and 19 genes respectively including PAFAH1B1 and PPP2R2B. PLAU, a gene responsible for creating suitable environment for embryo implantation and SCD, a gene responsible for fatty acids have a miRNA binding sites for bta-miR-181a. LDLR that has a demonstrated role in sperm storage tubules in hen  and its Bos taurus suggesting a cross talk between reproductive and immune related genes.The reproduction have several predominant biological processes like spermatogenesis, sexual development and pregnancy. The genes associated with these processes are co-expressed with genes associated with immunity in respective modules indicating interesting cross talk among reproductive and immune related genes. The reproductive driver genes PPP2R2B and PRM1 lies in brown module along with important immunity related genes including TREM-1, TNF and TRAF-6, whereas PAFAH1B1 lies in blue module along with immune related genes IL4, IL6 etc. in It is worth noting that the reproductive driver gene PAFAH1B1 is also associated with synaptic transmission along with other genes including MAOA, CACNB4, CHRNA1, FGF2 suggesting its pivotal role as neuro-transmitter that activates the receptors of another neuron indicating functional role in reproductive endocrinology.We found 6 reproduction related GO annotations  motif is found in eukaryotic intracellular proteins that is involved in microtubule dynamics, cell migration, nucleokinesis and most importantly in chromosome segregation when we look for its function in reproduction.Bos taurus and Sus scrofa. In our prediction, we used Methprimer [Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosine. In mammals, a methylating cytosine within a gene can change its expression pattern, hence regulating gene epigenetically. We searched CpG islands of PPP2R2B, PRM1 and PAFAH1B1 in thprimer  to identWe searched SNPs in PPP2R2B, PRM1 and PAFAH1B1 from dbSNP, shown in Table Protein structures of PAFAH1B1 (PDB ID: 3DT6), PPP2R2B (PDB ID: 4MEW) and PRM1 (PDB ID: 4Y0Q) were downloaded from PDB and docked against MPD3 database ready to dock library using MOE docking tool. Ten conformations were provided by MOE for each ligand. These conformations were arranged according to S score. From 2500 MPD3 database ligands, only top seven individual conformations for every ligand which showed minimum S score and RMSD value were chosen. Along with minimum S score, these selected ligands also showed strong binding with targeted proteins. Among the strongly bonded selected ligands Tannic Acid, 5,6,7,4'-Tetrahydroxyflavanone 6,7-diglucoside, Oolonghomobisflavan-A showed most favorable results with PAFAH1B, PPP2R2B, and PRM1 respectively. The detailed results of docking including information about interacting residues are shown in Table Bos taurus and Sus scrofa. Such an analysis has a wide application in animal reproductive biology where one can target specific set of genes as per own desire to generate desired phenotypes for higher reproductive performance, fertility or sex ratio control.The advances in a recent day molecular biology provides ample information suggesting that phenotypes are actually a result of several genes that works in a network. Reproduction is a complex biological process that involves a number of genes and pathways to work together in a coherent way to bring out the desired phenotypes or germ cells capable of fertilization. WGCNA has been widely used to find out candidate or signature genes for disease condition . We haveThe gene enrichment in co-expression network of genes in present study demonstrates 66 different biological processes including genes related to immunity indicating its connected role in reproduction. It is worth noting that the immunity genes co-express with reproductive genes provide a safe microenvironment for the sperm to develop as well as to fertilize oocyte in germ free environment. Another probable role of those genes could be assumed as generating a suitable environment in female reproductive tract to select specific X or Y bearing spermatozoa . Such geThere are mechanisms that favor the X chromosome bearing spermatozoa to fertilize oocyte. One such mechanism is that at time of spermiogenesis, the tail formation of sperm where certain gene expression benefits X bearing spermatozoa can make the female ratio higher than expected 1:1 , 23. In It is proposed in Mendel's first law that an XY cross will produce a 1:1 male to female ratio in sexually reproducing organisms as evolutionary forces favor parents that spend equal amount of resources on both sexes and an imbalance in this ratio will immediately counter balance the effect in favor of under-presented sex . It has Sperm needs to undergo many post meiotic changes of the spermatogenic process before it starts its journey into the reproductive tract of female. The haploid spermatid undergo striking morphological changes including restructuring and compaction of its chromatin material. This makes the almost complete substitution of histone based nucleosomal and histone based structure with protamine based structure. Protamine protects the paternal genetic material and remains key factor in male fertility . In our To further elucidate the important roles of PRM1, PPP2R2B, PAFAH1B1 we made genetic analysis of these genes. We made a phylogenetic tree, predicted important domains, carried out epigenetic analysis and mutation analysis. Furthermore, to regulate the expression of these gene we performed protein docking so that we can recommend chemicals or food items that are beneficial or harmful for reproduction.The Holstein sperm was separated by flow cytometry and after achieving purity of 95% and stored at -80 C. Then we extracted RNA according to manual performed RNA quality testing and then make the microarray geneChip.Bos taurus generated in our lab (only one sample for each chromosome). 2) NCBI raw transcriptome data of Sus scrofa with accession number of GSE47139 [Sus scrofa.). Both datasets were pre-processed by using R-language Affy package [The hybridized microarrays results in images that were used to generate datasets which were pre processed to get meaningful data sets for further analysis. A particular type of preprocessing is termed as normalization that accounts for systematic difference across different datasets. To identify reproductive driver genes, two datasets were included in present study: 1) X and Y chromosome Affimetrix transcriptome data of GSE47139  was down package , includiBos taurus and Sus scrofa respectively, based on weighed gene co-expression network analysis (WGCNA) algorithm, which is a typical system biology algorithm for constructing gene co-expression network [Bos taurus and Sus scrofa for WGCNA, the network building and module identification process steps in Bos taurus transcriptome were as follows:We constructed co-expression network and mined reproductive related modules in  network . We extrCorrelation coefficient of gene m and gene n was defined as:mn=|cor|, We calculated expression correlation coefficient of every two genes to make a whole correlation coefficient matrix.SWGCNA used power index adjacency function as measurement of correlated indicators, the function was defined as:mn, where, amn=power, then determined weighted factor β, we choose β above 0.9 as a power cutoff.aThe adjacency matrix amn was transformed into a topological matrix Ω=wmn, wheremn referred to adjacency coefficients sum of all connected nodes, km represented connection sum of gene m, dissimilarity of genes was defined as dmn, dmn = 1 - wmn.lmn defined before, tree branches represented different gene modules.We constructed hierarchical clustering tree based on dSus scrofa based on WGCNA with same processes and cutoffs as in Bos taurus described above. Then we calculated overlapped genes of each consensus modules in Bos taurus and Sus scrofa, the selected overlapped genes in consensus modules were used for further analysis.We also identified reproductive related modules in Bos taurus were analyzed with the test method in Limma package [Filtered expression data of  package , implemeWe calculated gene expression correlation coefficient of every two selected shared genes, and kept gene pairs with correlation coefficient higher than 0.8 to construct gene co-expression network. Network was visualized by Cytoscape 2.8.0 software .To further analyze the functions of network genes, we enriched genes in the co-expression network to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways by using clusterProfiler  with a cWe searched significantly related TF by using The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 , and extPhylogenetic tree was made using Mega 6 after aligning the downloaded sequences in ClustalW.We searchedwith InterProScan  in EBI, Molecular docking analysis were performed using MOE (Molecular Operating Environment) software . Protein


Acute coronary syndrome with precordial ST segment elevation is usually related to left anterior descending artery occlusion, although isolated right ventricular infarction has been described as a cause of ST elevation in V1–V3 leads. We present a case of a patient with previous inferior wall infarction and new acute ST elevation myocardial infarction (STEMI) due to proximal right coronary thrombotic occlusion resulting in right ventricular infarction with precordial ST elevation and sinus node dysfunction. The patient was treated with successful rescue angioplasty achieving resolution of acute symptoms and electrocardiographic abnormalities.  Several algorithms were established to locate the culprit vessel in ST elevation myocardial infarction (STEMI) with good accuracy –3, but iA 42-year-old male patient with history of hypertension, dyslipidemia, diabetes mellitus, and previous myocardial infarction (MI) presented to the emergency department with acute coronary syndrome. His past coronary event was an inferior MI which was treated with thrombolysis six years ago within 7 hours of onset. Catheterization at that time revealed anomalous right coronary artery originating from the left Valsalva sinus with moderate proximal obstruction and the patient received medical treatment. He had recently performed a clinical evaluation with a normal treadmill test and normal echocardiogram in another hospital.He presented to the emergency department of a non-24/7 primary PCI medical centre with acute onset (2 hours) of severe chest pain and diaphoresis. On admission, he was anxious, his blood pressure was 80/50 mmHg, and he had normal heart and lung auscultation. His first electrocardiogram  showed ePercutaneous coronary intervention was performed in the right coronary artery through right radial approach using Amplatz 1 (6 French) guiding catheter. He received 8,000 U of unfractioned heparin. The lesion was easily crossed with a Runthrough 0.014 guidewire and predilated with a 2,5 × 20 mm balloon with flow restoration. A Zotarolimus eluting stent (Endeavor) 2,75 × 24 mm was implanted at 12 atm and postdilated with a 3,0 mm balloon at 20 atm with clinical and angiographic success. At the end of the procedure, the patient was asymptomatic and ECG showed resolution of ST elevation.The patient had in-hospital course free of complications and predischarge evaluation revealed normal left and right ventricular function (ejection fraction 59%) and complete resolution of precordial electrocardiogram abnormalities and return to normal sinus rhythm . The patAcute myocardial infarction (AMI) with precordial ST segment elevation is usually related to left anterior descending coronary artery thrombotic occlusion and is rarely seen in proximal right coronary occlusion . ElectroSome case reports counteract the classical electrocardiogram finding of ST elevation in DII, DIII, and AVF for right coronary acute occlusion, with most of them being the result of isolated right ventricular myocardial infarction (RVMI) when precordial ST elevation can occur –7. InterIn the reported case, the patient had a mild cardiogenic shock presentation due to STEMI with failed thrombolysis and clear angiographic definition of RCA as the culprit vessel, without significant stenosis on the left coronary artery. Rather important too, the RCA occlusion was proximal to the RV branches and “isolated” RVMI in the setting of a previous inferior wall myocardial infarction was considered the cause of the precordial ST elevation without ST elevation in leads DII, DIII, and AVF. Also noteworthy, sinus node dysfunction was present as ectopic atrial rhythm was shown at presentation and normalized to sinus rhythm only after reperfusion. Some of the above-mentioned electrocardiographic features were present such as convex ST elevation and sinus node dysfunction but decreasing ST elevation from V1 to V3 was not seen in this case. Unfortunately, because our institution only performs right precordial leads on admission in inferior myocardial infarctions, we do not have data on V3-V4R leads which we believe would be showing ST elevation.Although uncommon in proximal RCA occlusion, the electrocardiographic pattern shown in this case may lead to erroneous culprit vessel definition and treatment delay in this severe clinical scenario. Thorough ECG evaluation and complementary quick bedside evaluation can improve diagnosis and shorten door-to-balloon time and result in better patient prognosis.


Cell division is a highly regulated process and involves sequential activation and deactivation of a number of proteins. Mitotic protein kinase MPS1) is a part of the spindle assembly checkpoint (SAC) activated during prometaphase and metaphase that prevents chromosome misalignment by arresting the cell in mid-mitosis until all of the chromosomes is properly attached to the mitotic spindle  is a par. OverexpInhibition of Mps1 activity can result in chromosome missegregation, aneuploidy, and eventually cell death. Thus, MPS1 may be an important target in cancer cells with dysregulated SAC and may be targeted via MPS1 inhibition. Recently several small molecule inhibitors of MPS1 have been developed and tested in pre-clincal models –10. Two Apart from being a major component of SAC machinery, MPS1 has been shown to be involved in turning on Smad signaling by phosphorylating Smad2/3 proteins . Our ownIn addition to regulation of the spindle assembly checkpoint (SAC), MPS1 may also participate in other stress responses. It controls DNA damage responses and genome stability by phosphorylating the Bloom syndrome helicase (BLM), CHK2 in the G2/M checkpoint and c-ABL after oxidative stress –15. MPS1in vitro and in vivo [Eukaryotic cells are subject to tens of thousands of DNA lesions per day. In order to overcome the accumulation of DNA damage and to maintain genomic integrity, cells are equipped DNA damage response machinery to detect and repair these lesions. Recently we published studies examining effect of MPS1 inhibition on glioblastoma (GBM) cell growth  in vivo . Inhibit in vivo . MPS1 inAlthough our research has shed light on the role of MPS1 in DNA damage responses, it raises an important question, “why would cells inhibit the recruitment of DNA repair factors during mitosis”? We know that NHEJ is active during mitosis; however does HR plays any direct role during mitosis? Clearly, additional research is required to further uncover the complex regulation of DNA repair during mitosis.Personalized medicine using small molecule cancer therapeutics is the major focus of current biomedical research. Low molecular weight inhibitors of protein kinases represent a very attractive family of cancer drugs. Taken together, small molecule inhibitors of MPS1 could add important therapeutic benefit for the treatment of various cancers.


ALK FISH probe is the FDA approved approach for detection of ALK rearrangements in lung carcinoma patients who may benefit from ALK kinase inhibitors. The FISH assay can be technically challenging and difficult to interpret. ALK immunohistochemistry and next generation sequencing have been proposed as alternative approaches. In this study, we compared various ALK –FISH patterns to next –generation sequencing (NGS) for gene fusion detection, ALK immunohistochemistry (IHC) and tumor responses to crizotinib. 72 (4%) of 2116 lung adenocarcinoma were positive by ALK- FISH. Of 28 ALK-FISH positive cases selected for the study, FISH patterns included 15 (54%) cases with split signal, 10 (36%) with single orange signal and 3 (10%) with “mixed pattern”. 12 (80%) cases with split signal and 4 (40%) cases with single orange signal were positive by NGS and IHC, while mixed cases were all negative. Mutation analysis of discordant cases revealed multiple mutations including oncogenic mutations in EGFR, KRAS, BRAF and ATM genes. All discordant cases in groups with split and mixed signal showed a lower number of cells with rearrangement . No statistically significant association between response to crizotinib and FISH patterns was observed (p=0.73). In contrast, NGS fusion positive cases were associated with more responses to crizotinib than NGS negative cases (p= 0.016). Our study suggests that ALK FISH alone may not be the most reliable assay for detection of ALK gene rearrangements, and probably should be used in parallel with ALK IHC and NGS for detection of gene fusions and mutations.Break-apart  ALK gene occur in up to 5% of non-small cell lung carcinoma (NSCLC), and are associated with an objective response rate of about 65% in patients treated with the ALK inhibitor crizotinib  after adapter sequences were removed by cutadapt. FastQC  was used for quality control of the raw FASTQ data, and alignStats and SAMStat were used to examine the quality of alignment. Visual inspection of the aligned reads for the fusions was performed in Integrative Genomics Viewer .Gene fusion detection was performed by sequencing of two RNA libraries using 20 ng of RNA (10 ng per amplicon pool) and Ion Total RNA-Seq kit  according to manufacturer's instructions. Briefly, RNA was reverse transcribed and amplified with the two multiplexed fusion primer pools. Primers were subsequently digested, followed by adapter ligation and emulsion PCR. Sequencing was carried out on an Ion Proton instrument. For detection of escribed . First, DNA sequencing was performed using the Ion AmpliSeq™ Cancer Panel  according to the manufacturer's instructions. Briefly, 10 ng of DNA was amplified by PCR using the AmpliSeq™ Cancer Panel Primers pool and Ion AmpliSeq™ Master Mix v2.0. Multiplexed barcoded libraries were enriched by clonal amplification using emulsion PCR on Ion Sphere™ particles (ISPs) (Ion PGM™ Template OT2 200 Kit or Ion PI OT2 200 kit v3) and loaded on an Ion 318™ Chip or Ion P1 Chip (Life Technologies). Massively parallel sequencing was carried out on a Personal Genome Machine™ Sequencer or Ion Proton . The raw signal data were analyzed using Torrent Suite (version 4.0.1) to generate BAM files after signal processing, base calling adapter trimming and alignment to the reference human genome (hg19). Variants were called with Torrent Suite Variant Caller, and were further analyzed using an internally created software suite. Analysis of copy number variations (CNVs) was performed as previously reported .ALK FISH and NGS gene fusion.Sequencing for mutations and CNVs was performed on all cases with discordant results between ALK FISH pattern. Fisher's exact tests were used to test for associations between the ALK FISH pattern and gender or smoking history. Kruskal-Wallis tests were used to test for associations between the ALK FISH pattern and age or disease stage at the time of diagnosis. Patients were classified as either responders  or non-responders (subjects with stable or progressive disease), and statistical association with ALK FISH pattern was tested using a Fisher's exact test. Maximal percent change in tumor size was compared between ALK FISH patterns with a Kruskal-Wallis test and between NGS fusion positive and negative cases with a Wilcoxon-Mann-Whitney test. Statistical analyses were performed using SAS version 9.4  and RStudio version 0.98.1062 using the ggplot2 package.Demographic and clinical characteristics were summarized for all 28 subjects, according to