Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of “type II” hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred. The expanding diversity of noncoding RNA discoveries is revealing a broader spectrum of roles RNA plays in cellular signaling and in biochemical functions. These discoveries in part are being facilitated by the expanding collection of genomic sequence data and by computational methods used to search for novel RNAs. In addition to searching for new classes of structured RNAs, these methods can be used to reevaluate the distributions of long-known RNAs. We have used a bioinformatics search strategy to identify many novel variants of hammerhead self-cleaving ribozymes, including examples from species in all three domains of life. New architectural features and novel catalytic core variants were identified, and the genomic locations of some hammerhead ribozymes suggest important biological functions. This ribozyme class promotes RNA cleavage by an internal phosphoester transfer reaction by using a small catalytic core. The simple sequence and structural architecture coupled with the general utility of RNA strand scission may explain its great abundance in many organisms. Hammerhead ribozymes 2+. Specifically, tertiary interactions form between the loop of stem II and either an internal or terminal loop in stem I that increase activity of the core by several orders of magnitude under low magnesium conditions. Identification of this tertiary substructure in high-speed hammerhead ribozymes Although a minimal three-stem junction constitutes the catalytic core of the ribozyme , additioSchistosoma mansoniArabidopsis thaliana Several searches for new examples of hammerhead ribozymes have been performed previously Using a combination of homology searches we found thousands of new hammerhead ribozyme sequences in all domains of life. These ribozymes are observed in the eubacterial and archaeal domains, as well as in fungi and humans. Moreover, many of the newfound hammerhead ribozymes exploit a pseudoknot interaction to form the tertiary structure necessary to stabilize the positioning of stems I and II. We also identified a number of active sequence variants that suggest the hammerhead consensus is more variable than previously thought.glmS ribozymes Although the biological functions of these hammerhead ribozymes remain unproven, some could be involved in gene regulation based on their genomic contexts, similarly to what has been proposed for the mouse hammerhead and human HDV ribozymes We used a comparative genomics pipeline Dolichopoda (cave crickets) Schistosoma mansoni (parasitic worm) Aedes aegypti (mosquito), Nematostella vectensis (sea anemone), Xenopus tropicalis (frog) and Yarrowia lipolytica (fungus) also appear to have hammerhead ribozymes associated with interspersed repeat elements, which are found in multiple copies in their genomes. Interestingly, we uncovered instances of this motif in humans, and the genetic contexts of two of these are conserved among many mammals.A large number of additional hammerhead ribozymes were identified in metazoans, including mosquitoes and sea anemones. While many hammerhead ribozymes associated with repeated elements were previously found in various species of Our search efforts also extended the range of known hammerhead ribozymes beyond the eukaryotic domain of life. At least three representatives are present in archaea and hundreds are present in bacteria , where mHowever, several novel features for this ribozyme class were observed among the expanded list of representatives. Among the notable variants are the first natural examples of “type II” hammerhead architectures , whereinThe type II hammerhead consensus identified in our bioinformatics search included a putative conserved pseudoknot linking the loop of stem I with the 3′ tail extending from the right shoulder of stem II . Indeed,On many occasions, multiple hammerhead ribozymes are arranged in close proximity to flank individual genes or short blocks of genes in prophage genomes, although the identities of these genes are not constant . Such arClostridium scindens, Azorhizobium caulinodans  for RNA cleavage of greater than 1.2 min−1 under single-turnover conditions and simulated physiological conditions . All deletions or other mutations that are predicted to disrupt the pseudoknot substructure drastically reduce cleavage activity  ribozyme sequence exhibits an observed rate constant  was removed to create a consensus catalytic core undergoes nearly quantitative self-cleavage during transcription (data not shown). Thus, the unusual insertion sequence in this saltern-derived hammerhead ribozyme impairs cleavage activity under standard assay conditions.To examine whether these alterations of the catalytic core reflect adaptations to hypersaline conditions, we prepared wild-type and mutant versions of HHmeta  derived kobs for self-cleavage of HHmeta in a high concentration of monovalent ions alone. HHmeta undergoes self-cleavage with a kobs of 4×10−4 min−1 in 4 M LiCl (data not shown). For comparison, a consensus hammerhead ribozyme catalyzes strand scission with a kobs of 0.17 min−1 under similar conditions kobs that is 425-fold faster than that of the saltern-derived variant.To test whether elevated salt concentrations can rescue this deficiency, we first determined the kobs values over a range of Mg2+ concentrations. The activity of the variant increases with increasing Mg2+ levels .Our homology searches reveal the presence of nine regions in human genomic DNA that conform to the consensus for hammerhead ribozymes  of C10orf118 , a negative regulator of certain metalloproteinases involved in tumor suppression RECK hammerhead also tested positively for cleavage in vitro , suggesting that this ribozyme may be active in vivo.The second human hammerhead we subjected to further analysis, termed “in vitro . AccordiThe application of increasingly powerful bioinformatics algorithms to the expanding collection of DNA sequence data is facilitating the discovery of novel noncoding RNAs and revealing new locations for previously known examples. A recent report Previous in vitro selection studies demonstrated that hammerhead ribozymes are among the first self-cleaving motifs to emerge from random-sequence populations Although the hammerhead consensus is highly conserved, there are rare instances in which the catalytic core is altered. Previous studies have established that mutations at most positions in the core resulted in drastic loss of activity The diversity of structural alternatives observed in our hammerhead collection hints at the inherent difficulty in any effort to comprehensively identify ribozyme representatives. Including more core variations or distal structure variations in search outputs will result in larger numbers of false positives. Given the simplicity of the motif, sequences that conform to the consensus are expected to occur by chance in large sequence databases, even if some of them might be incapable of folding into an active hammerhead.Aedes and Nematostella, organisms in which active hammerheads might require dimeric conformations . For most organisms, this divalent magnesium concentration is not attained. However, for microbes inhabiting certain environments, such as the Dead Sea or high salinity zones of solar salterns, growth has been reported in extracellular MgCl2 concentrations ranging from 0.6 to >2 M 2+, estimates of the intracellular Mg2+ concentrations range as high as 0.42 M For some hammerhead variants such as HHmeta, activity may be facilitated by extreme salt concentrations. In vitro assays reveal that HHmeta requires at least 75 mM MgClAlternatively, it is possible that HHmeta and related variants have been selected to function as gene control elements that modulate the expression of associated genes in response to fluctuating intracellular salt concentrations. HHmeta was identified in a metagenome survey as part of a short sequence fragment, and therefore its genomic context is unknown. However, the structurally analogous hammerhead ribozyme variant HHphage , which rDilution of the environment is highly toxic for obligate extreme halophiles. Accordingly, for certain bacteriophages that infect these organisms, virulence is tightly controlled in response to salt concentrations The discovery of thousands of new hammerheads in all three domains of life provides many opportunities to examine the functions and biological utilities of these ribozymes in their natural contexts. The activities of some representatives may be regulated by RNA folding changes induced by changes in protein, metabolite, or metal ion concentrations, similar to the structure modulation observed with riboswitches. Previous engineering efforts produced numerous examples of allosteric hammerhead ribozymes or other RNAs, establishing a precedent for ligand-mediated regulation of ribozyme function Type II hammerheads were uncovered by a comparative genomics method described previously i.e., N10.1–N11.1, N1.1–N2.1, and A15.1–U16.1) also led us to reject the hit. Finally, multiple mispairs or bulges in short stems resulted in candidate disqualification. The list of rejected hits consisted mainly of cryptic mutant hammerheads that are part of repeated elements, but those occurring in typical gene contexts  were often tested, as they were considered likely functional variants. Initially rejected hits were included in hammerhead alignments if activity could be measured.For final alignments, possible false positives were eliminated based on three criteria. First, any mutation in the core disqualified the hit. For this purpose, the consensus core was considered to be: C3, U4, G5, A6, N7, G8, A9, G12, A13, A14, A15, U16 and H17, where “N” means any nucleotide and “H” means A, C or U. Second, any mispairing directly adjacent to the core in stems I, II or III , Clostridium scindens (ATCC), PaP3 bacteriophage (kind gift of Professor Fuquan Hu) 2, 2 mM spermidine, 40 mM DTT, 2 mM of each ribonucleotide and 40 U µl−1 of purified T7 RNA polymerase. For ribozyme assays in trans, RNA was purified using denaturing PAGE, visualized by UV shadowing, and eluted in 200 mM NaCl, 10 mM Tris-HCl (pH 7.5 at 23°C), and 1 mM EDTA. RNA was then precipitated in ethanol, and the resulting pellet was rinsed in 70% ethanol and resuspended in water. Concentration was measured by UV spectrophotometry with a Nanodrop ND8000 (ThermoScientific).To produce in vitro transcription templates, PCR was performed using genomic DNA isolated from 32P]ATP according to the manufacturer's instructions. Labeled RNA was gel-purified as described, but visualized by autoradiogram.For 5′ labeling, RNA was dephosphorylated with calf intestinal phosphatase (NEB) according to the manufacturer's instructions. Following phosphatase inactivation at 94°C for 3 minutes, 1 pmole of dephosphorylated RNA was typically used for 5′-end-labeling with T4 polynucleotide kinase (NEB) and UTP and [α-32P]GTP. Incubations were continued at 37°C, and 1 µl aliquots were removed at various time points and added to 14 µl of stop buffer. Due to the initially low levels of incorporation of radiolabeled nucleotides, the earliest time point that can practically be assessed is 20 seconds. Note that, due to the requirements of T7 RNA polymerase, the Mg2+ concentrations used in these assays are considerably higher than those used for assays in trans. Note also that HHmeta, because of its requirement for particularly high Mg2+ concentrations, was able to be isolated in precursor form from standard in vitro transcriptions, and was subsequently assayed in cleavage assays in cis.To estimate Figure S1Clostridium scindens. Bands correspond to the expected sizes for hammerhead ribozyme cleavage . (B) Cleavage of internally radiolabeled RNA during in vitro transcription of PCR products from Azorhizobium caulinodans. Full length Azorhizobium caulinodans RNA is not detectable, presumably because of efficient ribozyme cleavage.Activities of RNA transcripts carrying multiple hammerhead ribozymes. (A) Cleavage of internally radiolabeled RNA during in vitro transcription of PCR products from (PDF)Click here for additional data file.Figure S2Agrobacterium tumefaciens multiple hammerhead ribozyme arrangement. (A) Genome context of ribozymes with annotations as follows: “tail” is a structural protein of the phage tail, “ardA” is an Anti-Restriction Defense protein, “marR” is a transcriptional regulation protein and “integrase” is a protein with predicted DNA integration activity. (B) Ribozyme cleavage during in vitro transcription using T7 RNA polymerase to produce internally-radiolabeled RNAs. Bands corresponding to expected sizes and compositions for hammerhead cleavage products are annotated.(PDF)Click here for additional data file.Figure S3Agrobacterium tumefaciens multiple hammerhead ribozyme activity in vivo. (A) Various RNA constructs expressed from plasmids carrying portions of A. tumefaciens hammerheads and expressed in E. coli BL21 cells. Various deletion or mutant constructs are expressed and examined by Northern analysis in the indicated lanes on polyacrylamide gel electrophoresis in panels .  Northern analysis of RNA products express in E. coli BL21 cells from the plasmids (pUC19 with the lac promoter removed) depicted in A. Transcription by T7 RNA polymerase was induced by IPTG and E. coli RNA polymerase was inhibited by rifampicin where indicated to maximize the amount of T7 RNA polymerase transcripts. After 2 h IPTG induction, RNA was extracted with Trizol according to the manufacturer's instructions (in the presence of EDTA). The RNA products were separated by denaturing agarose gel electrophoresis and the resulting gel was used for blotting. Probes for ORF 3, 2 and 1 were successively used to generate the images presented in , respectively. Because probes could not be entirely washed off the membrane between different probing experiments, there is some carry over from the probing in B to C to D. This explains why some bands corresponding to “ORF 2” can be seen in panel D for example.(PDF)Click here for additional data file.Figure S4Pseudoknot interaction between stems I and II. (A) Proportion of each type of hammerhead ribozyme that is predicted to form a pseudoknot. (B) Proportion of hammerhead ribozymes with a pseudoknot versus the length of stem II. (C) Proportion of ribozymes with a pseudoknot versus the difference in length between stems I and II (stem II number of base pairs subtracted from stem I number of base pairs). The highly repetitive type I hammerhead representatives are excluded from the analyses in B and C. Notes: There is some bias in the distributions of pseudoknots with the various hammerhead types and stem lengths. For example, stem length difference disparities between different types of hammerheads is especially striking in type III hammerheads where a two base pair difference between stems I and II commonly are associated with a pseudoknot, while other length differences are not. However, our data indicate that the pseudoknot contact generally is a structurally versatile way to constrain the locations of stems I and II.(PDF)Click here for additional data file.Figure S5Tertiary interactions between stems I and II. (A) Proportion of each type of hammerhead ribozyme that has predicted tertiary interactions noted in the diagrams . Notations: U, uridine; Nm, m number of any nucleotides; Y, pyrimidine; N, any nucleotide; R, pyrimidine; A, adenosine. (B) Proportion of each type of hammerhead ribozyme that have the tertiary interactions depicted in A either with or without a pseudoknot. Note that even when no tertiary interactions are predicted, some unknown interaction or a variation of a known interaction could exist.(PDF)Click here for additional data file.Figure S6Clostridium scindens hammerhead ribozymes are part of larger RNAs transcribed from PCR products. Non-native guanosine residues (lowercase) were added to facilitate transcription in vitro. Gene context is as follows: PaP3 and Bcep176 (phage intergenic regions); Clostridium scindens ; Solibacter usitatus .Complete sequences and secondary structure models of active hammerhead ribozymes that carry core variations. Positions diverging from the core consensus sequence are depicted in red. Variants were tested in trans or in cis as illustrated. Note that the RNA for the PaP3 bacteriophage and (PDF)Click here for additional data file.Figure S7Yarrowia lipolytica . Furthermore, some examples diverged from the consensus at more than one position .Secondary structures of examples of inactive hammerhead-like RNAs. Core nucleotides that differ from the consensus are depicted in red. Except for the (PDF)Click here for additional data file.Figure S8Aedes aegypti (A) and hypothetical dimeric conformation (B). Yellow box highlights loop III that can be completely base paired in a dimeric ribozyme (to form a 10 bp long stem). (B) Blue nucleotides correspond to one ribozyme and black to the other ribozyme. Green nucleotides correspond to one of the most common variants observed. (C) Palindromic character of stem III and loop III sequences for several ribozymes. The vast majority of these sequences would allow base-pairing in a dimeric hammerhead conformation like it has previously been described for ASBVd and newt hammerhead ribozymes Repeat-type putative hammerhead ribozyme arrangement and dimeric conformation. Secondary structures of a putative single hammerhead ribozyme of (PDF)Click here for additional data file.Figure S9C10orf118 expression analysis in human cell lines. (A) The C10 ribozyme is located within the first intron of the C10orf118 gene. The putative start codon of the protein is present at the beginning of the second exon. Several EST sequences have been identified within the first intron of this gene, as shown in the figure (short lines with accession numbers). (B) Expression pattern of EST sequences that map to the C10orf118 gene. %EST designates the proportion of C10orf118 EST sequences that are found in a tissue in comparison to all C10orf118 EST sequences. EST sequences were found in Genbank and GeneCards databases C10orf118 gene. (D) RT-PCR results showing expression of the C10orf118 mRNA in the different cell types indicated. The PCR primers were designed to anneal on the first exon of the gene.(PDF)Click here for additional data file.Table S1Primers used in this study.(PDF)Click here for additional data file.Text S1RNAMotif descriptors for hammerhead ribozymes.(DOC)Click here for additional data file.Dataset S1Multiple sequence alignments of all hammerhead sequences.(PDF)Click here for additional data file.


Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432), but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the ‘Golden Delicious’ genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of  Vitis vinifera L. Rubus ideaus L. Malus pumila L. Borkh. Brassica napus L. Citrus spp. Cynara cardunculus L. As genome and transcriptome sequence data become more accessible due to the availability of affordable second-generation sequencing technologies, the characterisation of single nucleotide polymorphisms (SNPs) has become routine. Thus, large genomic SNP data sets have been reported for many diverse plant species including grapevine  assays permit the economical and reliable genotyping in a semi-automated system, that permits SNPs to be multiplexed to include many tens or hundreds of thousands of markers per assay Zea mays L.) Prunus persica (L.) Batsch Solanum tuberosum L. Helianthus annus L. Solanum lycopersicum L. Malus pumila Mill.) and pear (Pyrus communis L.) using re-sequencing data from 27 Malus genotypes Malus and 921 Pyrus SNPs was developed as part of the International RosBREED SNP Consortium, and hence is denoted IRSC. The array was subsequently used to construct a comprehensive genetic linkage map for an apple rootstock mapping progeny M432 (‘M.27′×‘M.116′) by Antanaviciute et al The Illumina Infinium II technology has been extensively applied to plant species and arrays have been developed for grapevine Malus pumila Mill. is a diploid species (2n = 2x = 34), however, the extant genome of the species is known to have been derived from an ancient whole genome duplication (WGD) event Malus genome sequence, and that an unprecedented number of paralogous gene loci exist within the apple genome. The Infinium array technology generates a signal for SNP calling from the hybridisation of DNA to 50-mer oligonucleotide sequences. The presence of a high degree of paralogy within the Malus genome could lead to non-preferential binding of paralogous gene sequences to what were thought to be locus-specific probes on the IRSC array, causing shifts in clustering patterns obtained from SNP genotyping, and the erroneous scoring of segregating loci in the investigation of Antanaviciute et al V. vinifera) SNPs using other genotyping technology In this investigation, we re-evaluated data generated using the IRSC array for an apple rootstock progeny. We identified widespread incidences of incorrectly assigned genotypes following automatic genotype calling, in both the mapping progeny, and in data from a selection of 192 apple varieties. We attribute the incorrectly assigned genotypes to probe binding sequence paralogy and divergence. The data we present in this analysis demonstrate the potential difficulties that can be encountered when using high-throughput microarray-based genotyping applications in complex, heterozygous, genomes such as apple, in which recent WGD have occurred, but also in polyploid species.Malus genome, probe sequences from the IRSC array were used as queries to BLAST the ‘Golden Delicious’ genome sequence at two different levels of stringency. Only regions that returned a perfect match between the 50 nucleotides of the probes and the genome sequence were considered as positive for the first BLAST analysis, following the Illumina guidelines for probe development. Subsequently, 24 nucleotides from the 3′ end of the probe were used as queries, since this is the section of the probe sited closest to the SNP under assay and thus the most critical section in which to conserve sequence integrity. Due to the physical-chemical properties of DNA oligonucleotide binding and tolerances permitted in nucleotide divergence To determine the proportion of probes that could potentially anneal to paralogous regions of the Malus pumila)   varietieAA genotype, 1 for a BB genotype and 0.5 for an AB genotype. Data generated for SNPs heterozygous in the M432 progeny were exported in text format from GenomeStudio (Illumina). A custom Python script was written to hierarchically cluster these data with a simple agglomerative strategy based on the Euclidian distance of the normalised Theta and normalised R coordinates for each genotype at each locus. The Python script is available upon request from the corresponding author. Several clustering runs were performed with similarity thresholds between clusters of 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5. Clusters composed of fewer than three individuals were removed. The mean normalised Theta and R values for all markers that segregated in a 1∶1 or 1∶2:1 manner at a similarity threshold of 0.15, and for which BLAST data were available, were plotted according to whether the probe sequences returned a single BLAST hit to the ‘Golden Delicious’ genome, or multiple significant matches on multiple chromosomes, and the plots were compared.Single copy, heterozygous SNPs segregating in a diploid genome, cluster with mean normalised Theta values approximating to 0 for an Manually annotated markers were integrated into the consensus linkage map of Antanaviciute et al Of the 8,788 probes on the IRSC SNP array, 7,901 (90%) returned perfect matches to the ‘Golden Delicious’ genome sequence assembly ‘all contigs’ set when the full 50 nucleotides of each probe were used as queries; 6,767 (85.6%) of them returned a single significant association with the ‘Golden Delicious’ genome sequence, 44 (0.6%) returned multiple hits to a single linkage group, suggesting paralogous loci that may have arisen through copy-number variation (CNV) and 1,090 (13.8%) returned multiple hits to multiple linkage groups suggesting annealing to paralogous sequences . When thMalus SNP assays and the remaining 72 were derived from Pyrus SNP assays.Cluster data generated for the M432 mapping progeny using the IRSC array by Antanaviciute et al Malus SNP assays and the remaining 73 were derived from Pyrus SNP assays. Of the remaining 124 loci, 91 loci produced four clear clusters of genotypes, suggesting the segregation of three alleles at the locus (i.e. AB×AC) which could not be assigned genotypic classes for mapping, whilst 33 loci were not annotated because the number of genotype classes observed after cluster separation exceeded four, suggesting segregation of more than one heterozygous locus (i.e. ABAB×ABAB). Each of the misclassified loci for which clusters could be reliably differentiated, was manually annotated and each of the seedlings in the population was genotyped at each locus. Markers previously classified as monomorphic (i.e. AA×AA or BB×BB), and a number of those classified previously as segregating in a 1∶1 ratio (i.e. AA×AB or AB×BB) were reclassified variously as segregating either 1∶1 or 1∶2:1 for those displaying evidence of binding to paralogous sequences , and 1∶1Malus and 72 from Pyrus), 449 segregating approximating to a 1∶1 segregation ratio, 280 segregating approximating to a 1∶2:1 ratio and 68 segregating approximating to a 3∶1 ratio, mapped to one of the 17 linkage groups of the M432 map, bringing the total number of SNP markers mapped using the IRSC array in the M432 progeny to 3,069. The remaining 21 loci, all of which displayed cluster patterns suggesting annealing to paralogous loci, remained unlinked. The manually-annotated SNPs mapped across the 17 linkage groups of the M432 linkage map, and no clustering of re-scored loci was observed suggesting genome-wide incidences of probes binding to paralogous loci and reduced probe binding efficiency due to probe sequence divergence. All markers exhibiting recombination from previously mapped markers, and thus unique genotypes, were visually scrutinised to ensure no spurious genotyping calls had been made following which, a total of just 15.2% of the mapped SNPs revealed unique genotypes and thus map positions. The additional loci did not extend the M432 linkage map significantly, suggesting data for loci mapped in this investigation are robust and reliable. A total of 469 SNPs segregated approximating to a 1∶1 Mendelian ratio, 235 in the female ‘M.27′ genotype, 234 in the male ‘M.116′ genotype, whilst 68 and 281 SNPs segregated approximating to a 3∶1 and a 1∶2:1 Mendelian ratio respectively. Following co-segregation analysis with the data of Antanaviciute et al Of the 797 markers mapped in this investigation, 67 were not physically located to the ‘Golden Delicious’ genome. Of the remaining 730 markers, 470 (64.4%) mapped to the expected linkage group based on the physical location of the marker on the ‘Golden Delicious’ genome, 59 (8.1%) mapped to the identified homeologous linkage group BLAST and cluster analysis data were available for a total of 1,387 heterozygous marker clusters, of which 468 returned significant hits to multiple chromosomes in the ‘Golden Delicious’ genome sequence, whilst the remaining 919 returned a significant match to a single position. When the mean Theta and R values were plotted for each of these loci, clear differences were observed regarding the position of the clusters within graph space , with thMalus probes IRSC array, a total of 157 (19.7%) were derived from suspected divergence in the probe sequence binding site in the M432 parents, 92 of which created a null allele, whilst the remaining 568 (80.3%) SNPs annealed to paralogous loci in the Malus genome. In contrast, 45 (63.4%) SNPs derived from the 921 Pyrus probes on the IRSC array segregated due to heterozygosity due to suspected divergence in the probe sequence binding site in the M432 parents, 14 of which created a null allele, whilst just 27 (36.6%) annealed to paralogous loci  in the M432 progeny that displayed robust, reliable genotypes containing no null alleles and clustering in one, two or three distinct genotype groups. A further 285 (9%) probes displayed a low level of ambiguity that may have been the result of too few genotypes in a particular class and could have revealed clearer clustering with the inclusion of further cultivars in the analysis. The remaining 2,199 (72%) probes revealed cluster patterns containing more than three clusters and could not be assigned to a genotype Malus in which WGD events have occurred relatively recently, high degrees of paralogy in the genome and high levels of sequence divergence between genotypes within probe sequences will render the interpretation of a large proportion of the data generated from genotyping arrays problematic if they have not been taken into consideration during the initial design of the array.Genotyping using high-throughput microarray technology has the potential to fundamentally change the way genomes are genetically characterised and has vastly increased the level of resolution at which genetic analyses are performed. High and ultra-high throughput mammalian Infinium arrays are available for genotyping cows, dogs, pigs, sheep, and goats, and high-throughput Infinium arrays available for maize, peach Previously, using automatic genotype calling with GenomeStudio (Illumina), Antanaviciute et al Malus to which the cultivated apple belongs is a member of the Pyreae tribe of the Spiraeoideae subfamily of the Rosaceae Pyrus spp.), quince (Cydonia oblonga Mill.) and loquat (Eriobotrya japonica Lindl.) and ornamental genera such as the rowans (Sorbus spp.), all of which have the basic diploid genome configuration 2n = 2x = 17. However, the 17 chromosomes of the Pyreae tribe are derived from a recent WGD event as was demonstrated recently by Velasco et al Malus LGs 5 and 10; 3 and 11; 9 and 17; 13 and 16; 6 and 14, and more complex relationships between 4, 12 and 14; 1, 7 and 2; and 8 and 15. As a result of this recent duplication, the Malus genome contains the largest reported number of genes of any plant genome sequenced to date and as such, an exceptionally high number of paralogous loci have been identified in the sequence. Besides the more recent WGD, sequencing of the ‘Golden Delicious’ genome sequence also demonstrated the presence of other more ancient duplication events, including the ancient hexaploidisation event in the lineage of the angiosperms The genus Malus genome sequence aligned to two distinct regions, indicative of the WGD event that occurred 30–45 Mya in the evolution of the Pyrea tribe Malus genome following the WGD event, or possible inconsistencies in the ‘Golden Delicious’ genome sequence assembly. A minority of probe sequences returned significant alignments to between three and six Malus linkage groups, consistent with the paleohexaploidisation event shown to have occurred in the evolutionary lineage of the angiosperms, prior to the WGD event in the Pyrea tribe In line with the findings of Velasco et al. Malus genome. BLAST analysis at two levels of stringency of the 8,788 probes contained on the IRSC array clearly demonstrated the potential for paralogy to lead to non-specific annealing of probes within the Malus genome. This was evidenced by a three-fold increase in probe annealing site paralogy when the criteria for the BLAST analysis were changed to evaluate just the first 24 nucleotides of probe sequences. This study identified erroneously scored data for a total of 595 SNP loci due to shifts in genotyping cluster positions caused by the binding of probe sequences to paralogous loci within the Malus genome. However, a further 487 probe sequences successfully scored using the automated genotype calling function of GenomeStudio (Illumina) and previously mapped by Antanaviciute et al The recent WGD event, more ancient polyploidisation events, and instances of expansion of gene families have all contributed to the high degree of paralogy observed within the Vitis 9k array Gene duplication and thus the occurrence of paralogous loci within a genome is a phenomenon that occurs in all eukaryotic genomes Brassica napus L.), SNP assays contained on a WGG array developed using GoldenGate technology were demonstrated to have amplified two paralogous loci, both of which were heterozygous Malus genome has affected significantly more loci contained on the IRSC array than other polyploid species for which Infinium arrays are available.In contrast, in the polyploid species oilseed rape . However, for such analyses to be successful, it is vital that reliable genotypic data can be generated for many tens of thousands of loci, particularly in species such as Malus, and in maize, the rate of failure has been strongly correlated to the genetic distance of the genotype being assayed from the maize reference genome Mismatches between the IRSC probe sequences and the DNA of the progeny from the M432 mapping population, due to the presence of InDels and additional SNPs, interfered with hybridization signals and caused the failure of a hybridisation assay due to reduced hybridisation efficiency between the probe and the DNA being assayed. This phenomenon is not unique to apple and has been shown to occur in other crop species such as maize and grape SNP) from Malus to Pyrus of between 1.2 and 3.0% for P. pyrifolia and P. communis respectively using the SNPlex platform. Likewise Antanaviciute et al Pyrus SNPs to the M432 progeny when automatic SNP genotype calling was used. In this investigation, we demonstrated that manual annotation of genotype clusters produced from probes developed from Pyrus genomic sequences recovered a far higher percentage of heterozygous loci in Malus germplasm than would have been expected from TSNP values reported for transferability from Pyrus to Malus. This was due to the presence of other polymorphisms associated with the probe sequence and the existence of such variation has implications for the use of these probes in GWAS.Micheletti et al. Malus provides numerous experimental advantages over genotyping using traditional PCR-based assays analysed by electrophoresis Malus since both phenomena, especially when they occur together at a locus, make genotyping data difficult to score reliably. This has been demonstrated in this study through the genotyping of a set of Malus varieties from a diverse germplasm collection, where the majority of loci scored displayed evidence of these phenomena and rendered genotyping unreliable or impossible. For the development of a widely applicable tool for genotyping and marker-trait association, it is thus important to consider the degree of sequence divergence within the probe sequences by developing probes with the assistance of the largest re-sequencing resource possible, encompassing as diverse a germplasm base as possible. This will minimise the level of undetected probe sequence divergence, and thus the prevalence of null alleles and additional multi-SNP alleles within genotyping datasets.The development of array-based genotyping for Figure S1Erroneously scored genotype clusters annealing to multiple genomic regions. Examples of erroneously scored genotype clusters following automatic genotyping using GenomeStudio (Illumina)of SNP that target multiple sites. Red circles and genotypes written in red indicate genotype assignment by GenomeStudio: (a) Data scored as monomorphic AA clearly clustering in three genotype groups (AA/AA:AB/AA:BB/AA); (b) data scored as segregating 1∶1 (AA:AB) clearly segregating in three genotype groups (AA/AA:AB/AA:BB/AA); (c) Data scored as monomorphic AA clearly segregating in two genotype groups (AA/AA:AB/AA); (d) Data scored as monomorphic BB clearly clustering in three genotype groups (AA/BB:AB/BB:BB/BB); (e) data scored as segregating 1∶1 (AB:BB) clearly segregating in three genotype groups (AA/BB:AB/BB:BB/BB); (f) Data scored as monomorphic BB clearly segregating in two genotype groups (AB/BB:BB/BB).(PNG)Click here for additional data file.Figure S2Erroneously scored genotype clusters containing additional SNPs. Examples of erroneously scored genotype clusters following automatic genotyping using GenomeStudio (Illumina) containing additional SNPs (hereafter reported as B’) and null (n) alleles. Red circles and genotypes indicate scores assigned by GenomeStudio, blue circles indicate genotypes assigned ‘no call’ (NC) by GenomeStudio: (a) Data scored as monomorphic BB clustering in two genotype groups (BB’:BB) resulting from BB’×BB; (b) Data scored as BB clustering in three genotype groups (BB:BB’:B’B’) resulting from BB’×BB’; (c) Data scored as BB clustering in two genotype groups (BB or BB’:B’B’) resulting from BB’×BB’; (d) Data scored as BB clustering in two genotype groups (Bn:nn) resulting from Bn×nn; (e) Data scored as BB clustering in three genotype groups (BB:Bn:nn) resulting from Bn×Bn; (f) Data scored as BB clustering in two genotype groups (BB or Bn:nn) resulting from Bn×Bn.(PNG)Click here for additional data file.Figure S3Linkage map of newly-mapped SNP markers. The M432 consensus linkage map detailing the map positions of the 797 novel SNP loci mapped in this investigation following manual re-annotation of data generated with the IRSC array. The scale in centi-Morgans (cM) is given at the left edge of the figure.(PNG)Click here for additional data file.Table S1The names of the 192 Malus varieties genotyped with the IRSC array.(PDF)Click here for additional data file.Table S2The genetic positions, marker names, segregation ratios and physical positions on the ‘Golden Delicious’ genome sequence of the 797 SNP markers mapped to the M432 linkage map.(XLS)Click here for additional data file.


Background. Fibrin sealant became the first modern era material approved as a hemostat in the United States in 1998. It is the only agent presently approved as a hemostat, sealant, and adhesive by the Food and Drug Administration (FDA). The product is now supplied as patches in addition to the original liquid formulations. Both laboratory and clinical uses of fibrin sealant continue to grow. The new literature on this material also continues to proliferate rapidly (approximately 200 papers/year). Methods. An overview of current fibrin sealant products and their approved uses and a comprehensive PubMed based review of the recent literature  on the laboratory and clinical use of fibrin sealant are provided. Product information is organized into sections based on a classification system for commercially available materials. Publications are presented in sections based on both laboratory research and clinical topics are listed in order of decreasing frequency. Results. Fibrin sealant remains useful hemostat, sealant, and adhesive. New formulations and applications continue to be developed. Conclusions. This agent remains clinically important with the recent introduction of new commercially available products. Fibrin sealant has multiple new uses that should result in further improvements in patient care. Fibrin sealant is a two-component material consisting of fibrinogen and thrombin. In the presence of small amounts of calcium and factor XIII, the thrombin converts fibrinogen into insoluble fibrin, the final stable form of the agent. Fibrin sealant now has over a century of development and use. Bergel used fibrin first as a hemostat in 1909 , Matras Although straightforward, a few clear definitions are important in this area. A hemostat causes blood to clot and usually is not effective unless blood is present in the operative field. A sealant creates a sealing barrier that prevents the leakage of gas or liquid from a structure. It polymerizes on its own and is often most effective in a dry field. An adhesive is capable of gluing structures together. It is also self-polymerizing and usually most effective in a dry field. Both sealants and adhesives when applied to potentially leaking blood vessels may have a hemostatic effect by blocking holes in the vessel and preventing bleeding, but they do not necessarily cause blood to clot. Fibrin sealant is the only commercially available FDA approved material for clinical use in all the three of these groupings: hemostats, sealants, and adhesives .For a period of 25 years, it was my own good fortune to have the opportunity to perform both laboratory –24 and cA system of classification for the FDA approved hemostats, sealants, and adhesives has been developed and helps in understanding not only the available commercial products, but also the amazingly diverse abilities of fibrin sealant –45. ThisThe fibrin sealant category of hemostats  is dividThe safety of these liquid agents is most dependent on the fact that multiple donor plasma pools are used for their preparation  , 47. BotThe efficacy of pooled plasma liquid fibrin sealant as a hemostat has been demonstrated in at least 14 multicenter, prospective, randomized trials , 51–61, 2 for a gas source as it dissolves relatively rapidly in human blood and soft tissues as compared to air or nitrogen [Since its approval in 1998, liquid pooled plasma fibrin sealant has been relatively difficult to be reconstituted and used in the operating room . The neenitrogen . My own nitrogen , 51, 64 Liquid pooled plasma fibrin sealants are in the more expensive range of hemostats presently available. These commercial products cost about $50/mL of final mixed fibrin sealant .2 buffer) as well as the materials required to obtain plasma from patient's blood [This class of fibrin sealants contains one commercial product . This form of fibrin sealant comes as a kit containing bovine collagen (20 mg/mL) and thrombin  are similar to those of the pooled plasma liquid fibrin sealants discussed previously , 47 earlThe equine collagen as patch  may cause an allergic reaction in patients' sensitive to horse proteins  and the The fibrin sealant equine collagen patch  is presently FDA approved as a hemostat for use in only cardiac surgery. This patch has been evaluated in multicenter, prospective, randomized clinical trials in cardiac , hepaticThe fibrin sealant oxidized regenerated cellulose patch  is presently FDA approved as a hemostat only in soft tissue surgery. A multicenter, prospective, randomized trial of soft tissue application during retroperitoneal, intra-abdominal, pelvic, and noncardiac thoracic procedures has been completed with clinically and statistically significant benefit in achieving hemostasis in all four arms of the investigation .2 : Evarrest 7.8 mg and 31.5 U per cm2). The manufacturers recommend specific restrictions on the number of patches to be placed that depend on the size of the patch being used.A major advantage of both patches , 75 is tThese patches are the most costly hemostats presently approved by the FDA with prices reportedly approaching $800 per patch.The fibrin sealant category of sealants  consistsThe single product in this class  has been described previously in With respect to efficacy as a sealant, this particular form of fibrin sealant is approved by the FDA as a sealant to prevent leakage of bowel contents at the time of colostomy closure . Using tThe fibrin sealant category of adhesives  consistsThe single product in this class  consists of concentrated fibrinogen (85 mg/mL), thrombin (5 IU/mL), and synthetic aprotinin . It is similar in safety profile  to the pP < 0.0001) and the participating investigators noted statistically significant benefit using the fibrin sealant for graft: adherence quality, fixation method preference, satisfaction with fixation; and overall healing . Patient determined outcomes also favored fibrin sealant with less anxiety and a preference for use of the sealant over staples (both P < 0.0001). In the second study [P < 0.0001).The FDA has approved this adhesive for placement of skin grafts at burn debridement sites as a replacement for sutures or staples and for skin flap attachment at the time of rhytidectomy (face lift) procedures . The thrnd study  using faThe usability of this fibrin sealant adhesive is strongly affected by the additional time (60 seconds) available for manipulation of grafts or flaps in order to assure proper placement. The additional time to complete polymerization offers the advantage of allowing time to properly position grafts or flaps. It should be remembered, however, that this additional time to complete polymerization may allow more time for the liquid fibrin sealant to migrate to the most dependent portions of the wound before sticking particularly in wounds with large topographic differences resulting in an irregular final layer of application. After positioning of the graft or flap, gentle pressure is applied for 3 minutes to assure proper adherence. No additional manipulation of the graft or flap should then occur to prevent disruption of the fibrin sealant adhesive bonds as if this occurs the incidence of seroma formation and drainage volume will increase tremendously  as the fThis fibrin sealant is similar in cost to those commercial pooled plasma liquids approved as hemostats , 47, appA variety of methods for obtaining the fibrinogen and thrombin required for fibrin sealant are available although not FDA approved for any specific indications including ammonium sulfate  or cold A wide variety of devices are now available to assist with obtaining platelet-poor or platelet-rich plasma which can be used as a source of nonconcentrated fibrinogen (20–40 mg/mL) and combined with one of the commercially available free standing thrombins  to obtain fibrin sealant. The available devices  produce fibrinogen that when combined with calcium and thrombin produce fibrin sealant . CalciumThe fibrin sealant literature is quite extensive and continues to grow (approximately 200 papers/year). This literature in essence embodies not only the on-label applications , but alsThe latest laboratory work done on fibrin sealant consists of in vitro, ex vivo, and in vivo experiments and falls into two predominant descriptive bodies of research : evaluatThese papers consist of studies designed to identify the best composition of commercial or experimental fibrin sealant to maximize sealant/adhesive strength. The presently available commercial formulations appear to have some role as sealants but may need additional modifications to be successful in all of the applications that investigators and clinicians envision.P < 0.0001) in a rabbit, ileal intestinal, suture line model that included three lots of each sealant [α-chain cross linking on U-SDS-PAGE testing, and a longer lysis time on urokinase lysis.A study comparing aprotinin containing fibrin sealant (FS-apr) and plasminogen reduced fibrin sealant (FS-rplg) noted significant in-vivo increases in median bursting strength in favor of FS-apr  employed for hernia repair were combined with different commercially available fibrin sealants  to evaluate fixation strength. Researchers created an in vitro strength penetration test using a 45 mm defect in muscle tissue. It was determined by the test that each fibrin sealant had a particular best profile for which polypropylene mesh provided the strongest fixation: Tissucol—Optilene LP; Quixil—Optilene LP; and Evicel—Ultrapro. In addition, the three highest strength combinations of fibrin sealant and mesh were in descending order: Evicel—Ultrapro; Tissucol—Optilene LP; and Evicel—Optilene LP . These a2.A new test to evaluate the cohesive behavior of proteinaceous adhesives based on a “rigid double-cantilever beam” method was used to evaluate the strength of fibrin sealant and its components . This meP < 0.001) as well as TissuePatchDural (P < 0.05). Tachosil had higher burst strength than Tisseel (P < 0.001) and Evicel (P < 0.01) and Pleuraseal had higher burst strength than Evicel (P < 0.05). The authors determined that the liquid fibrin sealants used in this model had significantly less burst pressure than the Bioglue , Tachosil , and Pleuraseal (polyethylene glycol polymer) (P < 0.05).An ex-vivo model using porcine lungs was employed to determine burst strength of uniform pleural defects to compare a variety of potential sealants . Bioglue2) had higher adhesion strength than Quixil  (P < 0.002) or Beriplast  (P < 0.001) and that Tachosil  had higher adhesion strength than Quixil (P < 0.014) or Beriplast (P < 0.009). The authors suggest that the differences in adhesion strength may be influenced by the amounts of fibrinogen, thrombin, factor XIII, and collagen present in each product.Using a canine model of liver resection, comparisons of fibrin sealant adhesion strength were performed  which de23-gauge needle holes in expanded polytetrafluoroethylene (PTFE) grafts were employed as a model to compare a sealant made of gelatin (G) derived from porcine skin and cross-linked with glutaradehyde (GA) with fibrin sealant (FS) (Beriplast). This study  determinA computational model of fibrin sealant adhesion for cartilage implant fixation in the knee joint has been created which strongly suggests that fibrin sealant alone may not be strong enough to withstand in-vivo loading. However, the combination of fibrin sealant and chondrocytes may be more effective at preventing shear damage at the cartilage interface . The autβ chains resulting in the production of fibrin with an increased porosity, viscoelasticity, strength, and degradation resistance. This form of fibrin network may be more receptive to cellular infiltration and tissue regeneration avoiding the difficulties of reduced fibroblast ingrowth associated with the high fibrinogen concentration of commercially prepared fibrin sealants. This paper emphasizes the differences between fibrin monomer production and the assembly of the monomers into a final polymer. The latter appears to be particularly important for tissue regeneration and engineering.One additional publication  evaluateThese reports vary from evaluating the characteristics of autologous or salmon derived fibrin sealant to factors influencing fibrin sealant biodegradation.2 and ethanol to obtain thrombin [P = 0.041). Forty-one units of plasma were used  and each yielded a volume of components (12.4 ± 3.3 mL) including concentrations of fibrinogen (19.9 ± 4.6 mg/mL) and thrombin (25.7 ± 11.1 IU/mL). The study also reported that quality control requirements (5–20 mL yield) using the system during routine use over a three-year period (2008–2010) revealed success rates varying from 90.8 to 97.7%.Investigators evaluated fresh frozen apheresis plasma as a source of allogeneic single donor fibrin sealant using the methods provided in the Cryoseal System  which employs cryoprecipitation to derive fibrinogen as well as precipitation by CaClthrombin . EvidencThe antibody profile of Atlantic farm raised salmon derived fibrin sealant proteins was determined in a mammalian model using rats . IgG andP = 0.01).A report  evaluati125I) labeling at determining the level of fibrinogen in beagle blood following intraperitoneal injection of fibrin sealant (r2 = 0.989).Investigators  determinIt has been found that high levels of fibrinogen bound thrombin (≥820 IU/mL) in clots exposed to cultured cells not only decrease local proliferation of human keratinocytes but may also cause apoptosis .An in-vitro study determined that fibrinolytic activity in human bile caused by tissue plasminogen activator, bile acids, and other bile proteins produced a lytic effect on human fibrin sealant . This inP < 0.05) and increase the amount of vascularized connective tissue (P < 0.05) present.Using an isolation chamber model of an arteriovenous loop, it was reported that levels of fibrinogen in fibrin sealant have a significant effect on the amount of matrix degradation and vascular in growth . Lower lMultiple papers have been published in this area over the last year suggesting that this field of fibrin sealant investigation remains highly active and productive.P < 0.05). This finding was supported by a second publication [Five papers address the ability of fibrin sealant to successfully deliver stem cells as well as suggesting potential modifications in the fibrin sealant composition to improve its efficacy as a growth medium for tissue engineering. The first report found that human cord blood-hematopoietic cell derived three-dimensional scaffolds formed with fibrin (fibrinogen 20 mg/mL and thrombin 20 IU/mL) were most effective in both an in-vitro testing and an in-vivo murine transplantation model as compared to scaffolds formed from polycaprolactone or porous poly(lactide-co-glycolide) in terms of cell growth, maintenance of phenotype, morphology, and engraftment . A seconlication  that foulication . An extelication .P < 0.05) than the other mesenchymal cells. Another report [α to provoke inflammation, the amounts of proinflammatory cytokines were significantly (P < 0.05) reduced in the human and porcine IVD nucleus pulposus tissue embedded in fibrin sealant   suggesting a possible benefit to fibrin sealant injections to treat IVD pathology. Another group of investigators found that fibrin sealant with injectable calcium phosphate cement and bone morphogenic protein induced alkaline phosphatase and achieved stiffness similar to that of normal vertebral bodies at 8 weeks following implantation in rabbit model [P < 0.05) better than controls without stem cells at both bone length and formation in a rabbit tibial model designed to assess vertical bone augmentation [Several papers explored the use of fibrin sealant to facilitate bone growth or reduce inflammation. The first paper used in-vitro cell culture and in-vivo mice subcutaneous implantation experiments to evaluate bone tissue engineering potential created by human mesenchymal stem cells derived from skin, bone, and dental follicles when combined with demineralized bone matrix in fibrin sealant  scaffolds . The invr report  found thr report  used an it model . Similarit model . In dogsit model . Lastly,entation .P = 0.04, and day 7, P = 0.01) accelerate healing as measured by the degree of epithelialization in oral wounds in rabbits [Four other papers discuss the use of fibrin sealant in tissue regeneration using models in specific tissues. These include studies of using a fibrin patch to successfully deliver progenitor cells derived from cardiac adipose tissue into a murine model of myocardial infarction ; a cultu rabbits ; sheep r rabbits  and find rabbits .The new fibrin sealant clinical literature can be grouped into five  predominn = 50) of cases [P = 0.045); successful use [The number of contributions to the ophthalmology literature on fibrin sealant has been accelerating over the past 10 years with 21 papers published over the most recent year. The largest number of references concentrated on using fibrin sealant to assist with intraocular lens fixation. Successful use of fibrin sealant to secure positioning of a posterior chamber intraocular lens is a known technique in ophthalmology with the new contributions suggesting: reduced complications in a series  and flexural rigidity (P < 0.001) producing improvement in mechanical properties in terms of stabilizing and stiffening as well as bending rigidity which may allow for easier manipulation and less “rolling up” during selective corneal transplantation. Clinically, fibrin sealant  has been used to seal microperforations in Descemet's membrane occurring during deep anterior lamellar keratoplasty [An in-vitro study reported that fibrin sealant  Pte Ltd., Singapore) treatment of human Descemet's membrane by creating a network of fibers  significtoplasty  enablingP < 0.006) as well as assessed pain at week 1  and week 2  using fibrin sealant. Two dehiscences occurred in the sealant group (n = 28) and none occurred in the sutured group (n = 29).A variety of clinical applications of fibrin sealant in ophthalmology are focused on sealing of tissues and the avoidance of sutures. These include sealing scleral patch grafts or fistula at the time of drainage tube placement for glaucoma , 137, plP < 0.05) as well as postoperative pain and discomfort [n = 139) in whom conjunctival redundancy was removed using fibrin sealant in a “paste-pinch-cut” technique where placement of fibrin sealant is used to secure the remaining conjunctiva [Four additional reports described successfully using fibrin sealant in a variety of settings. The first was a prospective, randomized study showing that in amniotic membrane grafting for treatment of pterygium, fibrin sealant significantly reduced operative time  or transabdominal preperitoneal (TAPP) approach.n = 50) compared to conventional suturing (n = 52) in terms of operative time , numbness at week 1  and month 1 , analgesia usage (P < 0.05), and postoperative pain at week 1 (P < 0.05) and month 1 (P < 0.05). There was one dehiscence in the fibrin sealant treated group. Another multicenter, prospective, randomized clinical trial of fibrin sealant (n = 159) versus traditional suture (n = 160) of small to medium sized hernia repair was performed by different group of investigators [n > 600) single center retrospective nonrandomized series [P = 0.0344). When the one-year complication data was broken down into active  and retired  subpopulations, the findings favoring reduced complications in the retired fibrin sealant treated group lost their significance. Additionally, no significant differences were found between the fibrin sealant and sutured groups for pain, hernia recurrences, wound healing complications, duration of surgery, length of stay, return to work time, or quality of life evaluations. A nonrandomized series [n = 62) and sutures (n = 54) for mesh fixation in inguinal hernia repair found significant benefit in the fibrin sealant group for days to complete wound healing  and pain score 12 months following surgery (P < 0.001). A final paper [n = 38, 44%), coagulation pathology (INR > 2) , grade B or C child's cirrhosis , or dicumarol-treated cardiovascular disease in a trial of open hernia repair using mesh fixation with fibrin sealant. No complications including infection occurred and the majority of patients (85%) were same day discharges. However, disagreements between some authors continue over published results of fibrin sealant trials for inguinal hernia repair with respect to reductions in operative time, pain, complications, and recurrences [A multicenter, prospective, randomized trial  of fibritigators  because d series . Findingd series  includedd series  comparinal paper  enrolledurrences , 153.n = 25) with wrapping using a compression bandage alone (n = 25). There were significant benefits favoring the fibrin sealant group for the incidence of seroma at week 1  and at month 1 ; seroma volume at week 1 (P = 0.002) and month 1 (P = 0.001); and abdominal wall normalization as determined by patients input at week 1  and at month 1 . An in-vivo study in Wistar rats [P < 0.01). The methodology, however, included the placement of only one drop of fibrin sealant placed at the middle of the patches as opposed to the sutures that were placed at the 4 corners of the patches. Another in-vivo study in pigs using laparoscopic placement [P < 0.001) than butyl-cyanoacrylate (105.35 ± 22.59 N) or bovine serum albumin cross-linked with glutaraldehyde (131.69 ± 24.03 N), although all three glues were significantly better than no fixation at all .Reduction of seromas and adhesions in ventral hernia repair has also been a topic of investigation by multiple groups of investigators. A prospective nonrandomized single center study in patients undergoing laparoscopic repair  comparedtar rats  performelacement  of polyplacement  found thn = 44) over staples (n = 45) in terms of postoperative chronic pain and quality of life. Both groups had a low incidence of recurrence (one in each group). A similar study comparing self-gripping or fibrin sealant placed mesh found no significant difference in postsurgical pain at one and three months and that the operation was statistically significantly shorter by 4.5 minutes (P < 0.006) using the self-gripping mesh [In the setting of laparoscopic TAPP hernioplasty, a prospective randomize single center trial  found noing mesh .P < 0.0001) as measured by the visual analogue scale (VAS), mobility (P = 0.01), and quality of life (P < 0.001). A large review with meta-analysis of laparoscopic TEP found similar recurrence rates for staple and fibrin sealant mesh fixation and a decrease in postoperative chronic pain in the fibrin sealant group [n = 472) using endoscopic TEP repair of inguinal hernias employing fibrin sealant mesh fixation [n = 79) indirect inguinal hernias were specifically treated with an Endoloop  closure of the hernia sac and fibrin sealant mesh fixation with results described as: no postoperative recurrences, 1 seroma, 2 minor complications , and no chronic groin pain [Several papers report results treating inguinal hernias using laparoscopic or endoscopic techniques using TEP approaches with fibrin sealant mesh fixation. Laparoscopic technique was employed in 100 consecutive patients in one report  with no nt group . A singlfixation  reportedoin pain .A recent review of ventral and inguinal hernias  includinn = 10) or fibrin sealant (n = 10). Statistically significant benefit for the fibrin sealant group was found for a reduction in operative time . At the time of necropsy (30 days) all meshes were intact and there was no significant difference in mean peel force between the groups .Finally an in-vivo randomized prospective porcine model  was usedThe recently published papers concentrate on treatment using mesh with fibrin sealant of pneumo-, chylo-, or hydo-thorax as well as tracheobronchial injury.n = 377) treated for intraoperative air leaks undergoing resection for primary or metastatic tumors with fibrin sealant alone (n = 204) or with fibrin sealant plus a polyglycolic acid (PGA) sheet (n = 173). The fibrin sealant application was always done sequentially with rubbing of fibrinogen followed by rubbing of thrombin in the first group and rubbing of fibrinogen, application of the patch, rubbing of thrombin into the patch, and then rubbing of fibrinogen again on top of the patch in the second group. Statistically significant improvement in duration of pleural drainage  as well as incidence of prolonged (>1 week) air leak  was found using the second method. A second group of investigators [P < 0.05). This finding was also supported by a laboratory model that demonstrated a significantly higher seal breaking pressure (P < 0.05) for this third method of application as well. Another human study of sealing video assisted thoracic surgery (VATS) air leaks [n = 206). This group found that: the vast majority of patients (91%) had no air leaks allowing their chest tube removal on the first postoperative day; removal of chest tubes one day after cessation of any observed air leak (one day of prophylactic drainage) resulted in a low level of chest tube reinsertion for recurrent air leak (2.9%); and the need for recurrent chest tube insertion was greater in patients undergoing segmentectomy than lobectomy (P = 0.04). A case report of treating bilateral pneumothorax in a premature infant documented the use of a low calcium (0.59%) concentration fibrin sealant via the chest tubes to eliminate the air leaks and avoid reported complications such as hypercalcemia and bradycardia [A large sequential series over a total of 5 years  comparedtigators  performeir leaks  with fibdycardia . A rare dycardia . This tedycardia .A case report in a neonate with esophageal atresia and trachea-esophageal fistula who developed a chylothorax following primary repair and needed a repeat thoracotomy for treatment added to the existing literature supporting the use of fibrin sealant in this indication .Another report of successful treatment appeared for a case of hydrothorax secondary to peritoneal dialysis using VATS placement of mattress sutures as well as PGA felt and fibrin sealant application .Two reports of successfully treating membranous tracheobronchial rupture also appeared. One employed a combination of small pieces of adipose tissue and fibrin sealant delivered by a catheter through a bronchoscope  and the n = 91, control n = 94) undergoing major thoracic surgical procedures that revealed a mortality of 1.1% in the fibrin sealant group and 5.3% in the control group. There were no tangible differences in the incidence of adverse events between the two groups  including atrial fibrillation , hyperpyrexia , or thromboembolic events (none in either group). Thirty-seven percent of the fibrin sealant patients did develop antibodies to the bovine aprotinin contained in the fibrin sealant with no associated adverse events. There was a significant reduction in postoperative air leaks in the fibrin sealant group , but no significant reduction in the time to chest tube removal.With respect to possible complications of fibrin sealant use in thoracic surgery, a case report suggested that an eosinophilic postoperative pleural effusion (14% eosinophils) was caused by fibrin sealant used to seal a pulmonary bullectomy excision line as documented by a positive drug lymphocyte stimulation test . HoweverThis capability of fibrin sealant remains highly controversial. In my opinion the conflicting evidence relates to the concept of how to effectively employ fibrin sealant either as a sealant/adhesive to prevent serous drainage and seroma formation or as an antiadhesive that may actually increase serous fluid accumulation and seroma formation. The sealant/adhesive technique, although previously detailed in the literature , 32, 36 n = 25) versus quilting sutures with sprayed fibrin sealant (n = 21) conducted over a period of 5 months, significant reductions in the incidence of seroma , seroma volume , total drainage , indwelling period of drainage , and frequency of aspiration  was found in favor of using quilting sutures and fibrin sealant [n = 19) and a quilting with fibrin sealant group (n = 23) in favor of the quilting with fibrin sealant [P < 0.05), but not seroma rate . The quilting sutures were carefully placed and tied to take tension off of the wound closure prior to using the fibrin sealant. Wound closure was then performed after fibrin sealant application using the drip applicator up under the previously placed quilting suture fixed flaps. This method appears to reduce the likelihood of disrupting the fibrin sealant bonds during manipulation of the wound edges. Several reports described the lack of fibrin sealant efficacy including a nonrandomized study of patients by a single surgeon over 5 and 1/2 years [n = 23) was higher than in the fibrin sealant group (n = 23) until postoperative day 2 but beginning on postoperative day 3 and thereafter the control group had less drainage (P = 0.678) and the drain was removed earlier in the control group . In this study no description of the final skin closure and its possible movement of the fibrin sealant adherent skin flaps is made. It is possible that the fibrin sealant bonds were partially broken during skin closure producing an antiadhesive effect. In the second study [n = 53) and slow setting fibrin sealant (n = 52) treated groups powered for size effect, there were no significant differences with respect to the fibrin sealant and control groups, respectively, in seroma volumes , total drainage volumes , or seroma formation . Interestingly grade I/II skin necrosis trended towards reduction in the fibrin sealant group  following drain removal. Although the authors state that slow setting fibrin sealant was sprayed, the concentration of thrombin was reported as 500 IU/mL  making it unclear as to the type of fibrin sealant (fast or slow setting) actually employed. In addition, the authors state that two anchoring sutures were placed on either side of the skin closure about 2 cm away from the wound edge and were not tied until after the fibrin sealant was applied. This method again raises the issue as to whether the skin flaps were significantly manipulated after the fibrin sealant application during the tying of the two flap sutures up under the flaps causing a break in the fibrin sealant bonding of the flaps to the underlying tissues producing an antiadhesive fibrin sealant effect. These four papers appear to strongly reinforce the concepts previously discussed at the start of this section with respect to knowing the importance of how to use of fibrin sealant as a sealant/adhesive or as an antiadhesive.This past-year's literature on using fibrin sealant continues to reflect the controversy on its use to prevent serous drainage and seroma formation. At the latissimus dorsi harvest site during breast reconstruction following mastectomy, two papers support its use , 182 in  sealant . The aut/2 years  and a si/2 years . In the /2 years  as drainnd study  consistiP = 0.06; total amounts, 93.5 ± 30.7 mL versus 105.7 ± 31.2 mL, P = 0.05). The description of the operative procedure states that the fibrin sealant was sprayed, drains were then placed, and finally the wound was closed layer-by-layer suggesting a possible delay between sealant application and flap apposition as well as the potential for bonding disruption. Both techniques could have led to an antiadhesive effect of the fibrin sealant.Another report  describen = 33) versus the nontreated group (n = 37) included reduction in drainage time  and percent of patients without drains at day 21 . The authors point out that this patch was activated after 3–5 minutes of pressure. A case report of using lymphatic mapping, quilting sutures, and fibrin sealant to close a Morel-Lavallee lesion (persistent seroma formation following a degloving injury) of the thigh [Two additional reports of successful use of fibrin sealant to reduce serous drainage appeared. The first was a prospective, randomized, single center study of radical lymph node dissection  where a fibrin sealant coated equine collagen patch was placed over the entire wound prior to skin closure . Signifihe thigh  describen = 31) as compared to control (n = 29) to reduce seroma formation . The authors did note several findings in the fibrin sealant group: a baseline trend towards an increase in patient weight , a significantly reduced seroma aspirate volume , and a trend towards reduced day of seroma resolution . The fibrin sealant spray used was diluted twice (thrombin 125 IU/mL) and sprayed just prior to skin closure of a loose lattice work of sutures that was then followed by even pressure to the wound for 5 minutes. This method may have allowed the more slowly polymerizing fibrin sealant to run off to the more dependent portions of the wound before polymerizing resulting in an uneven seal. The meta-analysis [A prospective, randomized, single center report  performeanalysis  suggesteanalysis .n = 12) was superior to fibrin sealant (n = 12) at reducing seroma volume . A prospective, randomized, clinical study performed in a single center over a period of 7 months found the fibrin sealant spray group (n = 15) to have significantly higher seroma formation than the do nothing (n = 15) and quilting suture groups (n = 13) in abdominoplasty procedures [A rat study  found thocedures . No descn = 165). In this phase III trial, there was no statistically significant difference in the healing rate between the three groups 39.1%, 43.3%, and 37.3%, respectively, at 24–26 weeks (P = 0.79). At one year the rates were 57.1%, 52.4%, and 37.3% (P = 0.13) suggesting a possible trend towards a fibrin sealant benefit. Selective analysis of the initiating center's data alone found significant benefit to treatment with cells and fibrin sealant at 24–26 weeks with healing rates of 54.55%, 83.33%, and 18.18% (P = 0.007). A multivariate analysis revealed that treatment center and fistula severity were independent predictors of fistula healing (P = 0.024). Also analysis of those patients receiving cells with or without fibrin sealant versus fibrin sealant alone revealed a doubling of the healing rate (P = 0.048) in favor of those patients receiving at least some cells. A trend towards antibiotic use as being significant was also noted (P = 0.09). There were no significant adverse events. However a retrospective follow-up study [n = 25) using fibrin sealant or cyanoacrylate (cyanoacrylate in only the first two patients as vulvar ulceration was noted following use in second patient) persisted in only 26% of treated patients for more than 6 months using application via an endoanal ultrasonographic-assisted percutaneous transperineal approach . In the second study over a period of 6 years [n = 28) with transsphincteric cryptoglandular fistulas were treated in two stages. The first stage was a seton assisted fistulectomy of the main tract and the second stage involved insertion of fibrin sealant into remaining secondary segments of the tract. At a mean of 20.6 months, 67.8% had no recurrence (range 3–60 months) and none of these patients developed incontinence. A third large retrospective series [Only one multicenter, prospective, randomized, clinical trial involving the use of fibrin sealant for fistula closure appeared in 2012. It  involvedup study  from theup study , 195. Ine series  of patiee series . A finale series  continueRecent case reports of successful closure of intestinal tract fistulas using fibrin sealant include endoscopic instillation for treatment of esophageal perforation secondary to a fish bone , plateleMultiple additional papers address a variety of other topics and will be presented here in descending order of frequency  for the P < 0.02) increases in healing strength at 2 weeks [P < 0.001) [The platelet plasma literature has several articles discussing the need for consensus on naming the various potentially available products  and thei 2 weeks , skin fl< 0.001) , and too< 0.001) . Several< 0.001)  or PRP, < 0.001)  finding < 0.001) , general< 0.001) , gynecol< 0.001) , rhytide< 0.001) , and vag< 0.001) . Finally< 0.001) .n = 32) was superior to a fibrin sealant-equine collagen patch (n = 18) in terms of median time to hemostasis  during hepatic resection. A rabbit study [In hepatic surgery, fibrin sealant has been evaluated and found to be an effective hemostat particularly in large resections , 221, buit study  of tempoit study  with perit study .P < 0.05). Finally, an in-vitro report of using nimodipine polyglycolic acid microparticles suspended in fibrin sealant as a depot system to treat posthemorrhagic cerebral vasospasm was published [The neurosurgical literature continues to support the use of fibrin sealant for dural closure in specific instances including duraplasty after suboccipital decompression for Chiari 1 malformation in conjunction with autologous pericranium , repair ublished .n = 400) found a significant reduction in drain output using fibrin sealant, but no benefit on hemoglobin levels, transfusion rates, or cost [In orthopedic surgery with respect to joint replacement, the main issue is whether or not fibrin sealant is capable of reducing blood loss and cost of total knee replacement (TKA). Two papers describing prospective, randomized trials support the use of fibrin sealant to reduce bleeding , 235. Si or cost . A doubl or cost  found no or cost . A new m or cost  and in-v or cost .n = 40), patients treated with fibrin sealant coating of the tonsillar bed following tonsillectomy were found to have less postoperative inflammatory response as measured by circulating leukocytes, neutrophils, and cytokines [In head and neck surgery, two papers described use of fibrin sealant in conjunction with polyglycolic acid sheets as a method of oral mucosal repair following cancer resections in multiple individual patients , 243. Thytokines . Finallyytokines .P < 0.001) with no changes in early or late graft patency [P < 0.0001) [The literature on using fibrin sealant in vascular surgery suggests that it can be successfully used to augment two-suture fish mouth end-to-side microvascular anastomosis in rats with a significant reduction in operative time  . A case  0.0001)  as did a 0.0001)  in an esP = 0.049) as well as amylase drainage fluid levels . An animal study in rats of duodenal fistula found that fibrin sealant with gentamicin did not increase burst strength but did seem to have a trend towards a decrease in inflammation, adhesions, and abscesses on microscopic examination [n = 5) undergoing abdominoperineal resection had fibrin sealant used to reinforce the perineal closure with no wound complications at 6 months [The published literature in gastrointestinal surgery included two reviews on using fibrin sealant as a means of adjunctive anastomotic closure with the first suggesting possible benefit in ileal and gastric/bariatric anastomoses  and the mination . Finally6 months .n = 6) supported the use of fibrin sealant to close the mucosa in a model of transesophageal, endoscopic, esophagomyotomy (TEEM), although the authors suggested that the procedure was not yet perfected enough for use in humans [P < 0.010 and 19.7 ± 3.3 versus 34.6 ± 4.9 mmHg, P < 0.050), although no significant differences were noted in burst strength at days 5 and 7 .Several papers evaluated the use of fibrin sealant to seal the mucosa of the esophagus or at esophagogastric anastomoses. Reports of using fibrin sealant to repair an esophageal penetration at the time of endoscopic myotomy as confirmed on repeat endoscopy  and usinn humans . A rat mn humans  demonstrn = 51) undergoing reconstruction of bone defects following resection of benign tumor and tumor-like lesions when combined with a biphasic ceramic resulting in consolidation in 50/51 patients at 56 months of followup [P = n.s.) [In some additional new orthopedic surgery literature, fibrin sealant was found to be effective in a case report of arthroscopic, juvenile, allograft, cartilage implantation in the talus to secure the graft  and useffollowup . In an afollowup . On the  = n.s.) .n = 45) in whom autologous fibrin sealant was applied to the resection bed during laparoscopic partial nephrectomy [n = 12) or fibrin sealant  was applied to the resection bed finding that mean estimated blood loss was significantly less in the MPH group .With respect to renal surgery, two reports discuss the use of fibrin sealant in stone removal. The first one describes using fibrin sealant in 107 cases of tubeless percutaneous nephrolithotomy as a method of obtaining hemostasis in the access tract  with no hrectomy , and anohrectomy  where min = 42) into the bone marrow of sternotomy patients versus bone wax placement (n = 40) with statistically significant advantages to the fibrin sealant method ; the third and final report suggested that imbricated proximal sutures and fibrin sealant spraying during Bentall aortic root replacement are associated with low morbidity and mortality [In cardiac surgery, there were 3 reports of using fibrin sealant: the first was a case report  of usingortality .n = 10) serving as their own control that found significant benefits on the fibrin sealant side flap at gingival biopsy on day 8 ; the second again involved patients (n = 33) serving as their own controls to investigate using biphasic calcium phosphate and fibrin sealant as a bone substitute versus autologous bone in delayed dental implantation for bilateral maxillary sinus floor elevation [P < 0.05).The dental surgeons published 3 papers: the first was a trial of fibrin sealant versus sutures, respectively, for periodontal flap closure  performelevation  finding levation  finding The hemophilia literature remains concerned about achieving hemostasis during and following elective surgical procedures. A historical series of 76 patients undergoing circumcision over a 21-year period was reported and fibrin sealant application was employed as an integral part of the operation since 2000 allowing a reduction in factor concentrate administration from a previous high of 6-7 days down to only 3 days perioperatively . A case n = 15) prospective series of patients (n = 15) over 2 years, fibrin sealant was used as an adjunct to hemostasis on uterine closure sutures during laparoscopic myomectomy [P < 0.05), time to achieve complete hemostasis , estimated blood loss , and mean decrease in hemoglobin . Lastly, in an experimental endometriosis induction model using endometrial autografts [P < 0.05) and inflammatory reaction .In obstetrics and gynecology, a report of using fibrin sealant to facilitate full thickness skin graft attachment at the time of vaginal reconstruction for distal vaginal agenesis appeared . In anotomectomy . Signifitografts , fibrin n = 275) during open or laparoscopic distal pancreatectomy had no significant effect on reducing the rate of postoperative fistula formation, although the amount of amylase in the surgical drains was significantly reduced on the first postoperative day  [n = 57) of using external, anastomotic, fibrin sealant liquid application in pancreaticoduodenectomy found no significant benefit in terms of drain lipase levels, gastric or biliary leaks, wound infection, Clavien score of 3 or more, and hospital stay [n = 228) and pancreatectomy (n = 113) patients with and without fibrin sealant spray to the pancreas or pancreaticojejunostomy found no benefit in terms of reducing fistula occurrence [With respect to pancreatic resection, 3 papers appeared finding no reduction in the incidence of postoperative fistula following pancreatic resection. A large multicenter, prospective, randomized trial performed over 2 years found that a fibrin sealant equine collagen patch applied in patients (= 0.025) . Similartal stay . Finallycurrence .n = 9) was reported [n = 42) or without (n = 42) fibrin sealant found no benefit of using fibrin sealant in reducing recurrent postoperative CSF leakage [With respect to transsphenoidal surgical procedures, a series of patients  and with  local application of the antibiotic impregnated fibrin sealant and found significant reductions in the rates of surgical site infection in the antibiotic fibrin sealant hardware group  [Two papers described the use of fibrin sealant as a means of drug delivery, specifically antibiotics in orthopedic implant models. The first used cefazolin, fusidic acid, or 5-fluorouracil with fibrin sealant and found in-vitro agar plate bacterial killing with each drug. In addition, significant reductions in infection as measured by clip bacterial loads and local swelling with each drug were found in an in-vivo Staph. aureus contaminated rat model of titanium spine clip implantation . The sec 0.0003) .n = 344) suggested that fibrin sealant use based on multivariate analysis was associated with less delayed secondary hemorrhage following loop electrosurgical polyp excision (P < 0.01) [Two papers suggested using fibrin sealant during endoscopic polypectomy as a means of controlling hemorrhage. The first paper described methods of treating immediate bleeding that included hemoclips and fibrin sealant  and the  < 0.01) .n = 45) serving as their own controls was performed by using fibrin sealant to seal the skin to the underlying tissue on one side of the face and not the other [P < 0.0001). Patient evaluation of pain, numbness, and preference also favored fibrin sealant. A review article of using fibrin sealant in rhytidectomy also appeared emphasizing the importance of proper technique in assuring that fibrin sealant functions as a sealant/hemostat promoting reduction of fluid production in this setting and not as an antiadhesive potentially allowing for increased fluid formation [In cosmetic facial plastic surgery, a phase II, multicenter, prospective, randomized trial with the patients (he other . Signifiormation .P < 0.0001) [P = 0.22).Skull base surgery research on fibrin sealant has involved studies evaluating burst strength measurement of the best ratios of fibrinogen and thrombin as well as evaluating the best grafts and sealants. The best in-vitro ratio was found using a high fibrinogen concentration fibrin sealant with a 5 : 1 ration of initial fibrinogen  and the usual thrombin concentration (250 IU/mL) to maximize adhesion strength  while th 0.0001) . In the A single report appeared of two different cases of small bowel obstruction following use of fibrin sealant at a laparoscopic Roux-en-Y gastric bypass which both required reoperation for resolution . It was A comprehensive review article was published on the use of local and systemic hemostatic agents as an adjunct to control bleeding in trauma  that supLastly, the use of fibrin sealant and purified porcine small intestinal submucosa mesh has been described in urology to reinforce the repair for transsphincteric posterior sagittal treatment of rectourinary and rectovaginal fistulas .This spotlight paper has been designed to bring the reader up to date on the presently available forms of fibrin sealant as hemostats, sealants, and adhesives as well as to provide an organizational structure for contrasting these forms of fibrin sealants with other available products. In addition, a review of a year's publications on fibrin sealant has been provided based on both laboratory and clinical studies. These reports have been subdivided into areas that have been presented based on frequency and reported in order from most frequent to least frequent occurrence.


The exact pathophysiology of non-alcoholic steatohepatitis (NASH) is not known. Previous studies suggest that dietary advanced glycation end products (AGEs) can cause oxidative stress in liver. We aim to study the effects of dietary AGEs on liver health and their possible role in the pathogenesis of NASH. METHODS: Two groups of mice were fed the same diet except the AGE content varied. One group was fed a high AGE diet and the second group was fed a regular AGE diet. Liver histology, alanine aminotransferase, aspartate aminotransferase, fasting glucose, fasting insulin, insulin resistance and glucose tolerance were assessed. RESULTS: Histology revealed that neutrophil infiltration occurred in the livers of the high AGE group at week 26; steatosis did not accompany liver inflammation. At week 39 livers from both groups exhibited macro- or micro-steatosis, yet no inflammation was detected. Higher insulin levels were detected in the regular AGE group at week 26 (P = 0.034), compared to the high AGE group. At week 39, the regular AGE group showed higher levels of alanine aminotransferase (P<0.01) and aspartate aminotransferase (P = 0.02) than those of the high AGE group. CONCLUSIONS: We demonstrate that a high AGE diet can cause liver inflammation in the absence of steatosis. Our results show that dietary AGEs could play a role in initiating liver inflammation contributing to the disease progression of NASH. Our observation that the inflammation caused by high AGE alone did not persist suggests interesting future directions to investigate how AGEs contribute to pro-oxidative and anti-oxidative pathways in the liver. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver enzymes in the U.S. Evidence from animal models and human studies suggests that oxidative stress is one of the key factors in the disease progression of NASH In vitro experiments with a hepatic stellate cell line LI90 demonstrated that AGEs increase ROS production by NADPH oxidase and MRC in vivo study, in which mice fed a high AGE diet had lower levels of reduced glutathione/oxidized glutathione ratio and higher levels of 8-isoprostanes (lipid peroxidation products) than mice fed a low AGE diet Advanced glycation end products (AGEs) are potent trigger for ROS production in NASH livers ad lib with standard mouse chow  were used as reference animals. Fat contributed 12% of calories in TestDiet 58G7, compared to 18% in the standard mouse chow Teklad Global 18% Protein Rodent Diet. All mice in the study received the same calorie per gram body weight on a weekly basis.All procedures involving mice were reviewed and approved by the Institutional Animal Care and Use Committee of University at Buffalo. Four week old C57BL/6NHsd male mice were purchased from Harlan Laboratories . These mice were individually caged and provided free access to water. Mice were divided into two groups: 1) regular AGE group, mice were fed with TestDiet 58G7 ; 2) high AGE group, mice were fed with TestDiet 58G7 autoclaved at 120°C for 15 min, following a published method for the preparation of high AGE diet The concentrations of the major types of AGEs, carboxymethyl lysine and methylglyoxal, in mouse diets were determined by enzyme-linked immunosorbent assays as described previously 2 and 1mM MgCl2. Liver pieces of ∼5mm in all dimensions were taken from the mice and fixed in 4% formaldehyde for 15 min. The liver pieces were then sequentially equilibrated in 30% sucrose, 15% sucrose/50% OCT , and 100% OCT. The liver pieces were frozen in OCT before 10 µm sections were cut with a cryostat. The sections were stained with hematoxylin/eosin (H&E) or oil red O and visualized by light microscopy. A pathologist (R. K.) assessed each slide for steatosis, inflammation and fibrosis.Five mice from each group were sacrificed on weeks 13, 26 and 39. Whole body perfusion was performed via left ventricle with oxygenated phosphate buffered saline supplemented with 1mM CaClAnimals were fasted overnight before blood was collected via the submandibular vein with a sterile 5 mm point golden rod lancet . Glucose levels were measured using a FreeStyle Lite glucometer . Each reported value was the average of two readings. The remaining blood samples were incubated at 37°C for 40 min followed by centrifugation at 15,000g for 20 min at room temperature. Collected serum was aliquoted and stored at −80°C before further analysis. Insulin levels were determined with ELISA kits purchased from Mercodia  following manufacturer’s instructions. Insulin resistance (IR) was calculated using the homeostasis model assessment (HOMA) method (fasting blood glucose (mmol/L) x fasting serum insulin (mU/L)/22.5 Intraperitoneal glucose tolerance tests (IPGTT) were performed as described previously All analysis for statistically significant differences was performed with Student's t test with a two-tailed distribution. P values ≤0.05 were considered significant.To evaluate the effect of dietary AGE on mouse liver, a high AGE mouse diet was prepared by high temperature treatment. The AGE contents in the diets were measured by ELISAs with antibodies against two major types of AGE molecules, carboxymethyl lysine and methylglyoxal, respectively . These mTwo groups of mice were fed either a high AGE diet or a regular AGE diet. These two groups showed similar growth curves over the 39 weeks of the study; no significant difference was observed at any time point throughout the study . The groFive animals from each group were sacrificed at week 13, 26, and 39 to examine the possible effect of AGE on mouse livers. Cryosections of liver from each animal were stained with H&E and oil red O. Examination of the liver histology of all animals from the high AGE group and the regular AGE group showed no evidence of steatosis or inflammation at week 13 (data not shown). However, abnormalities were detected at week 26. Liver sections from two of the five high AGE-fed mice exhibited extensive macro- or micro-vesicular steatosis as indicated by oil red O staining . More imSerum ALT and AST levels were measured to evaluate hepatocellular damage. Normal ALT and AST values were observed in all animals at week 5 of dietary treatment . At weekBecause fat deposition in parenchymal cells  can contTo further examine glucose homeostasis, intraperitoneal glucose tolerance tests (IPGTT) were performed at week 39. All animals exhibited glucose intolerance as evidenced by persistent hyperglycemia 2 h after intraperitoneal glucose injection . No signMost fatty livers do not develop liver inflammation On the other hand, some mice at week 26 and all animals at week 39 developed macro- or micro-vesicular steatosis, and all animals exhibited glucose intolerance at week 39, but there was no progression to liver inflammation with these fatty livers. These results recall the observation that ob/ob mice develop steatosis and insulin resistance et al. also found that plasma 8-isoprostane, a measurement of systemic oxidant stress, were significantly elevated in the high AGE diet fed animals. One major difference between our experimental approaches is that high fat diets were used in the study of Sandu et al., while low fat diets were used in our study. Therefore, we speculate that dietary fat content differences contribute to the conflicting results. Perhaps, high AGE diet exacerbates the metabolic defect precipitated by a high fat diet. As such, it is of great interest to test whether low fat diets protect hepatocytes from the adverse effects of AGEs and prevented persistent liver inflammation. Another important question stemming from this discrepancy is whether a high AGE diet facilitates the development of liver inflammation on the background of a high fat diet and/or obese mouse model (db/db or ob/ob).Another possible explanation for this self-resolving inflammation is the low fat content in the diets of our study. One outstanding observation in our study was that fasting glucose, insulin, IR-HOMA and glucose tolerance were similar between high AGE and regular AGE fed mouse groups. This is in contrast to the observation of Sandu and colleagues AGEs have been implicated in the pathogenesis of various diseases including diabetes One of the striking and important aspects of our study is its length. For example, if this study had been terminated after a short period of time, as other studies of diet and liver injury have been, our observation that resolution of inflammation is possible would have been missed. This observation might lead to the understanding of mechanisms that could be used to mitigate harmful effects of diet on the liver.In this study, liver inflammation occurred before steatosis in the high AGE diet group. This phenomenon may add to the debate on whether steatosis is a consequence of liver inflammation or vice versa (reviewed in In summary, we demonstrate that a high AGE diet causes liver inflammation in the absence of steatosis. This finding is relevant to patients at high risk for NASH and other liver injuries. Our observation that the inflammation caused by high dietary AGE alone did not persist suggests interesting future directions to investigate how AGE induced pro-oxidative pathways are regulated and how they may be down-regulated by AGE-induced anti-oxidative pathway. The animal model we developed is a valuable platform on which a therapeutic and preventive agent against the adverse effects of AGEs could be identified and tested.