Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated. Through a complex network of mutualisticinteractions, the gut microbiota has a profound impact on the host's health byacting as a barrier against pathogens, contributing to the degradation of foodcomponents, stimulating the host immune system, and producing a series ofessential vitamins, enzymes, and short-chain fatty acids [The mammalian intestinal tract comprises a highlycomplex population of microorganisms reaching up to 10ntestine , 3. Follntestine . Triggerntestine –10. Thesty acids –14.Untila decade ago, knowledge on the taxonomic composition and metabolic activity ofthe intestinal tract microbiota was mainly based on the use ofculture-dependent techniques. Triggered by the growing awareness that only afraction of the gut microbiota is culturable under laboratory conditions,various culture-independent methods have been evaluated in intestinal microbialecology –19. DepeIn contrast to several of the aforementioned techniques that specificallytarget one or more autochthonous members of intestinal tract or that requireanalysis of large and complex datasets, population fingerprinting is auniversal concept that allows one to characterize and monitor intestinalmicrobiota without preexisting knowledge of its structure or composition. The most commonly usedfingerprinting techniques in the field of intestinal microbiology are based onthe sequence-dependent electrophoresis (SDE) principle and include denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),and temporal temperature gradient gel electrophoresis (TTGE). In contrast to conventionalgel electrophoresis based on fragment size, the SDE principle relies on thesequence-dependent electrophoretic separation of a mixture of equally sized PCRproducts in a polyacrylamide gel containing a linear gradient of chemicaldenaturants (DGGE) or a linear temperature gradient (TGGE and TTGE). This way,separation is achieved by the gradually decreasing electrophoretic mobility ofpartially melted, double-stranded amplicons in the denaturing gradient. PCRfragments equal in length but with different sequences have a different meltingbehavior and will stop migrating at different positions along the gel,eventually producing a banding pattern or fingerprint. To a lesser extent, alsosingle-strand conformation polymorphism (SSCP)  and termSince their introduction in microbial ecology in the early 1990s ,SDE finThis review will deal with all different aspects of SDE methodologyincluding its possibilities and limitations in terms of reproducibility,sensitivity, and data analysis. Through discussion of selected studies thathave contributed to the field, an overview will be presented of SDE-basedresearch approaches to study human intestinal ecosystems in relation to themicrobial ecology of healthy and disease-affected populations. The scope ofthis review excludes SDE applications dealing with the human uppergastrointestinal tract or with animal intestinal ecosystems.Tm). Thephysical denaturation of the dsDNA fragment is thus largely determined by its nucleotidesequence and %G+C content and proceeds in discrete portions of the fragment or the so-calledmelting domains. These domains interfere with the helical structure of the DNAmolecule and will eventually halt further migration. Amplicons that aredifferent at the sequence level are likely to display a different meltingbehavior and will, therefore, stop migrating at different positions along thelinear gradient of the gel, which upon visualization will result in bandprofiles representing the sequence diversity of the amplicon mixture.The principle ofSDE techniques relies on the electrophoretic separation of PCR amplicons withequal length in a sequence-specific manner in a polyacrylamide matrixcontaining a defined denaturing gradient of urea and formamide (DGGE) ortemperature gradient (TGGE and TTGE). The temperature gradient in TGGE iscreated along the length of the gel, whereas in TTGE a temporal temperaturegradient is gradually formed during the electrophoresis run. Theelectrophoretic mobility of double-stranded amplicons in a gel matrix with anincreasing denaturing gradientis retarded at a given chemical denaturant concentration or temperature thatcauses  melting of the sequence region with lowest melting temperatureextraction of total community DNA from the sample; (ii) PCR-controlledamplification using specific oligonucleotide primers; (iii) sequence-dependent electrophoreticseparation of the amplicons using either DGGE, TGGE or TTGE; and (iv)fingerprint processing and analysis.The endogenous microbiotadiffers along the length of the intestinal tract . In addiIn most studies, immediateprocessing of samples is not feasible due to the need for transportation and/or(long-term) storage of the specimen. It has been shown that storage of stoolsamples at room temperature and even at 4°C showed a substantial reductionin bacterial diversity and the degradation of bacterial DNA after 8 hours . TherefAn efficient,reproducible, and high-yield method for total DNA extraction is indispensablein order to obtain a representative view of the actual microbial composition ofan intestinal sample. The most crucial step in any DNA extraction procedure iscell lysis. A series of methods including commercial kits and inhouse laboratoryprotocols have been described and evaluated for the extraction of totalbacterial DNA or RNA from intestinal samples making use of chemical, mechanical, and/or enzymatic lysis –67. BecaDepending on the type of  application, high-quality communityDNA may need to be obtained from a range of different sample types such asdigesta, mucosal, and fecal samples. For this reason, the DNA extraction techniqueshould be carefully selected and possibly further evaluated or optimized withparticular attention for the type and number of specimens . In thisTm domain of the V region and on thenumber, resolution, and relative intensity of the bands in the resulting DGGEprofile, the V3 region was most preferred for analyzing intestinalmicrobiomes. In addition, the authors recommended touse the V3–V5 or V6–V8 regions if a longer amplicon is preferred. Next to the SSU rRNA gene, also its rRNA counterpart can be coextracted and used as PCR template in SDE analyses ofintestinal ecosystems when preceded by reverse transcription [Following DNAextraction and purification, multiple primer sets with different taxonomiccoverage can be applied for community PCR amplification. The use of universalPCR primers allows any microbial community to be analyzed, although inecosystems with a high diversity like the intestinal tract only the(pre)dominant constituents will in effect generate a visible band in SDE. Inorder to focus on a specific subpopulation within the totalcommunity, group-specific PCR primers can be used which allow detection ofbacterial taxa that are less prevalent in the intestinal tract. Traditionally, universal and specific community PCR primers for SDEapplications are designed using the 16S rRNA gene as a target molecule. This preferencestems from the fact that the SSU rRNA gene has a mosaic structure composed ofboth invariant, relatively conserved, and highly variable regions (V regions). In SDE-based population fingerprinting, primers are used that anneal toconserved sequence parts of the gene in order to cover one up to threehypervariable regions. In cription , 93, 95.Tm value close to 80°C whereasfragments with Tm > 80°C may require longer GC-clamps incombination with naturally occurring high-melting (thus GC-rich) domains [Anadditional 40-nucleotide GC rich sequence, the so-called GC-clamp, is usuallyattached to the 5′ end of one or both of the PCR primers and participates inthe PCR reaction. This way, the GC-tail generated at the end of the ampliconwill prevent complete denaturation of the product and is necessary to obtain astable melting behavior of the fragments during electrophoresis , 133. G domains .http://www.bio-rad.com/), INGENYphorU, and DGGEK-1001/2001/2401/4001/4801  appear to be most commonly used. The apparatus providedby Bio-Rad and Ingeny can also be applied for TTGE analysis whereas for TGGE atemperature gradient block should be integrated in the system. In case ahigh number of samples need to be analyzed such as in monitoring studies, the sample capacity of the system is animportant criterion. The maximum capacity per run for the three aforementionedsystems varies from 60 (DCode), 96 (INGENYphorU) to 128 (DGGEK-4801) samples.Essentially, aDGGE system consists of a heated buffer tank operated under strict control oftemperature and stable buffer circulation. Several systems are currentlyavailable, of which DCode . The optimal temperature gradient is theoretically delineated by the lowest andhighest Tm values obtained in the melting profile. The theoretical Tm values can be lowered by adding denaturing components to the gel, for example,one mole of urea will lower the theoretical Tm with 2°C [In the case of TGGE and TTGE, a linearly increasing temperaturegradient parallel to the electrophoresis direction or formed during the lengthof electrophoresis, respectively, is applied in combination with a uniform,high-denaturant polyacrylamide gel to separate PCR fragments. To determine thetemperature range for parallel TGGE or TTGE analysis, a melting profile of theDNA sequence can be generated using specialized software  that is visualized as a black precipitate. Potential drawbacks ofthis procedure include the fact that silver stained gels impede subsequentblotting experiments orband sequence analysis and the aspecific detection of protein components suchas BSA and Taq polymerase present in the PCR mix which may generateadditional background signals [Uponelectrophoresis, gels are stained and digitally captured for further analysis. Three staining agents are commonly used to visualize fragments. Originally, SDEgels were stained with ethidium bromide (EtBr) given its widespread use as anintercalating fluorescent dye used to detect nucleic acids. The next generationof fluorescent nucleic acid dyes such as SYBR Green and similar stains offer anincreased sensitivity compared to EtBr due to a lower overall background signalallowing detection ofDNA fragments at lower concentrations , 137. Ad signals , 137.http://www.applied-maths.com/), Quantity One and MolecularAnalyst (Bio-Rad Laboratories), GeneTools , andPhoto-Capt . These programs permit numericalanalysis of band patterns and usually also include statistical approaches fordata interpretation. Programs that have been used specifically for statisticalanalysis of SDE fingerprint data include R (http://www.r-project.org/) and DGGESTAT .Normalized SDE fingerprints can be analyzed visually and/ornumerically. Visual interpretation is attainable when only a limited number ofprofiles with low complexity are to be compared. However, once banding patternsbecome more complex such as those obtained from intestinal samples or when thenumber of profiles increases ,analysis of SDE fingerprints requires implementation of numerical methods. For thiMost commonly, numerical analysis of SDE profilesrelies on the use of diversity indices and/or cluster analysis. Diversity measuresfor fingerprintanalysis such as the Simpson index and the Shannon-Wiener/Weaver index express the degree ofecosystem diversity as a function of band profile complexity but fail toexpress similarity between profiles based on band positions. Hierarchicclustering algorithms such as unweighted pair-group method using arithmeticaverages (UPGMA) produce a visual representation of the similarity between SDEprofiles expressed as similarity indices, for example, using the curve-basedPearson product-moment correlation coefficient, the band-based Dice coefficient,or Sorenson's pairwise coefficient. Other authors have used multivariate ordination methods such as nonmetricmultidimensional scaling , 143, prBacteroides [Bifidobacterium [Bifidobacterium-specific PCR step followed by asecond PCR step in which both the V3 and V6–V8 regions of the 16S rRNA genewere amplified. A mix of both amplicons was analyzed on a DGGE gel, after whichband positions were compared with a user-generated database of referencestrains.Next to the firstSDE analysis level based on the use of diversity and similarity coefficients, asecond level can be defined that allows one to identify and monitor specificmembers of the intestinal ecosystem. Essentially, identification of individualbands in SDE fingerprints may be obtained by band position analysis (BPA)and/or through band sequencing analysis. Essentially, BPA relies on thecomparison of migration distances of band fragments from taxonomicallywell-characterized reference strains with those of unknown bands present in thesample profiles. BPA can either be performed by analyzing samples and referencestrains in adjacent lanes on the same gel  or bycomparing unknown band positions with those of reference strains present in auser-generated SDE database. In intestinal ecosystems, BPA-based identificationmay not always yield a conclusive result given the possibility that a singleband may consist of multiple amplicons from different species or that two ormore  species are characterized by the same bandposition in the sample profile. Ideally, each band position in a sample profileshould represent one species. In practice, however, the multioperon effectobserved for some taxa when using 16S rRNA gene primers may lead to anoverestimation of the number of predominant species in the sample  or GenBank(http://www.ncbi.nlm.nih.gov/genbank).Identificationresults from BPA can or even should be verified by band sequencing, and mayhelp to determine the phylogenetic affiliation of unknown bands. Variousprocedures have been described to excise and recover PCR fragments from thepolyacrylamide gel matrix ranging from conventional elution in electrophoresisbuffer to specialized protocols using diffusion buffers and commercial kits. A critiAsfurther discussed below, the significance of the obtained species informationis dependent on the length of the fragment and the hypervariable region itrepresents in the target gene. This sequence information can also be employedto develop probes for application in FISH and real-time PCR assays to detectand quantify the target organisms. Next to sequencing analysis, identities ofindividual bands in SDE profiles can also be revealed by Southern hybridizationwith taxonomic probes .The human intestinal tract harbors a highly dense and complex microbialcommunity which plays a pivotal role in maintaining the health status of thegut. Despite the fact that SDE-based methods only allow a superficial view onthe microbial diversity and population dynamics of what is considered thepredominant part of complex ecosystems, their use in the field ofintestinal microbiology has increased exponentially over the past 10 years. Thefollowing section aims at reviewing the main contributions of SDE populationfingerprinting to our current knowledge on the composition and ecologicalbalance of the human intestinal microbiota linked to health, disease, anddietary intervention.11-1012/gin the colon. Parallel to the increase in bacterial density, also the bacterialdiversity expands from the small intestine to the colon [Next to arelative minority of organisms belonging to other microbial domains, the humanintestinal microbiota mainly consists of bacteria. Although the major site ofmicrobial fermentation is the large intestine (colon), bacterial populationsare encountered along the total length of the digestive tract. Starting from the upperbowel, bacterial concentrations gradually increase up to 10he colon , 152. FrE. coli and Clostridium spp. were the main groups among the initial colonizers, which were rapidlyreplaced by a more complex microbiota consisting of Bifidobacterium, Clostridium, Enterococcus, Ruminococcus, Enterobacter, Streptococcus, Bacteroides, and Actinomyces. The diversity revealed by DGGEanalysis was fairly consistent with previous insights in infant successionpatterns based on traditional culture studies [Bifidobacterium-specificprimers [Bifidobacterium-related DGGE bands were observed which always included Bifidobacterium infantis. In another study, the dynamics of the developing bacterialcommunity in the neonatal intestinal tract of nine Japanese infants wasmonitored during the first two months of life [Pseudomonas were immediately replaced by facultative anaerobes including Enterococcus, Streptococcus, and Enterobacteriaceae during the first month. Finally, strictly anaerobic bifidobacteria and clostridia appeared. Theestablishment and succession of bacterial communities in hospitalized preterminfants tend to follow a different pattern compared to full-term infants [Cs) from 0 to 80%. In addition, alsothe interindividual Cs values increased (18.1 to 57.4%) all of whichindicated the acquisition of a highly similar bacterial community in theseinfants. In contrast, breastfed full-term infants showed a considerably lowerinterindividual Cs value (11.2%). The strikingly high similaritybetween bacterial communities from different preterm infants was considered tobe associated with hospitalization because the major bacterial groups identified by DGGE BPA belongedto taxa that are routinely isolated in baby care units such as E. coli, Enterococcus spp., and Klebsiella pneumoniae. This finding thus indicatesthat the initial colonization of the newborn's intestinal tract is highlydependent on the immediate environment of the individual. In anotherstudy assessing the global diversity of the fecal microbiota of preterm infants (n = 16), aremarkably low-species diversity and high-interindividual variability werereported [Enterobacteriaceae family and of the genera Enterococcus, Streptococcus, and Staphylococcus. Seven out of 16 preterm infants were colonized byanaerobes, of which four infants were shown to harbor bifidobacteria.Several studieshave used SDE-based techniques to monitor the development of the newborn gutmicrobiota in humans , 107. At studies , 7. In aprimers . Wherea of life . Althoug infants . Fecal reported . The lowBacteroides represent the main constituents [E. coli, Ruminococcus spp., and several Bifidobacterium species including B. longum, B. infantis, and B. breve. Both for the bacterial and bifidobacterialTTGE profiles, distance analysis indicated the expected maturation of the faecalmicrobiota between 5 and 20 weeks of age, but did not reveal any correlationwith the dietary supplementation. Despite a high-interindividual variability,it was observed that the composition of the faecal microbiota appeared morehomogenous after weaning which may suggest a correlation with the cessation ofbreastfeeding. In another study, 65 10-month old infants were included in arandomized dietary intervention study that compared the effect of cow'smilk (CM) with infant formula (IF) with or without fish oil (FO) supplement onthe diversity of the fecal microbiota [n-3polyunsaturated fatty acids, respectively, but further indepth analysis of theDGGE profiles in combination with other molecular tools is required tosubstantiate this hypothesis.Several studies havedocumented that bifidobacteria predominate in the fecal flora of breastfedbabies, whereas informulafed infants, other bacterial groups such as coliforms, enterococci, and tituents , 153. Intituents . In the crobiota . Based on = 13), fraternal twin pairs (n = 7), andunrelated control pairs (n = 12) were compared both visually and numerically. Althoughthe community fingerprints of each individual were unique, increased levels ofsimilarity were found between TTGE profiles of genetically related individuals,with the highestsimilarity values obtained for genetically identical twins (median Cs of82%) which was significantly different from fraternal twin pairs (median Cs of 68%) and from the unrelated control group (median Cs of 45%). Theresults of this TTGE study thus suggested that host genetics can have an impacton the composition of the predominant fecal bacterial community in children. Likewise, DGGE analysis of the dominant intestinal microbiota amongst adultsdisplaying varying degrees of genetic relatedness showed that the host genotypehad a significant effect on the species composition of the intestinal community[Besides theinfluence of environmental and dietary factors, also the host genotype may havea significant effect on the species composition of the intestinal microbiota. Stewartet al.  used TTGommunity.Lactobacillus-Leuconostoc-Pediococcus-Weissella-group tended to show strongtemporal variations. Among other autochthonous groups such as the Bacteroides fragilis subgroup, however, DGGE profiling using group-specific primersdid not reveal such variations. Importantly, the specificity of thesegroup-specific primers was only validated using a set of taxonomic referencestrains. A more elaborated strategy was followed in the validation of DNA- andRNA-based DGGE protocols specifically designed to assess the diversity andstability of the Clostridium coccoides-Eubacteriumrectale  group in fecal samples[Clostridium cluster XIVa. The authors concluded thatthe members of this cluster, representing one of the most dominant bacterialgroups in the normal intestinal microbiota, followed the same pattern ofrelative stability as the total predominant population in 12 healthy Finnishadults during six months to two years. Although using protocolsdiffering in sample type, SDE method and primer target, the current viewon the uniqueness and temporal stability of the predominant intestinal flora inadult individuals has also been confirmed in other human volunteer studiesusing SDE-based analyses of fecal samples [Upon succession, it is thought that a relatively stableintestinal community is established in the adult intestine that appears to bespecific for each individual. Zoetendal et al.  were the samples. In that samples , 154 and samples , 98, 123Although the vast majority of SDE-based studies in intestinalmicrobiology rely on direct DNA extraction from human samples in order toobtain a culture-independent inventory of the microbial diversity, there hasalso been interest in using DGGE and related fingerprinting techniques tospecifically explore the composition of culturable intestinal subpopulations. For instance, DGGE analyses of resuspended bacterial biomassobtained from agar plates of different media selective and nonselective forlactic acid bacteria (LAB) have been used to evaluate the choice of medium andincubation conditions on LAB recovery and to gain insight in the diversity of culturablefecal LAB in healthy adults .Lactobacillus, Leuconostoc, Weissella, Pediococcus, and Aerococcus produced relativelycomplex DGGE profiles that varied between hosts and between sampled sites inthe colon. In contrast, Zoetendal et al. [The different physicochemical conditions such as pH and concentration offermentation products prevailing in the ascending, transverse, and descendingparts of the colon  suggest l et al.  obtainedGiven the fact that each individual displays a uniquefecal SDE fingerprint , 95, invA number of studies have also investigated to whatextent the composition of the fecal microbiota reflects the composition of themucosa-associated colonic microbiota , 123. InThe pathogenesis of many chronic intestinal disordersand even a number of nonintestinal diseases is believed to be directly orindirectly linked to somemembers of the indigenous microbiota. Several studies have implementedan SDE-based approach to analyze and monitor the composition and temporalstability of the intestinal microbiota of patients suffering from gutdisorders. As an initial approach, SDE techniques permit a rapid and global assessment of microbial diversitywithout previous knowledge of the composition and are well suited to analyzeintestinal microbiota in relation to different experimental conditionsand parameters such as healthy versusdisease status, active versus quiescent disease phase, different segmentsof the intestinal tract and response to nutritional or therapeuticinterventions. Moreover, the combined use of SDE techniques and quantitativeassays such as real-time PCR and FISH that allow to determine the relativeconcentration of specific indicator organisms offers great potential in this type of studies. Thefollowing sections of this review are based on a selected number of studiesthat have implemented SDE-based methods to assess the potential role of theintestinal microbiota in the (etio)pathogenesis of chronic intestinaldisorders.Although theexact etiology of inflammatorybowel disease (IBD) is not known to date, it is generally assumed toresult from an inappropriate response of the mucosal immune system to thenormal enteric microbiota in a genetically susceptible individual . It hasClostridium spp., Ruminococcus torques, and E. coli in samples from CDpatients (n = 19) compared to healthy specimens (n = 15) [Faecalibacterium prausnitzii was morefrequently encountered in the latter group. Overall, DGGE fingerprints ofmucosal CD populations displayed a higher patient-to-patient variabilitycompared to healthy subjects. The authors postulated that this difference mayreflect the difficulty of patients genetically predisposed to CD to maintainand regulate a stable intestinal microbiota. A study of Bibiloni et al. [n = 20) and UC patients (n = 15) and healthysubjects (n = 14). Biopsies collected from inflamed and noninflamed sites of the terminal ileum andvarious colonic regions were analyzed by DGGE, 16S rRNA gene clonelibraries, and qualitativeand quantitative PCR for detection of selected bacterial groups. DGGE profiles of universalV3-16S rRNA gene amplicons were very similar within each subject (mean 85.0 ± 2.4%),irrespective of the intestinal region. However, enumeration by quantitative PCRrevealed approximately double numbers of biopsy-associated bacteria for UC patients than CD patientsand healthy subjects. In addition, the clone library composition indicated that the composition ofbiopsy populations in UC and CD patients (P < .05), and those from healthy subjects(P = .05) were statistically different. This comparison highlighted a significantly higher prevalenceof unclassified members of the phylum Bacteroidetes in CD patients, which may indicatethat UC and CD are bacteriologically distinct diseases.The majority of SDE-based studies on IBD have primarily attempted to finddifferences between CD/UC and healthy fecal or mucosal populations. As such,V3–V5-16S rDNA DGGE profiling and subsequent band sequencing analysis of freshmucosal biopsy samples revealed a significantly higher prevalence of (n = 15) . In turni et al. showed tn = 8),patients in remission (n = 9), and healthy volunteers (n = 16). TTGE profiles of universal 16S rRNA gene V6–V8 amplicons were very stable over time in the healthy controls but varied markedly for a number of patients(n = 4) who were monitored during both active and quiescent phase of CD. FecalTTGE profiles of these four patients revealed only a slight decrease in thenumber of bands during the active phase (meanloss of 1.7 ± 2.7 bands), which indicated that the predominant fecal microbiotaretained a high degree of diversity in both phases. Based on TTGE band profile composition, no specificbacterial groups could be assigned to active or quiescent CD state. Incontrast, quantitative dot blot hybridization of stool samples showed that the fecal microbiota in patients with CD (both active and inactive)differed considerably from those of healthy subjects. Both the Bacteroides group and the bifidobacteria tended to be less represented in CD patients whereassignificantly more enterobacteria could be detected. In addition, approximately30% of the endogenous microbiota of CD patients did not belong to the dominantphylogenetic groups commonly found in healthy controls.Depending on theindividual effectiveness, IBD patients undergoing immunomodulatorytherapy continuously balance between active disease and remission status. However, it is unclear if and inwhat way the intestinal microbiota of these patients undergoes compositionalchanges during these subsequent transitions. In this context, Seksik et al. monitoreAlthough currentlyavailable data from SDE profiling and other molecular tools implicate a role ofintestinal bacteria in CD pathogenesis, a detrimental effect of localizedqualitative dysbiosis in CD-associated ulceration has so far not bedemonstrated by community fingerprinting. TTGE analysis of biopsy samples ofulcerated and adjacent nonulcerated mucosa of 15 patients with active CD didnot reveal qualitative differences in the dominant bacterial populationprofiles (V6–V8 region of the 16S rRNA gene) within a given patient although a high biodiversity was retained in both cases . Mean sin = 9) in comparison with that of healthy subjects(n = 9) by applying DNA- and RNA-based TTGE analysis of V6–V8 ribosomalamplicons. Thenumber of bands in DNA-derived TGGE profiles were significantly higher than in RNA-derived profiles for UCpatients  but not for controls  which indicated a reduction in thebiodiversity of the active portion of the fecal microbiota in UC patientsrelative to healthy controls. Irrespective of the initialtemplate (RNA or DNA), Pearson-UPGMA clustering analysis of TGGE profilestended to group the samples on the basis of their clinical affiliation (UCversus controls) suggesting that each group has its specific bacterialsignature. Interindividual comparison of the “active” microbiota (RNA-derivedprofiles) revealed a band that was significantly associated with UC patients(89% versus 22% for controls). Sequence analysis attributed this band to E. coli or related enterobacteria. Clearly, the possible pathophysiologicalrole of this overrepresentation in the active microbiota of UC patients shouldbe further assessed during remission and within the mucosa-associatedmicrobiota.Whereas most studies concentrated on theinventorization and monitoring of bacterial groups potentially associated withCD, very few studies aimed to address the same question within themetabolically active compartment of the gut microbiota. Sokol et al.  analyzedC. histolyticum, C. coccoides-Eubacterium rectale, C. lituseburense, and C. leptum, were shown to represent the dominant fecal microbiota in 26of the 32 subjects under study, contributing altogether 29–87%. The proportion of the C. coccoides-E. rectale group was found to be significantlylower in the constipation-type IBS subjects compared to the controls. Although DNA- andRNA-derived predominant community profiles showed considerable biodiversity andsubject-specificity, RNA-based DGGE profiles contained significantly feweramplicons (16 ± 5 compared to 22 ± 5 amplicons). In addition, only RNA-based DGGE profilesof the IBS subjects indicated higher instability of the bacterial populationcompared to the control subjects. Although intraindividual temporal instability of thepredominant microbiota was observed in both IBS and control subjects (with bothDNA- and RNA-based DGGE), only RNA-derived DGGE profiles of IBS subjects showeda broader range in similarity values (39–95%) compared to control subjects (68–94%). When considering symptomaticIBS subgroups, thelargest intraindividual variability in DGGE similarity values was observed inthe diarrhoea-type subgroup. These observations suggest that clostridial microbiota, inaddition to the instability of the active predominant fecal bacterialpopulation (RNA-derived profiles), may be involved in IBS. For futureresearch, the use of group-specificprimers in SDE analysis focussing on apparently affected groups  could be a valuable and effective approach toidentify potential IBS indicator organisms.Irritable bowel syndrome (IBS) is an intestinaldisorder that is characterized by bowel dysfunction and pain , 162. IBTheuse of SDE-based methodologies to determine the diversity and stability ofmicrobiota in inflammatory diseases has meanwhile expanded from IBD and IBS toother intestinal diseases in which dysbiosis of the human microbiome is thoughtto play a role such as neonatal necrotizing enterocolitis  and coelApart fromcomponents naturally occurring in a normal diet, also functional foods(including pre- and probiotics) and antimicrobial agents are able to inducebeneficial or detrimental changes in intestinal ecosystems. Starting from thefirst weeks upon birth, the human diet is able to modulate the composition andbalance of the intestinal microbiota , 153. ThLactobacillus spp. Aprebiotic, on the other hand, is a nondigestible selectively fermented compoundthat induces specific changes both in the composition and/or the activity ofthe gastrointestinal microbiota thereby conferring benefits upon hostwell-being and health [The fact that diet is a major factor controlling thehuman intestinal balance has triggered the development of a new generation of foods specifically designedto strengthen the gut microbiota via modulation. Functional foods include foodsand food products with a clearly identifiable health benefit in addition totheir basic nutritional value . In funcd health . Essentid health –178.® (http://www.vsl3.com/) consisting of three Bifidobacterium strains and five LAB strains  in patients with active mild to moderate UC. DGGE analysis of V3-16S rRNAgene amplicons generated from biopsies collected from seven patientsbefore and after 6-week VSL#3 administration revealed considerable variation ofthe predominant microbiota in four out of five patients in remission (mean dice similarity coefficient (Ds) of 69.9 ± 12.7%). Incontrast, the DGGE profiles of the two patients with continued activedisease remained relatively stable after VSL#3 consumption (mean Ds of 92.3 ± 4.1%). Importantly, it should be noted that the study did not report on the temporalstability of biopsy profiles in the absence of probiotic treatment. In anotherstudy, the effect of a 4-week administration of the candidate prebiotic di-D-fructofuranose-1,2′ : 2, 3′-dianhydride (DFA III) on human fecal microbiota was studied by DGGEanalysis using universal V3-16S rRNA primers and Bacteroides fragilis subgroup-specific primers [Bacteroides spp. [Clostridium coccoides-Eubacteriumrectale group remained relatively stable during the study period. Incontrast, the Lactobacillus group showed temporal variation whichconfirms previous observations under basal conditions [SDE-based methods haveplayed a key role in human dietary intervention studies aiming at demonstratingthe efficacy of functional food components and to substantiate potential healthclaim. A selection of relevant studies that have contributed to this field is listedin  primers . Visual des spp. . In a stnditions .Lactobacillusparacasei NFBC 338and B. animalis subsp. lactis Bb12in ileostomy effluents of two infants without a history of probiotic intake. In this context, it shouldbe noted that SDE fingerprinting is not the most optimal tool for detection of administeredstrains because of the relatively poor detection limit  and the lack of resolution to discriminate the introducedstrain(s) from other strains of the same or highly related autochthonous memberspecies of the intestinal microbiota. More suitable approaches are those applying strain-specific primers or probes which will not only provide a higher sensitivity but may also allow relativequantification of the probiotic target [In the course of probioticintervention studies, DGGE and related fingerprinting techniques have been usedto verify if the administered strain(s) is (are) detectable in intestinalsamples , 179. Bac target –184. On Bifidobacterium adolescentis and/or Collinsella aerofaciens. In contrast, DGGE analysis usinggenus-specific primers derived from the transaldolase gene generated relatively stable profilesfor fecal bifidobacteria. Although the taxonomic composition of the bifidobacterial populationwas not substantially different and both DGGE and FISH revealed that the Bifidobacterium and Collinsella populationsremained relatively unchanged, rRNA-DGGE provided evidence of increasedmetabolic activity inresponse to prebiotic consumption. A combination of DGGE and FISH wasalso used to investigate the effect of black tea drinking on the fecalmicrobiota of healthy volunteers with hypercholesterolemy [Bifidobacterium, Bacteroides and Prevotella, Clostridium phylogenetic clustersIV and XIVa, Atopobium group, Faecalibacterium-likespecies and E. coli), it did decrease the total amount of bacteriadetected by the universal bacterial probe. In a study that combined the use ofTGGE and FISH analysis, it was demonstrated that isoflavone supplementation with and without pro- orprebiotics induced significant dynamic changes on the composition of the dominantintestinal microbiota of 39 postmenopausal women [Bifidobacteriumanimalis DN-173010) and theprebiotic (FOS) test groups, respectively. In addition, FISH results showed a bifidobacterial increase following prebiotic supplementation, oftenreferred to as the bifidogenic effect. Amongst others [In recent years, SDE-based community fingerprinting has been integratedin larger polyphasic studies in combination with conventional culture methodsand/or with other molecular culture-independent methods to detect and monitorchanges in human intestinal ecosystems upon administration of probiotic,prebiotic, or other (in)organic compounds with claimed functionalities. Assuch, DGGE and FISH approaches were combined with selective culture methods toevaluate the impact of a 3-week diet supplementation with prebiotic GOS or FOS on the composition andactivities of the fecal microbiota of 15 healthy human volunteers . V3-16S terolemy . DGGE ofal women . Resultst others , 180, thSaccharomyces boulardii), and their synbiotic combination [Bacteroides fragilis subgroup, the genus Bifidobacterium and the Clostridium lituseburense and Clostridium coccoides-Eubacterium rectale groups remained fairly stable, one pronounced change wasobserved in the universal fingerprint profiles after lactulose ingestion. The DGGE band appearing or intensifying in 27of the 30 subjects could be assigned to Bifidobacterium adolescentis by band position analysis and bandsequencing. In subsequent real-time PCR analysis, this finding wascorrelated to a statistically significant stimulation of total bifidobacteriaand of B. adolescentis. In contrast, the probiotic yeast S. boulardii did notdisplay any detectable universal changes in the DGGE profiles nor influencedbifidobacterial levels. In a double-blind crossover study on the qualitativeand quantitative effects of fresh and heat-treated yogurt on the bacterialintestinal microbiota from healthy subjects [Clostridium perfringens and a significant decrease in the density of Bacteroides after consumption of both types of yogurt.Next to FISH, also real-time PCR has been used in combination with DGGEto verify and substantiate compositional changes in a semiquantitative manner. The latter two methods were used in an integrated approach to monitor andquantify pronounced changes in fecal microbiota of healthy subjects uponlong-term administration of a prebiotic (lactulose), a probiotic  and wereonly minimally affected following one week ingestion of ciprofloxacin (Ds of 73%). In contrast, clindamycin markedly reduced the microbial complexity (Ds of 11–18%). However, onceclindamycin therapy ceased, recovery of some intestinal groups was evident within daysas indicated by the increasing similarity indices when compared to the patternprior to antibiotic treatment (Ds of 36–44%). In three otherpatients, cefazolin caused onlyminimal alteration of V3-16S rRNA gene patterns (Ds of 81–83%) whereasamoxicillin/clavulanate triggered marked changes in profile compositions (Ds of 19–42%). Overall, therelative degree of alterations in the universal DGGE patterns tended tocorrespond to the relative activity spectrum of the antimicrobials against intestinal anaerobes.Not suprisingly, it has been shown that the human adult intestinalmicrobiota is affected to a different degree during antimicrobial therapydepending on the type and/or activity spectrum of the therapeutic component. In the Lactobacillusacidophilus LaCH-5 and Bifidobacterium animalis subsp. lactis Bb-12 from the administered commercialpreparation Trevis were recovered from fecal samples,and phenotypically and genotypically characterized for their tetracycline (Tc)resistance. The Tc-susceptible strain LaCH-5 remained so during therapy,whereas recovered isolates of the Tc-resistant strain Bb-12 containing the tet(W)resistance gene were not found to have acquired additional Tc resistance genes. Although these observations evidence the stability of the probiotic strains assuch, however, the authors did not investigate the possible effect ofintroducing a tet(W)-carrying strain during doxycycline therapy on thedissemination of this gene throughout the intestinal microbiota.In order to reduce the possible side-effects of antimicrobial therapy,probiotics are commonly administered in combination with antimicrobials duringand after the period of intake . In suchDespiteits increasing use in the field of molecular microbial ecology, it is clearthat SDE-based community profiling has a number of limitations that do notallow indepth analysis of microbial communities as complex as the humanintestinal tract. Some of these limitations, such as detection level andtaxonomic resolution, can be regarded as potential pitfalls and should becarefully taken into account during protocol development and data analysis. Infact, many of these critical factors are situated along the stages prior to theactual SDE step such as sampling and sample processing, nucleic acid extractionand community PCR, and deserve specific attention when troubleshooting SDEproblems.As discussedabove, the choice of an efficient and reproducible nucleic acid extractionmethod ensuring optimal cell lysis and maximal removal of various PCRinhibitors present in intestinal samples is highly crucial. Likewise, possiblebias introduced during PCR amplification by differential or preferentialamplification of target genes from complex communities may prejudice theanalysis . As a reBifidobacterium adolescentis ATCC 15703T obtained with Bifidobacterium-specific PCR primers Bif164-f andBif662-GC-r  be present in a single copy in the bacterial genome; (ii)contain conserved regions among the members of the population to allow rationalprimer design; and (iii) comprise regions with sufficient sequence variationamongst the members of the population to produce a fingerprint revealingmaximal diversity. Although the 16S rRNA gene is the prototype target in SDEapplications based on the above criteria, it should be kept in mind that thepossible occurrence of intraspecific multicopy operon heterogeneity  andthe 662-GC-r . Furtherus group  and the us group , Table 2RNA gene , 198. Alne [rpoB –202 havene [rpoB is the oSeveral authors have identified cases of comigrationin SDE analysis of amplicons showing clear sequence variation –206. EveEspecially for SDE profiles from complex ecosystems such as theintestinal tract, band sequencing analysis may prove to be less straightforwardas anticipated for several reasons. First of all, there is the possibility ofmultiple sequences being present in a single band due to comigration. In thiscase, a cloning step should be introduced prior to actual sequencing of thefragments. Furthermore, it has been reported that excised DNA fragments arecommonly contaminated with ssDNA originating from other organisms present inthe sample resulting in genetic contamination of the sequence profile. Eliminationof the ssDNA products through mung bean or S1 nuclease treatment of theeluted DNA prior to amplification (and cloning) can increase the success rateto obtain a pure DNA sequence of the SDE band target , 211. AnTaq DNApolymerase to synthesize a complete amplicon. Another observation potentiallyhampering the resolution of SDE analysis is linked to the phenomenon ofextended fuzzy bands. The source of this inconsistency is the existence ofmultiple melting domains (MMDs) in the amplified fragments which results in astepwise increase in retardation and ultimately leads to the visualization of awide and diffuse band. Little is known about the distribution of MMD which isdependent on the target fragment and the phylogenetic group. This phenomenonhas been observed when using universal 16S rRNA gene primers in SDE analysis ofdifferent types of environmental samples including feces [Nextto their relative electrophoretic position and sequence composition, also theintensity and sharpness of SDE bands require special attention. Artifactualdouble bands, that is, the situation where each prominentband is accompanied at close distance by a second, often less intense band,have been reported in several SDE applications. Janse et al.  suggesteng feces , water [ng feces , and soing feces . Weak, fng feces .6 CFU/g feces (wetweight) as the detection level that could be reached by DGGE for predominantmembers of the fecal microbiota. In this context, it should be stressed thatthe detection limit is a relative value that may strongly depend on severalparameters including the taxonomic complexity of the ecosystem present in thesample, the efficiency of DNA extraction, the total number of bacteria, and therelative concentration of each organism in the sample. Human stool usuallycontains 1010–1012 CFU/g feces, and it is thus possiblethat the detection limit may improve if 1010 CFU/g compared to 1012 CFU/g is present due to alower competition among the constituting DNA templates during PCRamplification. Ingeneral, the potential to detect a specific taxon can be improved by usinggroup-specific primers that can narrow the size of the target population. Even when using agenus-specific primer, however, the template DNA ratio may still affectthe DGGE-based detection of certain species that are underrepresented in amixed community sample [The detectionlimit of SDE-based methods, that is, the minimum (relative) concentration ornumber in which any given member of a complex bacterial ecosystem needs to bepresent in order to be visualized in the corresponding community fingerprint,was initially estimated to approach 1% of the total population . Thisesy sample . Althouy sample , and they sample .This review has highlighted the broad applicationspectrum of SDE-based techniques in the field of intestinal microbiology,ranging from primary assessments of the bacterial complexity and diversity ofintestinal community structures to the monitoring of compositional changes atdifferent population levels upon dietary or therapeutic interventions. In moreadvanced approaches, additional tools such as band sequence analysis, bandposition analysis, and blotting analysis permit further taxonomic explorationof the microbialcommunities present in the gut. Overall, SDE techniques are technically fairlysimple, fast, flexible, and reproducible. Because they allow simultaneousanalysis of multiple samples, SDE-based methods may be highly suitable in theselection of candidate subjects for human metagenome studies. Taken togetherwith the ability to visualize poorly or as yet unculturable bacterial groups,these features have contributed to the current fame and reputation of SDEtechnology.http://www.transgenomic.com/), DHPLC analysis offers several advantages overconventional SDE analysis including the lack of gel preparation, the higherthroughput, and the possibility to automatically collect sample fractions forfurther (sequence) analysis. DHPLC has been successfully used to analyzemicrobial communities with a low [As is the case with any other methodology, however,also the SDE approach has a number of intrinsic limitations. Besides thegeneral biases associated with sampling (including sample size), total DNAextraction and PCR amplification, also more specific restrictions such as intraspecies16S rRNA gene operonheterogeneities, limited fragment length, or fuzzy bands can limit the applicability of SDE. On the otherhand, it is important to always consider the significance and possibleconsequences of these drawbacks in the context of the study because somelimitations will not be equally important when monitoring community stabilitycompared to when assessing biodiversity. One of the most important steps in thedefinition of a new SDE protocol is the choice of the primer target which canalready prevent several potential drawbacks related with SDE. A careful selection of thetarget fragment with regard to sequence variability and the distribution ofmultiple melting domains and a clear focus on the phylum of interest isconducive to achieve the desired resolution. In addition, also the SDEtechnology itself is constantly developing. Denaturing high-performanceliquid chromatography (DHPLC) has relatively recently been introduced to detectgenetic variation based on the SDE principle, but employs an HPLC columninstead of a polyacrylamide gel matrix for amplicon separation . Wheninth a low , 219 or th a low  complexith a low . Othereth a low  may offehttp://www.nihroadmap.nih.gov/hmp/) may open newavenues for the development of SDE primers targeting specific functional genes. Considering all upcoming technological developments, it is expected that SDEcommunity profiling will maintain and even reinforce its position in the largespectrum of molecular approaches currently employed to unravel host-microbe andmicromicrobe interactions within the human microbiome. The successfulincorporation of DGGE profiling in the recently launched concept of functionalmetagenomics [In contemporary intestinalmicrobiology, SDE-based methods are rarely used as a single or end-pointapproach but are usually combined with culture methods and/or other molecularmethods such as clone libraries, FISH, real-time PCR, and microarrays in a complementaryresearch strategy. It is beyond doubt that these polyphasic study designsshould be further pursued and developed to broaden current insights in themicrobial diversity, dynamics, and interactions within the intestinal tract. Inthis regard, one of the major challenges ahead lies in the combined analysis ofmicrobial presence and microbial activity. As an example of such an integratedapproach, parallel DGGE analysis targetting the 16S rRNA gene as taxonomicmarker and the adenosine-5′-phosphosulfate reductase subunit Agene as functional gene has been used to study the succession anddiversity of sulfate-reducing bacteria in the mouse gastrointestinal tract. In thisgenomics , that is


Bos taurus. Reliable identification and annotation of entire sets of tRNA genes allows the evolution of tRNA genes to be understood on a genomic scale.Detailed information regarding the number and organization of transfer RNA (tRNA) genes at the genome level is becoming readily available with the increase of DNA sequencing of whole genomes. However the identification of functional tRNA genes is challenging for species that have large numbers of repetitive elements containing tRNA derived sequences, such as B. taurus genome using bioinformatics and comparative genomics approaches to catalogue and analyze cow tRNA genes. The initial analysis of the cow genome using tRNAscan-SE identified 31,868 putative tRNA genes and 189,183 pseudogenes, where 28,830 of the 31,868 predicted tRNA genes were classified as repetitive elements by the RepeatMasker program. We then used comparative genomics to further discriminate between functional tRNA genes and tRNA-derived sequences for the remaining set of 3,038 putative tRNA genes. For our analysis, we used the human, chimpanzee, mouse, rat, horse, dog, chicken and fugu genomes to predict that the number of active tRNA genes in cow lies in the vicinity of 439. Of this set, 150 tRNA genes were 100% identical in their sequences across all nine vertebrate genomes studied. Using clustering analyses, we identified a new tRNA-GlyCCC subfamily present in all analyzed mammalian genomes. We suggest that this subfamily originated from an ancestral tRNA-GlyGCC gene via a point mutation prior to the radiation of the mammalian lineages. Lastly, in a separate analysis we created phylogenetic profiles for each putative cow tRNA gene using a representative set of genomes to gain an overview of common evolutionary histories of tRNA genes.In this study, we explored the The use of a combination of bioinformatics and comparative genomics approaches has allowed the confident identification of a set of cow tRNA genes that will facilitate further studies in understanding the molecular evolution of cow tRNA genes. Transfer RNAs (tRNAs) are members of a family of ubiquitous molecules that provide the essential link between the genetic code and amino acids. tRNAs are a central component of ribosomal protein biosynthesis that acts by decoding template messenger RNA (mRNA) into the amino acid sequence of the encoded protein. Although the mechanistic role of tRNA in protein synthesis is well understood, evidence illustrating the complexity of evolution and expression of tRNA genes continues to emerge -3.The tRNA multigene family usually comprises 20 amino acid accepting groups; each group may contain one or more tRNA members that recognize different codons commonly referred to as tRNA isoacceptors. Generally, the number of tRNA isoacceptors is highly variable amongst different genomes . While v4 [Bos taurus genome is predicted to comprise of SINEs containing tRNA-derived sequences [With the advent of multiple genome sequencing projects, entire tRNA repertoires have been identified in many genomes. While features of tRNA genes are well defined, predicting functional tRNA genes in eukaryotic genomes can be difficult. Sequencing of vertebrate genomes revealed that tRNA-derived sequences are often contained within retrotransposon insertions that give rise to different families of short interspersed elements (SINEs) . In the 4 . For exaequences . These cequences ,13.Identification of functional non-coding RNAs in genomic sequences depends heavily on the analysis of conserved RNA secondary structure in order to make accurate predictions . The mosB. taurus genome, we were challenged by a large number of tRNA-like sequences due to SINE activity. In order to provide a comprehensive annotation of functional tRNA genes in the B. taurus genome we used a combination of approaches which involved tRNAscan-SE, RepeatMasker and comparative genomics to overcome the issue of SINEs. We then conducted cluster analyses on our set of putative tRNA genes, to gain insight into the evolutionary histories of cow tRNA genes. This in turn will help further the understanding of tRNA gene evolution.For our analysis of the The initial analysis of the cow genome using tRNAscan-SE identified 191,073 and 29,978 tRNA-like sequences positioned evenly across assembled cow chromosomes and unordered genomic contigs respectively . In contrast, subfamily 2 tRNA-GlyCCC genes appear to be only present in the mammals as no orthologs were detectable in the current assemblies of the chicken, lizard and fugu genomes.The set of 158 unique tRNA gene sequences was examined using cluster analysis. tRNA genes that carry the same amino acid generally formed clusters with ≥ 0.95 posterior probability (PP) Figure . This obs Figure . The firWe created phylogenetic profiles of cow tRNA genes to identify the presence or absence of tRNA gene homologs in other genomes. We compared the cow tRNA gene repertoire to a representative set of genomes from mammals , fish , reptile (green anole lizard), bird (chicken) and invertebrates . To be comprehensive we used the set of 31,868 cow putative tRNA genes identified using tRNAscan-SE and another 328,857 putative tRNA genes from the other 20 genomes . This observed tRNA-GlyCCC appears to have arisen in the ancestor of the eutherian mammals and marsupials by a mutation in the anticodon of a member of the tRNA-GlyGCC family, as this new family was not identified among the non-mammalian genomes we investigated  between vertebrate and invertebrate genomes. The number of cow tRNA orthologs among vertebrates is 2–3 folds larger than the number among invertebrates. Expansion of vertebrate genomes resulting in emergence of paralogous copies of tRNA genes may explain the larger numbers of tRNA orthologs observed in vertebrates. This is in line with the observation that the number of tRNA genes in a genome is positively correlated to the genome size . HoweverB. taurus genome. Our analyses revealed that while most tRNA isoacceptors seem to have evolved from a common ancestor, some may have different evolutionary histories. Our additional analysis of the glycine tRNA genes revealed two distinct families of tRNA-GlyCCC genes, one of which seems to have been formed via a point mutation in the anticodon of a member of the tRNA-GlyGCC family just prior to the radiation of mammals. Finally, our phylogenetic profiles of cow tRNA genes shows a large core set of tRNA genes conserved among vertebrate genomes, which highlights the importance of vertebrate genome expansion in relation to tRNA gene sets in genomes.We have identified a set of highly conserved tRNA genes among vertebrate genomes as part of our analysis of the Homo sapiens), chimpanzee (Pan troglodytes), dog (Canis familiaris), mouse (Mus musculus), rat (Rattus norvegicus), chicken , and fugu (Takifugu rubripes) tRNA gene sequences predicted by tRNAscan-SE and their respective tRNAscan-SE output files were downloaded from the genomic tRNA database [th January 2008. The cow (B. taurus) (August 2006), horse , opossum , platypus , green anole lizard , medaka , stickleback , tetraodon , zebrafish  and honey bee  genome assemblies and sea urchin  scaffolds were downloaded as unmasked fasta files from the UCSC genome browser [Petromyzon marinus, March 2007) contigs, Pacific sea squirt  reftigs and lancelet  scaffolds were downloaded as unmasked fasta files from the UCSC test genome browser [Human   running Phylogenetic profiles describe the presence-absence of homologous genes in genomes . We consWe inferred cluster relationships of all cow tRNA genes with orthologs in human, chimpanzee, horse, dog, mouse, rat, chicken and fugu. We aligned all unique tRNA sequences using T-Coffee  running DTPT performed detailed data analysis and wrote the manuscript. SM and WCB assisted with data analysis. BPD initiated this study and participated in its design, and the preparation of the final version of the manuscript. EAG coordinated this study, assisted with data analyses and drafted the final version of the manuscript. All authors have read and approved the final manuscript.Figure S1 – Bioinformatics pipeline for prediction of functional cow tRNA genes. The pipeline shows the various stages of tRNA filtering for the cow genome using bioinformatics and comparative genomics. Each process is indicated by a rectangle and each data store by a slanted rectangle. For more details for each process please refer to the Methods section.Click here for fileBos taurus genome compared against tRNA gene numberTable S1 – Codon usage in . The number of cow tRNA genes was predicted using our comparative approach (see methods). Codons that have been highlighted in bold are generally decoded by a single tRNA species due to wobble. Codon frequencies were obtained from .Click here for filebtau3.1_tRNA.fa – Fasta file of our confident set of cow tRNA genes. The fasta file is annotated in the same format as seen in [ seen in .Click here for fileFigure S5 – Cluster relationships of cow serine tRNA genes. Relationships between cow serine tRNA genes and a histidine tRNA gene, which is used as an outgroup. Posterior probability scores are shown for each cluster to support the strength of the respective clusters.Click here for fileFigure S6 – Cluster relationships of cow valine tRNA genes. Relationships between cow valine tRNA genes and a histidine tRNA gene, which is used as an outgroup. Posterior probability scores are shown for each cluster to support the strength of the respective clusters.Click here for fileFigure S2 – Multiple sequence alignment of predicted cow glycine tRNA genes. Multiple sequence alignment of all glycine tRNA genes with 20 bp of flanking sequence identified in the cow genome. BoxA (corresponding to nt 8–19) and BoxB (corresponding to nt 52–62) sites constitute internal promoter sites for RNA polymerase III. Conserved nucleotides are highlighted and indicated by asterisks.Click here for fileTable S2 – Summary of BLASTn matches and number of tRNA gene matches at a 95% similarity threshold. Cow tRNA genes were blasted against the full set of tRNA genes from a respective genome. BLAST results were parsed using two different thresholds, an e-value of 0.0001 and using our comparative approach.Click here for fileTable S3 – Summary of most abundant phylogenetic profiles and the bovine tRNA genes constituting the profile. The number of cow tRNA genes that make up the most abundant phylogenetic profiles are shown. Cow tRNA genes are grouped according to their amino acid specificity and anticodon.Click here for fileFigure S3 – Predicted secondary structure of the consensus Bov-tA2 sequence (downloaded from RepBase). Repetitive elements derived from tRNA maintain sequence similarity as well as structural features. Here we observe the similar stem loop structure of Bov-tA2 to tRNAs. Prediction of the secondary structure was done using tools available at Genomic tRNA database website [ website .Click here for fileFigure S4 – Phylogenetic relationships of human, chimpanzee, rat, mouse, dog, horse, cow, opossum, platypus, chicken, lizard, tetraodon, fugu, stickleback, medaka, zebrafish, lamprey, lancelet, sea squirt and sea urchin. Phylogenetic relationships of many vertebrate genomes based on NCBI taxonomy.Click here for file


Despite advances in treatment, cancer remains the 2nd most common cause of death in the United States. Poor cure rates may result from the ability of cancer to recur and spread after initial therapies have seemingly eliminated detectable signs of disease. A growing body of evidence supports a role for cancer stem cells (CSCs) in tumor regrowth and spread after initial treatment. Thus, targeting CSCs in combination with traditional induction therapies may improve treatment outcomes and survival rates. Unfortunately, CSCs tend to be resistant to chemo- and radiation therapy, and a better understanding of the mechanisms underlying CSC resistance to treatment is necessary. This paper provides an update on evidence that supports a fundamental role for CSCs in cancer progression, summarizes potential mechanisms of CSC resistance to treatment, and discusses classes of drugs currently in preclinical or clinical testing that show promise at targeting CSCs. Individualized cancer treatment has been an attractive concept since the beginning of cancer research. Breakthroughs in research have allowed the characterization of malignancies according to their unique gene expression, which has allowed the pragmatic targeting of many cancer types based on their specific gene expression patterns. For example, trastuzumab improves the overall and progression-free survival in human epidermal receptor 2- (Her2-) positive breast cancer –3. The rIndividualized profiling and targeting systems provide novel tools to improve both prognostic accuracy and individualized treatment for patients . In addiHowever, despite this progress, cancer remains the 2nd most common cause of death in the United States . Our conA paradigm shift in our understanding of cancer tumorigenesis emerged in 1994 when John Dick and colleagues demonstrated that human acute myeloid leukemia (AML) has a hierarchical organization that originates from a primitive hematopoietic cell . This poDespite controversies surrounding the CSC hypothesis , 21, suben masse within such cancer types may limit disease progression. Alternatively, suppressing residual CSCs after initial tumor debulking may sustain remissions and extend the progression-free survival of patients receiving CSC suppressive therapy. Considering these distinct therapeutic potentials of targeting CSCs, it appears that CSC-targeted therapies could be an effective complement to traditional treatment approaches such as surgery, chemotherapy, and radiation therapy. Indeed, it is possible that these traditional strategies leave behind residual CSCs which are capable of spreading and regenerating tumors, leading to cancer recurrence and metastasis. Moreover, these recurring tumors often acquire resistance to chemotherapy and radiation [In cancer types where neoplastic growth and differentiation depend on CSCs, complete eradication of this population may be curative. Furthermore, agents that force CSCs to rapidly differentiate adiation , 37. MulEvidence suggests that CSCs are highly heterogeneous , 39. ThiAs previously mentioned, cancer recurrence may be partly due to the fact that conventional therapies such as chemo- and radiation therapy fail to specifically target CSCs. Instead, these therapies likely enrich CSC populations by preferentially killing differentiated cancer cells that had little potential to sustain cancer growth. Numerous studies indicate that CSCs are resistant to chemo- and radiotherapy and are therefore preferentially preserved when cancer cells are targeted by these approaches , 42–45. in vitro and differentiating into tumors in vivo. Matsumoto et al. elucidated a mechanistic relationship between CD133 and the hypoxia-inducible factor-1α (HIF-1α), a downstream molecule in the mammalian target of rapamycin (mTOR) cell signaling pathway, suggesting a role for mTOR in the regulation of CD133 expression [In recent years, an effort has been made to successfully identify stem cells in multiple human malignancies, including hematological, breast, colorectal, brain, pancreatic, and maxillofacial cancer , 48–50. pression . In addition to cell-surface markers, many investigators have focused on the selective overexpression of certain genes normally present in progenitor cells. Leukemia cells, which have been transformed from the normally present “partially committed” cells responsible for physiological cellular maintenance, undergo mutations that result in self-perpetuated renewal capabilities. These cells can be identified by selective gene overexpression , 53–57. Interestingly, Glinsky has developed a novel clinical model to assess the relative “stemness” of cancer cells by quantifying gene expression signatures, and he has shown that this model may predict therapeutic outcomes. The “BMI1 pathway” algorithm is based on a collective signature of 9 individual gene characteristics: TEZ, EED pathway, Suz12/POLII, Suz12, Nanog/Sox2/Oct4, PcG-TF, BCD-TF, ESC pattern, and BMI1 pathway. This multifactorial model allowed the stratification of patients into high-risk and low-risk groups in a retrospective analysis of large cohorts of breast, prostate, lung, and ovarian cancer patients . It remaCSCs have been found to exhibit a number of genetic and cellular adaptations that confer resistance to classical therapeutic approaches. These include relative dormancy/slow cell cycle kinetics, efficient DNA repair, high expression of multidrug-resistance-type membrane transporters, and resistance to apoptosis . Cancer Normal, noncancerous stem cells exhibit well-fortified DNA mutation defense systems that typically serve to prevent mutation into carcinogenic CSCs. Unfortunately, when mutations that create CSCs do occur, the inherent defense systems of stem cells serve to protect them from DNA-targeting chemo- and radiation therapy. The chemo- and radioresistance of CSCs has now been demonstrated in numerous experiments , althougOne potential modulator of CSC resistance to DNA-targeting agents is the family of checkpoint kinases 1/2 (Chk1/2 kinases), which become activated after genotoxic stress and arrest the cell cycle to allow DNA repair. These kinases have higher basal and inducible activities in CSCs than in nonstem cells . SupportIn addition to augmented DNA repair systems, CSCs may also exhibit changes in telomerase function, which allows resistance to chromosomal degradation in these rapidly dividing cells. Telomerase is a complex ribonucleoprotein enzyme that synthesizes and maintains telomeric repeats at the ends of chromosomal strands . SustainAn important component of the DNA integrity defense systems in normal stem cells is the relatively high expression of efflux transporters from the ATP-binding cassette (ABC) gene family . These pResistance to therapy might also be conferred to CSCs through the activation of the Akt pathway , 76 and The mitochondrial pathway of apoptosis is triggered by cytochrome c release and second mitochondria-derived activator of caspase (Smac) activation . Smac, iκB (NFκB). NFκB is a transcription factor believed to be intricately involved in the development and progression of certain cancer types [κB, is an antiapoptotic transcription factor that is activated in leukemias [κB inhibitors include NPI-0052 , which is in phase I clinical trials, and TDZD-8 (parthenolide), which is still in preclinical testing.Another promising molecular target to promote apoptosis in CSCs is nuclear factor er types . Nucleareukemias , 87, paneukemias , and meleukemias . Althougin vivo, and the clinical significance of this remains unknown. Still, we speculate that radiation might be more effective in treating cancer if it is administered before any antiangiogenic chemotherapies are applied. Oxygen is a well-known radiosensitizing agent due to its ability to form radiation-induced reactive oxygen species that can damage DNA. Accordingly, radioresistance in breast CSCs may be mediated by increased production of free-radical scavengers . ConsideVermeulen and colleagues recently discovered another interesting role of the microenvironment, specifically in the promotion of cancer cell stemness. They found that high Wnt pathway signaling functionally defines colon CSCs . ImportaAn important result of the well-documented CSC resistance to radiation and chemotherapy is that these therapies often serve to enrich the resistant CSC subpopulation, perhaps even selecting for more resistant clones within a heterogeneous CSC population. Evidence of radiation-induced enrichment has been shown in both brain , 45 and Additional strategies to overcome therapeutic resistance during cancer treatment are as follows.It is now well established that combination therapy helps prevent the development of cancer resistance, except in a select group of cancer types where a single pharmaceutically correctable mutation exists . For exaAs new data supports a role for the CSC hypothesis in solid tumors in addition to hematologic malignancies, outcomes following the surgical resection of solid tumors may significantly improve. If induction approaches can be effectively augmented with anti-CSC therapies, then followup surgical resection may show improved curative outcomes. Theoretically, CSC-specific induction chemotherapies should offer an immediate reduction in CSC metastatic potential and should reduce any hematogenous and lymphatic CSC micrometastases that would otherwise diminish the efficacy of surgical resection. Considering its powerful therapeutic potential, CSC-targeted therapies may be particularly valuable in surgically challenging malignancies such as pancreatic  and braihttp://clinicaltrials.gov/). In order to more effectively target CSCs, molecular proliferation and survival mechanisms of CSCs must be better understood. Many institutions have developed large banks of malignant tissues with coordinated clinical data, and this resource is being actively mined. Techniques for concentrating, isolating, and enriching CSCs from resected tumors are also rapidly evolving, and cell culture and xenograft models that allow us to transplant and sustain CSCs are maturing. Finally, these advances have been translated into the development of several therapeutic opportunities. Here, we will review some of the prominent classes of drugs that will potentially yield clinical benefits in the near future. An update of clinical trials assessing these targets is illustrated in β-catenin [Developmental pathways that direct the differentiation of normal stem cells represent attractive targets for drug discovery. In particular, the roles of Notch and Wnt/-catenin  signalin-catenin , 101. Fo-catenin . The non-catenin .β-catenin pathway promotes genomic instability and DNA damage tolerance that may be enhanced by DNA damage in CSCs [β-catenin pathway may promote genomic instability after irradiation, thus allowing tumor cells to both survive after irradiation and to develop additional adaptive mutations. Wnt inhibitors have been designed to therapeutically prevent this possibility and include ICG-001, fungal derivatives PKF115-854 and CGP049090, as well as monoclonal antibodies against Wnt-1 and Wnt-2 [The Wnt/ in CSCs . The pat in CSCs  and poss in CSCs . Moreove in CSCs . It has nd Wnt-2   can be induced by histone deacetylase inhibition to inhibit the growth of and induce the differentiation of Glioblastoma neurospheres and xenografts . This prThe hedgehog signaling pathway may represent an important modulator of CSC carcinogenesis with significant therapeutic implications –117. Sim+ cells from brain tumor biopsy samples were able to reform tumors in in vivo mouse models [+ and CD133− glioblastoma (GBM) cells. A mixture of CD133+ and CD133− cells were then exposed to near-infrared laser light, and the CD133+ GBM cells were selectively destroyed. They found in vivo benefits of this technique as well in mouse models [As mentioned previously, the cell-surface molecule CD133 is believed to be a stem cell marker for multiple cancer types . Its tume models . A recene models .en masse as they do in the treatment of APML. It is important for future studies to focus on the discovery of new molecular targets for the development of better pharmaceutical agents to eliminate or differentiate CSCs and that these agents be studied in tandem with traditional cancer therapies.With the advent of multidisciplinary approaches to cancer therapy, significant strides have been made in the treatment of cancer. Now with new discoveries relating to CSCs, we have yet another mechanism of therapeutic arsenal that may prove beneficial in combination with current therapeutic modalities. The basic foundations for CSC-targeted therapy are actively being discovered, and there are already several pharmacologic agents available that are capable of specifically modulating CSC intracellular signaling. Still, much remains unknown about the basic signaling mechanisms of CSCs that confer resistance to treatment, and better methods for the disruption of CSC signaling must be developed to fully integrate the CSC hypothesis into our treatment paradigms. Interestingly, CSCs may not necessarily need to be eradicated to prevent cancer progression if they can be forced to differentiate down their lineage


Head and neck (HN) cancer represents one of the most challenging diseases because the mortality remains high despite advances in early diagnosis and treatment. Although vaccine-based approaches for the treatment of advanced squamous cell carcinoma of the head and neck have achieved limited clinical success, advances in cancer immunology provide a strong foundation and powerful new tools to guide current attempts to develop effective cancer vaccines. This article reviews what has to be rather what has been done in the field for the development of future vaccines in HN tumours. The concept that a vaccine could be useful in the treatment of cancer diseases is a long-held hope coming from the observation that patients with cancer who developed bacterial infections experienced remission of their malignancies. In 1896, New York surgeon William Coley locally injected streptococcal broth cultures to induce erysipelas in a patient with an inoperable neck sarcoma, obtaining a tumour regression. Although the therapy was toxic, the patient's tumour ultimately regressed, and he lived disease-free for 8 years before succumbing to his cancer . NevertThe discovery of so powerful TAAs in HNSCC is giving substantial basis for efficacious and less toxic treatments, but in the mean time HNSCC as other tumours participates in tumour immune escape through various mechanisms:i) it disrupts antigen processing and presentation machinery by altering the MHC class I and TAP 1–2 expression;ii) it recruits immunosuppressive Treg to dampen effector T-cell activity,iii) by chemokine production it alters T-cell homeostasis increasing the sensitivity of effector T cells to apoptosis.in vitro the ability of specific CTLs to recognize their tumour cell targets and subsequently to lyse them . Chiiew, see ,25; co-aiew, see ,27. Receiew, see ,29The results of clinical trials indicate that peptide/protein vaccination has low toxicity but a strong discordance exists between immune and clinical responses, reinforcing the need of further improvement to the vaccination by the utilization of peptide-pulsed dendritic cells, the addition of helper peptides, and depletion of Treg. Several phase I clinical trials using antigenic peptides derived from HPV E6/E7 have been so far conducted as well as multivalent peptide-based vaccination against p53 -32 with DNA vaccines have been used in the clinical arena to elicit antigen-specific immune responses. Although nucleic acid vaccines do not appear to induce as vigorous immune responses as live viral vaccine vectors, they have several advantages. Naked DNA is relatively safe, stable, cost efficient, and able to sustain reasonable levels of antigen expression within cells DNA-based plasmid vectors remain stable in a wide range of conditions over great lengths of time, and they can be delivered with little risk to individuals who are immunosuppressed. In addition, since DNA vaccines do not elicit neutralizing antibodies in the vaccinated patient, they can be repeatedly administered with similar efficacy.The cellular machinery is needed to generate tumour antigens and other necessary proteins are provided by the host and not required to be incorporated into the vaccine itself. Finally, the DNA backbone of the injected plasmid contains its own cognate immunostimulatory sequences, which have been shown to activate innate responses . HoweverA clinical trial with the administration of liposome-encapsulated plasmid IL-2 in combination with chemotherapeutics, was conducted and robust IFN-γ and IL-12 titers were detected in patients with advanced HNSCC .Similarly, phase I clinical trial using a naked DNA vaccine encoding the HPV-16 E7 gene linked to M. tuberculosis HSP70 (pNGVL4a-Sig/E7(detox)/HSP70) is conducting at the Johns Hopkins Hospital (USA) in patients with advanced HPV-16 associated HNSCC. The DNA vaccine was well tolerated and a subset of the vaccinated patients demonstrated detectable systemic levels of E7-specific CD8+ T cell immune responses .Bacteria, such as Listeria monocytogenes, Salmonella, Lactococcus lactis, Lactobacillus plantarum, Bacillus Calmette-Guerin, and several viral vectors, including vaccinia virus (VV), adenovirus, adeno-associated virus, alphavirus, and its derivative vectors, such as sindbis virus, semliki forest virus, and venezuelan equine encephalitis virus have been used to deliver genes or proteins of interest to elicit antigen-specific immunotherapy .Among the bacterial vectors, L. monocytogenes has emerged as a promising vector, because in animal models it is able to induce both CD8+ and CD4+ immune responses to elicited regression of established tumours, and to overcome central tolerance by expanding low avidity CD8+ T cells specific for E7 .Among viral vectors, VV was historically one of the first viral vector employed in clinical trials of therapeutic vaccines against HPV-associated cancer . To dateSince ancient times plants have been used for therapeutic purposes, mostly by providing medicinal compounds that have been extracted and used to treat illness. Nowadays, plant molecular farming provides new therapeutic possibilities combining the innovations in medical science and plant biology to create affordable pharmaceutical products. Many methods are available for the antigen production and all the TAA antigen in principle can be obtained with the available technologies .(N. benthamiana) and utilized as therapeutic lymphoma vaccine in subcutaneous immunization, induced an anti-idiotype immune response and protected mice from challenge by a lethal dose of syngeneic tumour cells. Interestingly, mice that received the scFv alone, without adjuvant, showed a high degree of protection . GM-CSFex vivo activation and expansion of tumour-reactive lymphocytes which are then returned to the host.All the above mentioned vaccine preparation can reach a strong CTL stimulation in vaccinated animals or humans. However, even high levels of CTL did not correlate with the presence of active effector cells within the tumours as the tumour escaping mechanisms are actively fighting the CTL induced by the TAA utilised for immunotherapy. The adoptive transfer of active T cells may overcome the immunotolerance obstacle. This technique relies on the Poorly immunogenic established tumours have been cured by ACT in murine models -66. ConsAlthough these studies, for the first time in humans, demonstrate that ACT is a viable therapeutic strategy, it has to be pointed out that results were achieved in the setting of a combined approach with chemotherapy as immunomodulatory agent, indicating the pivotal role of modulating T-reg and suggesting that in this setting even other vaccine therapy could be efficacious. Furthermore for the treatment or prevention of HNSCC it is important to note that ATC as well as DC strategies require cellular products that are subject to individual patient variability, and the differences in culture methods, loading strategies, and injection techniques render these approaches hard to be transferred to phase II/III studies and posing formidable challenges to large-scale clinical implementation.Targeting HNSCC cell surfaces with high-affinity antibodies is a total different approach that is emerging as advantageous strategy in the development of immunotherapies. mAb therapy is based on multiple mechanisms of action including: inhibition of ligand induced activation; induction of receptor degradation or complement-mediated/antibody-dependent cellular cytotoxicity; activation of tumour-specific CTL via cross-priming of lysed tumour cells; and finally delivering of a conjugated chemotherapeutic toxin to the tumour bed when linked to the antibody -78.To date, most of the mAb therapies target the EGFR as this receptor is overexpressed in more than 90% of HNSCC .Cetuximab, a chimeric IgG1 isotype murine/human epidermal growth factor receptor-specific monoclonal antibody, as well as has Panitumumab, a fully humanized IgG2 isotype monoclonal antibody, have been approved by the US Food and Drug Administration, and their clinical efficacy is well documented . It is pAntibodies that may have an immunostimulatory component have been developed against another overexpressed tumour antigen, the vascular endothelial growth factor (VEGF) which is a tumour secreted molecule that stimulates angiogenesis and lymphangiogenesis. High expression of VEGF and its receptor was detected and associated with poor survival in patients with head and neck cancers . BevacizFinally antibodies can be targeted to molecules involved in immune modulation. In mice antitumour immunity was achieved by antibodies against the CTLA4 molecule that is a ligand expressed by T lymphocytes that functions to inhibit T-cell activation by binding to B7.1 and B7.2, thereby preventing CD28 from binding to B7 .The brilliant results of a phase 1 clinical trial using a fully humanized antagonistic CTLA4 monoclonal antibody highlight the potential immunotherapeutic value of antibody-based therapies for cancer .The introduction in the clinical practice of two highly efficacious preventive vaccines ,85   the pessData of phase I-II trials with these large arrays of therapeutic vaccines indicate their efficacy in elicit some immunological response, and only few phase III trials reported success in the therapy having the RECIST as end point.In a recent reviews for all type of tumours a percentage of only 2.9% of clinical response to therapeutic vaccines was reported ,89. HoweTherefore the pessimistic and simply conclusion that cancer vaccines have been tested and failed may be wrong. Only in relative short time the knowledge on immunotolerance and tools to overcome it have been achieved, emphasizing the need for profound changes in the application of immunotherapy.Firstly, investigators have to concentrate their efforts in:• Generating antitumour CD4+ cells that enhance antitumour reactions and sustain the activation and survival of CD8+ cells.• Activating innate immunity by new toll-like  receptor• Targeting TAA that are essential for tumour cell survival.• Stimulating inflammatory response/environment at the tumour site.• Utilising adjuvated antigens to activate quiescent cells (e.g. with costimulatory molecules).• Blocking negative costimulatory molecule .• Finally eliminating both tumour and Treg-mediated immune suppressive mechanisms without adversely affecting effector cells, that recent evidence indicates as the most importantly achievement .Secondly, wide-scale evaluation and clinical application of cellular-based vaccines are limited by factors such as product uniformity and the significant resources necessary for successful production. Efforts must be done in order to overcome the technology obstacles limiting the development of T-cell based vaccines as standardized reagents. Moreover even the other immunotherapeutic approaches need the development of standardized procedures and vaccines to be evaluated in multi-institutional studies. The future success of immunotherapy will depend mostly on standardization.Thirdly, when used in the therapeutic setting, it is now clear that antitumour immunity can be augmented by ancillary approaches such as prime-boost strategies, or multivalent vaccines, or the use of chemotherapeutics or molecules which regulate costimulatory functions or different route of delivery. The last issue may hold promise as a mean of enhancing vaccine efficacy. Classical antimicrobial vaccination strategies have relied on subcutaneous or intramuscular injections to stimulate long-lasting immunity. However, it is now clear that the route of vaccination impacts both the potency and location of immune response generated. DNA immunization elicits completely different response if the same antigen encoding plasmid is injected intradermally, subcoutaneously or intramuscularly. In mouse model, subcutaneous injection of DC causes the T-cell responses and the localization of DC into the draining lymph nodes whereas intravenous administration does not . IntratuWhile immune therapy for the treatment of cancer holds promise, current cancer vaccines have broad limitations and few objective clinical responses.Nevertheless immunotherapy can be successful in cancer patients and thus increased effort in the development of cancer immunotherapy is needed. Further clinical studies should utilize standard criteria for clinical response and require validation in increased numbers of patients.Now, where are we? We have climbed the K2 mountain (the individuation of useful TAA and of vaccine settings able to induce CTL response) and we are climbing the Everest mountain (the tumour immunotolerance and immune escape).serological analysis of recombinant cDNA expression; TAA: tumour associated antigen; TAP: transporters associated with antigen processing; TSA: tumour-specific antigen; TMV: tobacco mosaic virus; Treg: regulatory T cells; VEGF: vascular endothelial growth factor; VV: vaccinia virus; WHO: World Health Organization.ACT: adoptive cell transfer; APC: antigen-presenting cell; APM: antigen-processing machinery; BCG: Bacillus Calmette Guerin; CCR7: chemokine receptor 7; CTL: Cytotoxic T cell; CTLA4: anti-CTL antigen-4; DC: dendritic cell: GITR: glucocorticoid-induced tumour necrosis factor receptor family; GM-CSF: Granulocyte-macrophage colony-stimulating factor; GMP: good manufacturing practice; HLA: human leukocyte antigen; MHC: major histocompatibilty complex; HN: head neck; HNSCC: head neck squamous cell carcinoma; HPV: human papillomavirus; HSP70: heat shock protein 70; IFN: interferon; RECIST: Response Evaluation Criteria in Solid Tumours; SEREX: The author declares that they have no competing interests.


Even using intensive statin monotherapy, many patients fail to achieve all the desired lipid goals and remain at high residual risk of cardiovascular events. In view of the still unproven decisively intensive "statin as monotherapy" strategy and "residual risk" concept, it is logical to ask whether other strategies, particularly fibrate/statin combination therapy, could be more beneficial and safer. A clear benefit of fibrate monotherapy did emerge previously among patients with atherogenic dyslipidemia (particularly high triglycerides and low high density lipoprotein cholesterol [HDL-C]) typically present in the metabolic syndrome and type 2 diabetes. In contrast, in patients without atherogenic dyslipidemia this favorable effect was not demonstrated.The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study investigated whether combination therapy with a statin plus a fibrate, as compared with statin monotherapy, would reduce the risk of cardiovascular disease in patients with type 2 diabetes mellitus. However, relevant patients with atherogenic dyslipidemia represented less than 17 percent of the ACCORD Lipid population (941 out of 5518 patients). In this prespecified subgroup, the patients benefited from fenofibrate therapy in addition to simvastatin similar to the previous "fibrate's as monotherapy" trials: the primary outcome rate was 12.4% in the fenofibrate group, versus 17.3% in the placebo group . Among all other 4548 patients without atherogenic dyslipidemia such rates were 10.1% in both fenofibrate and placebo study groups. Authors concluded that in the overall cohort of patients the combination of fenofibrate and simvastatin did not reduce the rate of the cardiovascular events as compared with simvastatin alone. Thus, their results do not support the routine use of combination therapy with fenofibrate and simvastatin to reduce cardiovascular risk in the general patients with type 2 diabetes. A recent large meta-analysis regarding effects of fibrates on cardiovascular outcomes noted greater effect sizes in trials that recorded a higher mean baseline triglyceride concentration (p = 0.030). As expected, in a so called "general population", reflecting a blend of effects in patients with and without atherogenic dyslipidemia, a mean "diluted" effect of fibrate therapy was reduced, but still producing a significant 10% relative risk (RR) decrease in major cardiovascular events (p = 0.048) and a 13% RR reduction for coronary events (p < 0.0001).It should be pinpointed that the epidemiological characteristics of the ACCORD Lipid study depart from those seen in real clinical practice: among people with type 2 diabetes, there is a high prevalence of atherogenic dyslipidemia and metabolic syndrome. For example, an analysis of NHANES III data in adults aged ≥50 years showed that approximately 86% of patients with type 2 diabetes also had the metabolic syndrome. Therefore, an importand finding of ACCORD Lipid study was the observation that fibrates may lead to cardiovascular risk reduction in patients with atherogenic dyslipidemia not only as monotherapy but in combination with statins as well.In conclusion, in patients with atherogenic dyslipidemia (high triglycerides and low HDL-C, fibrates -- either as monotherapy or combined with statins - were associated with reduced risk of cardiovascular events. In patients without dyslipidemia this favorable effect - as expected - was absent. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study investigated whether combination therapy with a statin plus a fibrate, as compared with statin monotherapy, would reduce the risk of cardiovascular disease in patients with type 2 diabetes mellitus who were at high risk for cardiovascular disease . In thisDespite the aforementioned conclusion, an in-depth examination of the ACCORD study itself and of several other mainstay trials concerning the pharmacological management of dyslipidemia, yields a completely different clinical picture. Namely, fibrates -- alone or combined with statins -- emerge as a proficient therapeutic tool leading to an improved cardiovascular outcome. This appraisal is depicted in the present commentary.Even using intensive statin monotherapy, many patients with atherogenic dyslipidemia fail to achieve all the desired lipid goals and remain at high residual risk of cardiovascular events . In addiDirect testing of varying degrees of low density lipoprotein cholesterol (LDL-C) lowering by using of active comparators (statin vs. statin) has been tested in 4 large outcomes trials-8: PROVEThe 'scores' of these trials where 'positive' means 'in favor of intensive LDL-C-lowering strategy using statins as monotherapy' are as follows:1. PROVE IT--TIMI 22: high-dose strong atorvastatin (80 mg/day) demonstrated a modest 16% relative reduction in the risk of death and major cardiovascular events vs. medium-dose gentle pravavastatin (80 mg/day), which was observed over the subsequent 2 years following an acute coronary syndrome. It is a "positive" result - in favor of the concept of intensive statin therapy, but it was based on very strange study design.2. the A to Z trial compared early intensive  versus a delayed conservative strategy  in patients with acute coronary syndromes. It was a "negative" study which did not achieve the prespecified end point.3. The Treating to New Targets (TNT) trial: There were reductions in cardiovascular death, myocardial infarction (MI), need for revascularization, and stroke with use of high-dose vs. standard-dose atorvastatin. Although the trial results were consistent with the concept that for cholesterol, "the lower the better", concerns were raised regarding a nonsignificant difference in total and noncardiovascular death in favor of less intensive statins therapy. In other words, the most important issue for both patients and clinicians, a hard end-point -- total death -- moved in a wrong direction in this "positive" study.4. The Incremental Decrease in End Points Through Aggressive Lipid Lowering (IDEAL) trial. In this study of patients with previous MI, intensive lowering of LDL-C  did not result in a significant reduction in the primary outcome of major coronary events vs. usual-dose simvastatin . There were no differences in cardiovascular or all-cause mortality. Patients in the atorvastatin group had higher rates of drug discontinuation due to nonserious adverse events. "Negative" results.So, out of the 5 trials which investigated intensive vs. standard statin regime, we have 2 "positive" with reservations (PROVE IT--TIMI 22 and TNT), 2 "negative" (A to Z and IDEAL) and 1 "missed" (SEARCH - suggestively negative). Alas, until now the bitterest comparator for the "intensive" statin monotherapy was 'usual-dose" statin monotherapy... then, in view of the lack of a proven effect of the current intensive "statin as monotherapy" strategy and "residual risk" concept, it is logical to ask whether other strategies could be more beneficial and safer .Fibrates have been used in clinical practice for more than four decades due to their ability to substantially decrease triglyceride levels, increase HDL-C levels and in addition moderately but significantly reduce LDL-C . Due to Although less clinical intervention studies have been performed with fibrates than with statins, there were clear evidences that two of the fibric acid derivates -- gemfibrozil and bezafibrate -- reduce the risk of cardiovascular disease -20. InteThe primary-prevention trial Helsinki Heart Study (HHS) showed that treatment with gemfibrozil led to a significant reduction in major cardiovascular events . In the In the Bezafibrate Infarction Prevention (BIP) study an overall trend of a 9.4% reduction of the incidence of primary end point  was observed. The reduction in the primary end point in 459 patients with high baseline triglycerides (above 200 mg/dl) was significant . Recent Therefore, a clear benefit of fibrate therapy did emerge among patients with atherogenic dyslipidemia (particularly high triglycerides and low HDL-C). This is the subgroup of patients for which fibrate treatment is indicated under current guidelines , this is the subgroup of patients in whom fibrates are typically prescribed in clinical practice and this is the subgroup of patients which should be investigated in the large clinical trial. So, it remains embarrassing that after all the achievements, mistakes and lessons of the previous fibrate's studies, an appropriate trial design has still not been utilized for fenofibrate.The researchers who planed the ACCORD Lipid study faced a conundrum when starting the trial , questioObviously, the epidemiological characteristics of ACCORD Lipid study depart from the real clinical practice: among people with type 2 diabetes, there is a high prevalence of atherogenic dyslipidemia and metabolic syndrome ,34, whicIn patients with atherogenic dyslipidemia (high triglycerides and low HDL-cholesterol) fibrates both as monotherapy and as combination with statins were associated with reduced risk of cardiovascular events. In patients without dyslipidemia this favorable effect was absent: don't prescribe fibrates for these patients, please.BIP: Bezafibrate Infarction Prevention study; CHD: coronary heart disease; HDL-C: high density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol; MI: myocardial infarction; PPARs: peroxisome proliferators-activated receptors.The authors declare that they have no competing interests.Both authors have equally contributed in the conception and drafting of the manuscript.