Ceratitis capitata, is one of the factors that has led to its success as an invasive pest species. To identify the molecular changes related to maturation and mating status in male and female medfly, a microarray-based gene expression approach was used to compare the head transcriptomes of sexually immature, mature virgin, and mated individuals. Attention was focused on the changes in abundance of transcripts related to reproduction, behaviour, sensory perception of chemical stimulus, and immune system processes. Broad transcriptional changes were recorded during female maturation, while post-mating transcriptional changes in females were, by contrast, modest. In male medfly, transcriptional changes were consistent both during maturation and as a consequence of mating. Of particular note was the lack of the mating-induced immune responses that have been recorded for Drosophila melanogaster, that may be due to the different reproductive strategies of these species. This study, in addition to increasing our understanding of the molecular machinery behind maturation and mating in the medfly, has identified important gene targets that might be useful in the future management of this pest.Sexual maturation and mating in insects are generally accompanied by major physiological and behavioural changes. Many of these changes are related to the need to locate a mate and subsequently, in the case of females, to switch from mate searching to oviposition behaviour. The prodigious reproductive capacity of the Mediterranean fruit fly,  Ceratitis capitata (Wiedemann) , is a highly invasive agricultural pest insect that has spread from its native range in East Africa and has now attained an almost worldwide distribution The reproductive biology of medfly has been well studied at the physiological and behavioural levels Drosophila melanogaster, Apis mellifera and Anopheles gambiaeDrosophila brain are responsible for sexual behaviours and several sex-differentiation factors are expressed therein Drosophila Sex peptide (Acp70A) have not yet been identified, they have been implicated in female sexual refraction and the switch in the female olfactory preference from male pheromones to fruit odours Bactrocera tryoni, radioactively labelled products of the male accessory glands were shown to be transferred during copulation and to migrate throughout the body of the female, including the head, within a few hours after mating D. melanogaster large amounts of male accessory gland proteins bind to the female suboesophageal ganglion and the base of the antennal nerves The molecular basis of reproductive behaviour has been studied in the model insects C. capitata transcripts Using previously identified The experimental approach followed in this paper covered the following phases: (i) creation of the microarray; (ii) selection of flies; (iii) completion of microarray assays; (iv) data analysis; (v) validation of the transcript abundance data derived from the microarray analysis using a real-time quantitative PCR; and (vi) further analysis of immunity-related genes expressed in both the head and abdomen of immature, mature and mated males and females.C. capitata sequences obtained from GenBank, formed the basis of the custom microarray. Assuming that the medfly possesses a similar number of genes as D. melanogaster, these sequences could represent about 80% of the medfly transcriptome. Up to two independent unique 60-mer oligonucleotides were designed for each sequence using the Array designer software (Premier Biosoft). Oligonucleotides were designed to be complementary to the 3′ end of each sequence where feasible. Microarrays were constructed by Agilent Technologies Sure Print Technology on glass slides.A total of 11885 assembled sequences derived from 21,253 expressed sequence tags (ESTs) from two medfly cDNA libraries from male and female heads (0–8 days old) and from 0–36 h embryos C. capitata, established in Pavia in 1979 from a colony started in 1968 at the European Community Joint Research Centre, Ispra, Italy, with wild flies from Sicily and Greece, was used in this study. Standard rearing methods were used The ISPRA strain of 3 cage shortly after lights-on. Mating in our laboratory strain of C. capitata begins soon after lights-on. As copulating pairs were observed they were isolated and removed from the cage in small vials. Only those pairs that remained in copula for at least 100 minutes were used to avoid false matings, i.e. those with little or no sperm transfer To obtain mated flies, approximately 100 three day old virgin flies of each sex were introduced into a 30 cmTotal RNA (3 µg) was used to synthesize double-stranded cDNA using an oligo d(T)-T7 promoter primer. Complementary RNA (cRNA) probes labelled with Cy-3-dUTP and Cy-5-dUTP fluorescent nucleotides were synthesized from this double-stranded cDNA using the Agilent Technologies low-input linear amplification kit. Unincorporated fluorescent nucleotides were removed using the Qiagen RNeasy kit (Qiagen) and probe quality determined spectrophotometrically.The experiments performed and the labelling of the cRNA probes were: 1 d virgin female head (Cy-5) against 4 d virgin female head (Cy-3); 1 d virgin male head (Cy-5) against 4 d virgin male head (Cy-3); 4 d mated female head (Cy-5) against 4 d virgin female head (Cy-3); 4 d mated male head (Cy-5) against 4 d virgin male head (Cy-3). Three biological replicate assays were performed for each experiment.in situ hybridization kit. After washes the microarrays were dried using pressurised air. Microarrays were scanned using an Axon GenePix 4200AL scanner using a 10 µm pixel size and adjusting the PMT to maximise effective dynamic range. Spot size, location and quality were assessed using the GenePix Pro 6.0 software (Axon Instruments) and hybridization artefacts and damaged spots were manually flagged for removal from subsequent analyses. The dataset was filtered using the TIGR MIDAS 2.19 software Hybridisations were performed over-night at 60°C according to the Agilent Technologies The normalised Cy-5/Cy-3 ratios from replicate assays were used as input for BAGEL  D. melanogaster transcripts. Gene Ontology (GO) analyses were performed using GO Slimmer Drosophila genes were obtained from FlyMine While the putative function (where known) of ESTs used in the microarray had been previously determined takeout, smell-impaired 35A, male-specific serum polypeptide-γ1, Defensin, Relish, Ptp61F, PGRP-LC and five odorant binding protein genes Obp69a, two Obp19d homologues (HS3757 and HC2265), Obp83a and Obp28a using cDNA from immature, mature virgin and mated male and female heads.To validate the transcript abundance data derived from the microarray analysis, real-time quantitative PCR (qRT-PCR) was independently performed on 12 genes  datasets was performed using Pearson correlation analysis.The quantity and quality of DNase-treated RNA was assessed using a Nanodrop ND-1000 spectrophotometer . Synthesis of cDNA was performed using 250 ng RNA in 20 µl reaction volumes using a mixture of oligo (dT) and random hexamer primers (Bio-Rad). Real-time quantification was performed using the iQ SYBR Green Supermix kit (Bio-Rad) and MiniOpticon (Bio-Rad). Cycling conditions involved an initial 95°C for three minutes, 40 cycles of 10 seconds at 95°C, 30 seconds at 57°C and 30 seconds at 68°C. A fluorescence reading was made at the end of each extension step. Three replicates were performed and specificity of the amplification products was assessed by melt-curve analysis. PCR efficiencies were above 88% for all primer pairs . StatistCecropin 1 and Ceratotoxin ADrosophila genes involved in the immune response  on the heads and abdomens of virgin and mated males and females. Three replicate assays were performed for each experiment and differences in the relative transcript abundance of each gene in 1 d flies and mated 4 d flies for both sexes were separately compared against transcript abundance in 4 d virgin flies of the same sex using 2-tailed t-tests.To confirm the influence of maturation and mating status on the expression of immune response related genes, qRT-PCR assays were performed using two previously identified medfly immune response genes, We analysed a total of 11,885 transcripts, of which 811 displayed changes in transcript abundance during female maturation and 301 during male maturation. Transcripts that showed changes in abundance related to mating amounted to 264 in males and only 32 in females.D. melanogaster genes and their associated ontologies. A total of 349 transcripts were more abundant in mature females, of which 137 (39%) were assigned putative functions. Of these transcripts those that are related to reproduction, behaviour, chemoreception and immune processes are listed in Out of 811 transcripts (6.8%) that displayed significant transcriptional changes between immature and mature virgin female heads, almost half (49%) did not share any significant similarity with known genes. As many as 462 transcripts displayed a greater abundance in immature females and of these, 188 (41%) were assigned putative functions based on their sequence similarity with Statistically significant  enriched biological process GO terms in the immature females included processes involved in development, regulation of growth, morphogenesis, nervous system development and activation, response to stimuli, movement, oogenesis and immune effector processes. Significantly enriched terms in the mature female included lipid, sugar and amino acid metabolic processes, the biosynthesis and regulation of hormones and pheromones, and sensory perception of chemical stimuli .A total of 301 transcripts displayed differential abundance between immature and mature virgin males. Of the 119 transcripts that were more abundant in the immature males, 59 (50%) were assigned putative functions and of the 182 transcripts that were enriched in mature males, 124 (68%) were similarly annotated . In immaObp19d enriched 1.76 fold), and one to reproduction . The enriched biological process GO terms in the mated females included regulatory processes related to sensory perception of chemical stimuli, amino acid and protein metabolism and transport  were less abundant in the mated female head compared to the mature virgin female head . Of the ransport . Only twIn males, by contrast, mating induces a massive wave of transcriptional changes. A total of 264 transcripts displayed changes in abundance, 79 enriched and 185 with lower abundance in the mated male, compared to the virgin male. Thirty-eight (48%) of the enriched and only 39 (21%) of the lower abundance transcripts were assigned putative functions . EnricheAs a confirmation of the validity of the microarray results, a highly significant positive correlation  was found between the microarray and qRT-PCR datasets derived from the abundance of twelve transcripts in immature, mature and mated males and females .In the microarray analyses no evident mating-induced activation of immune-response related genes was observed. This apparent lack of an immune response after mating in the medfly might, however, be the result of the expression of these genes being limited to the reproductive tract or fat body, rather than in the head. To test this hypothesis, qRT-PCR analyses were performed on heads and abdomens of immature, mature and mated individuals of both sexes in order to determine whether genes related to the immune response show changes in transcriptional activity in relation to sexual maturation and mating status.Defensin gene transcript with respect to their immature counterparts, while Ceratotoxin A and Attacin A transcripts showed strong increases  in the mature virgin female abdomen with respect to the immature females  in immature, mature and mated individuals of both sexes. Transcripts of Cecropin 1 and PGRP-LC showed 15- and 3-fold lower abundance, respectively, on maturation in female abdomens, whereas in the male abdomen the abundance of these gene transcripts increased 1.6-fold and 2.6-fold, respectively, during maturation. A modest 2-fold increase in the transcript abundance of Relish was evident only in the mature virgin male abdomen with respect to the immature male. Thus, during maturation, massive changes in the transcriptional activity of immune-response-related genes were evident predominantly in the female abdomen  levels of the  females . Apart f abdomen .Ceratotoxin A and Cecropin 1 showed significant, but very modest increases in transcript abundance in the female abdomen (1.5-fold). The only other immune-related genes to show significant changes in transcript abundance after mating in the female were Defensin which was less abundant, particularly in the abdomen (10-fold), and Attacin A that was less abundant in the female head, compared to virgin females, although the extent was difficult to quantify due to the very low transcript abundance (Cecropin 1 transcript abundance in the head and abdomen (1.6- and 1.9-fold respectively) and a decrease in PGRP-LC abundance in both body compartments (1.6- and 1.7-fold) compared to virgin males. Therefore, after mating, apart from a large reduction in Defensin transcript abundance in the female abdomen, only modest changes of transcript abundance were evident for the other immune-related genes . In D. melanogaster, Obp8a has been shown to be involved in the response to the aliphatic fruit odorant, hexanol Obp19d is associated with nutrient sensing, synaptic transmission and resistance to starvation stress Obp genes may be related to the localisation of food resources during foraging activity. Apart from these chemosensory-related transcripts, three male-specific serum polypeptide (MSSP) transcripts were enriched up to almost nine-fold. These MSSP genes were initially thought to be expressed only in the fat body of adult males, however, subsequently low levels of transcripts and their products were identified in the fat body of females and in the midgut of both sexes. They belong to the Minus-C subfamily of odorant binding proteins and are thought to be involved in the transport of volatile substances or other hydrophobic molecules With regard to chemoreception, transcripts of two odorant binding protein (Obp) gene homologues were enriched, Relish that induces the synthesis of the antimicrobial peptides Defensin, Cecropin, Diptericin, Attacin and Metchnikowin in DrosophilaCecropin 1, thiolester containing protein IV and Dicer-2A number of genes related to immunity displayed enriched transcript abundance in immature females compared to mature females. Among these is the Imd pathway transcription factor Upon reaching sexual maturity, and having located appropriate plant hosts, the female's movements become less dispersive and she becomes receptive to courting males. In the meantime, foraging continues to be a major activity to sustain the energetic requirements of continuous egg development and oviposition armadillo, spindle-B and Cdc42. Of these, Cdc42 is involved in the regulation of cell shape both in follicle cell development, the nervous system and the eye Vg2) precursor genes show relatively large increases in transcript abundance in mature females. These genes are expressed by the fat body, secreted into the haemolymph and subsequently sequestered by oocytes Vg transcripts were detected therein. Indeed, expression of vitellogenin genes and their yolk protein homologues have been described in the An. gambiae and D. melanogaster head respectively As in the immature females, many of the enriched transcripts in mature females are implicated in oogenesis and include desat1 gene showed enriched transcript abundance in mature females. In Drosophila desat1 is expressed in the fat body and is involved in the biosynthesis of unsaturated hydrocarbons on the cuticle of mature flies of both sexes takeout (to). The to gene possesses an insect juvenile hormone (JH) binding domain to enrichment, may be independent of any requirement for mating. In addition, to regulates feeding behaviour in a circadian sensitive manner Homologues of the Obp19a, Obp19b, a second homologue of Obp19d (HC2265), Obp56h and two homologues of Obp83a (HS1065 and HC2536), may be related to host location, feeding and detection of the male pheromone. In Drosophila, Obp19a and Obp19b are involved in the response to aromatic and aliphatic odorants present in fruits Obp56h may have both olfactory and gustatory roles as it is expressed in the antenna and the pharyngeal organs Obp83a homologues (HS1065 and HC2536), which incidentally also display enrichment in mature virgin males , may be related to the perception of pheromones emitted by courting males. In Drosophila, Obp83a is expressed in the antenna and exhibits sexual dimorphism in its response to odorants Pinocchio (Pino) is another chemosensory-related gene, which contributes to early chemical recognition processes in the olfactory and immune system and may mediate the removal of xenobiotics such as odorants and bacterial toxins Pino is thought to negatively regulate the expression of Obp8a, as Pino-deficient Drosophila have elevated Obp8a expression Pino transcripts had lower Obp8a transcript abundances.The enrichment of six odorant binding protein transcripts, Defensinthiolester containing protein II (TepII) Mature females also showed higher transcript levels of immune-response related genes active against bacterial and fungal infections, α-actinin, and the tropomyosin 1 genes, which are expressed principally in the indirect flight muscles and are essential for muscle contraction Obp8a and Obp19d (HS3757), showed changes in transcript abundances that mirrored those found in immature females , again suggesting their involvement in the location of food resources. Similarly, smi35A and crammer, both involved in the development of the nervous system, memory formation and smell-recognition, showed similar changes in transcript abundance as in their female counterparts.During their brief maturation period, before they become sexually active As mature males are actively involved in lek formation, which involves the emission of pheromones and the courting of females, it is of no surprise that they show significant enrichment of GO terms involved in numerous metabolic and biosynthetic processes including pheromone synthesis. These processes are an indication of the high energetic investment required for flight to the lek site, courtship, and pheromone production. The enrichment of terms related to eye pigmentation is also interesting, given that the sexually dimorphic bright colouring of the male's eyes are thought to send visual signals to the female during courtship behaviour vitellogenin genes 1 and 2 were observed in mature males compared to their immature counterparts. This differs from the expression of Yolk proteins 1 and 2 in D. melanogaster, which are limited almost exclusively to females Significantly higher transcript abundances of Obp83a (HC2536 and HS1065). Changes in the abundances of these transcripts may be correlated to the onset of participation in leks and active courtship behaviour. As the sex pheromone emitted by males attracts not only females, but also other males to participate in the lek As in their female counterparts, mature males displayed enrichment of two transcripts with significant sequence similarity to Defensin, CG16799, Spn27A, TepII and modSP were more abundant in mature male individuals compared to their immature counterparts.Transcripts related to the innate humoral response system tended to increase in abundance with male maturation. As in the mature females, the transcripts of protein tyrosine phosphatase 61F (ptp61F) and a homologue of Obp19d (HS3757). The altered abundances of these transcripts may be the result of the action of male accessory gland proteins, and/or sperm ptp61F transcripts may be related to behavioural changes in the response of mated females to courting males as, in Drosophila, the protein associates with dreadlocks an adaptor protein involved in axon guidance, GPCR protein signalling pathway and the insulin receptor signalling pathway Upon mating, medfly females tend to become unreceptive to courting males, although a proportion of females do remate Drosophila females down-regulate expression of Obp19d after mating Obp19d in the medfly may contribute to post-mating changes in the females' attraction or receptivity to courting males Obp19d may also be implicated in the detection of the host-marking pheromone deposited on the fruit by other females after oviposition, that acts as a deterrent to further oviposition Obp19d homologue (HS3757) was also enriched in immature medfly individuals of both sexes and also in mated males  it is more likely, as previously suggested, that this gene is related to foraging behaviour. No immune-related genes showed changes in transcript abundance between mature virgin and mated females.Unlike the medfly, Ptp61F transcripts responsible for changes in the synaptic organisation in the nervous system related to learning, perhaps as a result of stimuli received during the courtship process derailed, involved in synaptic growth and synapse-specific modification, was also enriched double-time influences the oscillations of the clock genes period and timelessMale medfly can potentially continue to court and mate throughout their life. Courtship involves a complex series of activities such as lek participation, male-male conflict, pheromone emission and complex wing movements, all of which may eventually lead to mating that can last several hours. After this intense energy expenditure, the male needs to replenish his depleted reserves by foraging Basigin (Bsg), which in Drosophila is principally expressed in the fat body, has been implicated in inter-male aggressive behaviour Basigin in Drosophila is associated with increased inter-male aggression Male medfly display a variety of aggressive behaviours, related to competition for access to females and to the defence of favourable positions in the lek CDP diglyceride synthetase (CdsA) gene were less abundant in mated males. This gene is under the influence of the photocycle and is thought to promote daily variations in visual sensitivity Given the importance of visual cues in medfly courtship behaviour, it was interesting that transcripts of the D. melanogasterDrosophila the immune response is activated in mated females by the sperm and seminal fluid components. It has been suggested that this immune response is part of a sexually antagonistic arms race in which the male produces increasingly potent signal molecules that modify the behaviour and physiology of the female away from reproductive receptivity towards fecundity. For the females these signalling molecules may induce suboptimal reproductive behaviour as they may favour short-term fecundity over long-term reproductive investment. Thus, the female immune system may have been co-opted to sequester the male signalling molecules to limit their effects In our transcriptome analyses, although changes in transcript abundance of immune-response related genes were observed during the maturation of both sexes, no evident mating-induced activation effect was observed. This is in contrast to previous studies on Ceratotoxin A and Cecropin 1 showed significant but very modest enrichment in the female abdomen. This Ceratotoxin A expression profile is congruent with that described in a previous study in which it was shown to be expressed in the accessory glands of sexually mature medfly females and to be enhanced by mating Ceratotoxin A is also present on the surface of laid eggs it has been suggested that it may function in protecting early larval development from bacterial infection. The only other immune-related genes to show changes after mating in the female were Defensin and Attacin A which displayed lower transcript abundances, particularly in the abdomen and head, respectively. In males there was only a modest enrichment of Cecropin 1 and reduced abundance of PGRP-LC transcripts in both body compartments after mating.The apparent lack of an immune response after mating in the medfly might, however, be the result of the expression of these genes being limited to the reproductive tract or fat body, rather than in the head. To test this hypothesis, qRT-PCR assays were performed on six genes involved in the immune response on heads and abdomens of immature, mature virgin and mated males and females . AlthougA. gambiae and A. mellifera, which also lack this post-mating immune-response, than to D. melanogasterThis, albeit limited survey, suggests that mating in medfly does not appear to activate immune gene expression. In this sense the medfly displays a greater similarity to its more distant evolutionary relatives, D. melanogaster, where the transfer of accessory gland proteins (Acps) to females during copulation is essential for egg development, egg maturation and the induction and maintenance of egg laying Drosophila, C. capitata male accessory gland proteins transferred during copulation have little or no effect on egg production. The medfly is an r-strategist and the selective benefits to a virgin female having mature eggs ready for fertilisation and oviposition immediately upon mating may counter-balance the costs of egg production in the unlikely event that the female remains unmated C. capitata to remating DrosophilaA substantial number of transcripts displayed significant changes in abundance during maturation in both sexes and these changes generally mirrored the physiological and behavioural changes that accompany maturation in male and female medfly. Despite extensive post-mating transcriptional changes in the male, changes in the female were surprisingly modest. The current study was restricted to the head transcriptome which may be a limitation, but the observation that unmated medfly females are just as prolific egg-layers as their mated counterparts Knowledge of the intricacies of the processes related to maturation and mating provides powerful targets for manipulating the reproduction of these insects in wild populations. As the medfly represents a model species for tephritid fruit flies, which include numerous important invasive agricultural pests throughout the world, the potential benefit of this knowledge will not be restricted to the medfly alone. In addition to increasing our understanding of the molecular machinery behind these biological processes, our findings may provide insights into the evolutionary processes that have led to the invasive success of this tephritid species.Figure S12 ratio) for 12 genes from the four assays Click here for additional data file.Table S1Primers used for qRT-PCR analyses.(DOC)Click here for additional data file.Table S2Transcripts that change in abundance in mature virgin females compared to immature virgin females.(DOC)Click here for additional data file.Table S3Significantly enriched biological process gene ontology annotations among transcripts that showed changes in abundance in mature virgin female heads compared to immature female heads.(DOC)Click here for additional data file.Table S4Transcripts that change in abundance in mature virgin males compared to immature virgin males.(DOC)Click here for additional data file.Table S5Significantly enriched biological process gene ontology annotations among transcripts that showed changes in abundance in mature virgin male heads compared to immature male heads.(DOC)Click here for additional data file.Table S6Significantly enriched biological process gene ontology annotations among transcripts that showed changes in abundance in mated female heads compared to mature virgin female heads.(DOC)Click here for additional data file.Table S7Transcripts that change in abundance in mated males with respect to mature virgin males.(DOC)Click here for additional data file.Table S8Significantly enriched biological process gene ontology annotations among transcripts that showed changes in abundance in mated male heads compared to mature virgin male heads.(DOC)Click here for additional data file.Table S92 transformed fold expression).Correlation of microarray changes of transcript abundance with real-time qRT-PCR for 12 genes (log(DOC)Click here for additional data file.


Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.in situ hybridization. We also show that interfering with the function of two genes, f11r and cd9b, defects in primordium migration are induced. Finally, pathway construction revealed functional relationships among the genes enriched in the migrating cell population.Through the combined use of transgenic fish, Fluorescence Activated Cell Sorting and microarray analysis we identified a repertoire of key genes expressed in the migrating primordium and in differentiated neuromasts. We validated the specific expression in the primordium of a subset of the identified sequences by quantitative RT-PCR, and by Our results demonstrate that this is a robust approach to globally analyze tissue-specific expression and we predict that many of the genes identified in this study will show critical functions in developmental events involving collective cell migration and possibly in pathological situations such as tumor metastasis. The formation of an embryo and its organ systems requires the coordination of diverse cellular behaviors to achieve proper development of form and function. Cells must migrate, often collectively, and proliferate in a regulated way, while simultaneously carrying out specific developmental programs. A full characterization of these events requires a description of the cellular histories (lineage) and knowledge about the molecules that regulate these processes.Active cell movements take place not only during the development of organisms, but also in processes that occur during adult life. For example, in the immune system, an effective immune response depends on the regulated traffic of its cellular components. Disrupted cell migration also contributes to several important pathological processes, including cancer and chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis.in vitro studies of single cells in two-dimensional cultures. These studies have allowed great progress on the intracellular events that take place during cell motility, elucidating the details of the cellular machinery driving migration. However, in vivo models of collective cell migration have been less intensely studied because of the inherent difficulty in undertaking such analyses. Recently, the migrating primordium of the zebrafish posterior lateral line has emerged as an attractive system for genetic analysis of cell migration and tissue organization and for understanding how these processes are controlled [To date, most knowledge about cell migration is based on ntrolled -9.The lateral line is a mechanosensory system present in fish and amphibians that responds to water movements and is involved in a large variety of behaviors such as predator avoidance, prey detection, or swimming in schools ,11. Thisfgf3 and fgf10, are essential for the internal organization, patterning and migration of the primordium and therefore for correct neuromast deposition [One of the essential features of the migrating lateral line primordium is that its cells must become organized in order for them to migrate coherently and produce a functional organ. Today, it is known that the primordium of the LLP contains mesenchymal-like cells at its leading edge and, towards the trailing edge, 2 to 3 groups of rosette-shaped polarized cell clusters are formed, each corresponding to a proneuromast. Moreover, recent studies have shown that ligands of the FGF family, position ,5. Theseposition . Both thposition ,21. Whilatonal homolog 1a (atoh1a), is expressed in the center of the rosettes that form successively within the primordium [delta and Notch participate in cell type specification in neuromasts and are detected in presumptive neural and accessory cell progenitors, respectively [As the PLL primordium migrates, cells are simultaneously dividing and undergoing developmental programs that will build the functional lateral line. Hair cells and accessory cells derive from migratory primordium cells and their fates become apparent prior to neuromast deposition. The proneural master control gene for hair cell fate, imordium ,23. Likeectively ,24.The identification of new molecules that control the directional movement of the primordium, cell proliferation and subsequent differentiation of hair cells and other cell types will contribute to a better understanding of the processes leading to development of the mechanosensory lateral line, which shares many physiological properties with the mammalian auditory system. More importantly, formation of this organ shares molecular mechanisms with processes involved in cancer metastasis  and immucldnb:gfp) [In this study, we carried out a global survey of genes expressed in the PLL primordium and in maturing neuromasts. We have taken advantage of the ClaudinB:GFP transgenic zebrafish line (dnb:gfp) , in whiccldnb:gfp transgenic zebrafish line expresses membrane-bound GFP in the PLL primordium and neuromasts, in the olfactory system, the ear, branchial arches, and a lower amount of expression in the pronephros and skin in embryos and larvae [cxcr4b [epcam [cldnb [in situ hybridization to detect expression of eya1, a gene specific to the primordium at this stage [The d larvae . As we w [cxcr4b , epcam [b [epcam  and cldnm [cldnb   and a muscle-specific gene (myod), which should be enriched in the GFP- fraction. As expected, the epcam and cldnb mRNAs are enriched in the GFP+ population of cells while myod is overrepresented in the unlabeled collection of cells  using primers specific for genes known to be highly expressed in this tissue . This preparation was carried out in order to have a sample of RNA obtained from the same tissue but at a different developmental stage. Given that these cells are beginning to differentiate and they are no longer migratory, comparison with the primordium sample would allow us to discriminate between genes involved in the different cellular processes occurring during lateral line development. As in the earlier stage, RNA was prepared from GFP+ and GFP- cells obtained from 48 hpf embryos.A parallel experiment was carried out using 48 hpf For isolating lateral line-specific transcripts, we reasoned that we could subtract the transcriptome obtained from the sorted GFP- cells to the one obtained from GFP+ cells by carrying out a co-hybridization experiment using microarrays. The sorted GFP- sample contains unlabeled cell types belonging to many different tissues of the same embryos and should thus allow us to remove most housekeeping transcripts from the lateral line transcriptome. We obtained RNA from GFP labeled and unlabeled cells from two independent tail sectioning experiments for each stage analyzed. From each of these samples, cDNA synthesis was carried out and four rounds of dye labeling (including one dye swap) were done for probe generation. "In house" microarray slides printed with 34,000 oligonucleotides that correspond to 19,000 zebrafish-expressed sequences were co-hybridized with probes prepared from GFP+ and GFP- cDNA from each stage analyzed. Differential expression was considered significant when the average hybridization signal from all 4 hybridizations differed by a factor of more than 1.5 (p < 0.05) in either direction . We identified 4,449 (36 hpf set) sequences enriched in the primordium, that represent 3,505 known genes and 944 ESTs that do not map uniquely to a single gene; and 3,425 genes and 575 ESTs 48 hpf set) in the neuromasts  and grouped some of the sequences obtained according to known biological processes or developmental pathways Table . Genes icldnb:gfp transgenic line and thus would not have been purified during the cell sorting procedure. In our database survey, we found 372 genes in which specific expression in the PLL primordium or neuromasts has been convincingly described  to identify all genes previously known to be expressed in the lateral line system (primordium and/or neuromasts). We excluded genes expressed only at the placodal stage (PLL precursors), in neural components  and in glial cells. These cell types do not express GFP in the To provide an independent verification of our results, we randomly selected 15 different cDNAs from sequences showing higher than 1.5-fold enrichment in the GFP+ cells in our 36 hpf set. We performed qRT-PCR to confirm the presence of these transcripts in the purified primordium cells Figure . For 12 in situ hybridization. Specific label was detected in 36 hpf embryos with seven of these probes in the PLL primordium and/or neuromasts  is a transmembrane adhesive glycoprotein that participates in numerous cellular adhesive process including leukocyte migration, intracellular junction assembly and the regulation of cell morphology and motility. In endothelia and epithelia, JAM-A localizes at cell-cell contacts with the tight junction proteins occludin and claudins . In canccldnb:gfp transgenic zygotes and examined the development of the PLL system from 24 to 48 hpf. Neither of the two MOs elicited gross developmental abnormalities, as was the case for the corresponding control . In early LL development, the overall migration process seemed unaffected in either f11rMO or in cd9bMO injected embryos  directed against the ATG region of each transcript. We injected s Figure  and in 3e Figure  at 36hpfe Figure . Differecxcr4, cxcr7, fgfr, egfr and matrix metalloproteases [In order to determine possible pathways and biological relationships in our 36 hpf data set, we searched for functional interactions among enriched genes using the GeneGo Bioinformatics software. Of the primordium-enriched set of 4,451 zebrafish genes, we identified 1, 911 human orthologs. We focused on the Gene Ontology (GO) terms for the following 5 categories: cell-cell adhesion, guidance, collective cell polarization, EMT remodeling and signaling Figure . All of roteases . We alsoInterestingly, all the enriched genes captured in these categories have demonstrated roles in cancer progression. Furthermore, the many interactions found between these groups of genes, corroborate that collective cell migration results from a complex molecular cascade where dynamic processes occur in a coordinated fashion.Additionally, we compiled the complete list of genes with known functions in cell adhesion, migration and cancer metastasis provided by GeneGo Figure  to identThus, we propose that the PLL migrating primordium is a relevant model in which to study the diverse processes that occur during collective cell migration in normal and pathological conditions.cldnb:gfp transgenic line, which expresses GFP in all of the cells derived from the posterior lateral line primordium. GFP positive cells were isolated through cell sorting and the mRNA prepared from these cells was reverse transcribed and probes generated; GFP negative cells were treated similarly to generate probes for subtraction in microarray hybridization experiments. Our reasoning was that this strategy would reveal a collection of genes whose expression was specific to lateral line cells. While this experimental design allowed us to detect genes highly expressed in primordium cells in relation to other tissues -and to discard housekeeping genes-, it was obviously limited by the fact that genes expressed both in the lateral line cells and at the same time in one of the GFP negative cell types, would go undetected. Indeed, as our results show  in these organs and thus may have specific roles in development of the lateral line. Our estimates, comparing publicly available data with our results, show that we have detected between half and two thirds of the genes expressed specifically in the lateral line primordium and neuromasts. We speculate that the remaining genes are either expressed simultaneously in other tissues, their expression levels are only slightly elevated in the primordium and neuromasts vs. other cell types, or the appropriate oligonucleotide is absent from the gene array that was used in this study. Despite these caveats, we believe that we have successfully identified a significant fraction of the genes expressed at two different stages of lateral line development: primordium migration and neuromast formation. Moreover, the recovery of genes previously known to be expressed in the LL indicates that the sorting process itself has relatively modest effects on their normal expression profile. Again, this result indicates that the approach followed in this work is able to correctly identify lateral line-specific genes.Our aim was to uncover a set of genes expressed in the lateral line primordium and in developing neuromasts, the principal components that form the mechanosensory lateral line system in the zebrafish. We took advantage of the cldnb:gfp transgenic line. In the trunk and tail of the transgenic embryos there is detectable expression of the reporter in the pronephros and in skin cells and, given the wide selection window used in the cell sorting procedure and the sensitivity of this technique, there is likely to be contamination of our sorted cell sample with these other cell types. Further exploration of the identified gene collection by in situ hybridization will determine the extent of false positive sequences incorporated in our sample. One strategy we followed for increasing the likelihood of identifying lateral line-specific genes was to consider only those overrepresented in both the primordium and neuromast cell selection experiments . Additionally, it may be possible to obtain a superior level of enrichment if a transgenic GFP line with higher expression specificity becomes available. Moreover, a different gene identification approach might be warranted, such as deep sequencing of cDNA tags  [Despite the evidence described above, it is also likely that a number of genes identified in our screen are not expressed in the lateral line system and represent false positive identifications. These genes could be expressed in some of the other cell types that also express GFP in the ., SAGE) , which dThis study provides a novel set of candidate genes potentially having roles in primordium organization, migration and lateral line differentiation. Formation of the lateral line system is a highly complex and coordinated process in which cell migration, morphogenesis, neural specification and cell differentiation occur simultaneously. Thus, this system offers a unique opportunity to analyze these biological processes in a simple and accessible manner, and reaffirms the conviction that the study of the general principles governing cell migration and fate determination can be studied using the lateral line of the zebrafish. Our study further opens the door for molecular dissection of these processes by increasing the repertoire of genes that are known to be expressed during lateral line development and relate these genes to diverse biological functions Table .e.g., mmp9 and mmp14) [e.g., adam9 and adam23) [c-met, were detected in this study. C-met itself is strongly expressed in the migrating primordium [gab1, shp2, stat3, pi3k, pak1 and Mitogen-activated protein kinases [For example, genes implicated in cancer progression and metastatic behavior such as matrix metalloproteases (d mmp14) , the ADA adam23)  and the imordium  and we a kinases .f11r, cd9, cd99 and members of the tight junction Claudin family genes were also amply represented in our collection. These molecules may be important for maintaining internal tissue organization, coherence of the primordium, pro-neuromast deposition, and/or allowing coordinated multicellular movements [Cell adhesion molecules such as ovements .in vivo small molecule drug screens [The collective migration of primordium cells has been the focus of intense study and others have noted the parallels of this cellular behavior with that of invasive cancer cells ,8,26. In screens .cxcr4 and cxcr7, we detected lect1 (Leukocyte Cell Derived Chemotaxin 1), scyba and a sequence with homology to human CXCR6 (Gene ID: AW232037), suggesting that the high level of complexity required for directional migration of the PLL primordium may involve additional molecular components.Another biological process that shows molecular parallels with the migrating primordium is immune cell migration. The involvement of chemokines and their receptors in regulating guidance of the primordium cells is well documented. These molecules were first identified in mammals due to their involvement in homing events in the immune system though lymphocytes migrate singly rather than as ensembles of cells. In addition to the chemokine receptors NFkappab (nfkbiaa), Interferon Regulatory Factor 5 (irf5), 7 (irf7) and 11 (irf11), and ticam1 . Likewise, we found members of the prostaglandin family, such as ptgs1, ptgs2, ptgds and ptgisl, that regulate diverse functions of many cell types of the immune system, including T and B lymphocytes and dendritic cells [Interestingly, a number of proteins that regulate the innate immune response were detected in our experiment. Downstream components of the transcription factor NFkB, key regulator of innate immune response, were also expressed in the primordium and/or neuromasts such as Inhibitor of ic cells . It is nic cells .apc, axin2, dickopf, frizzled 7a, lef1 and sfrp1a have been described [wnt4b, a Wnt ligand, could be implicated in segregation of the different compartments of this group of cells. Likewise, Fgf signaling has been shown as a key regulator during lateral line development [fgf3 and fgf10, guide the tissue compartmentalization in the primordium for proper rosette formation, required for efficient migration [fgf4, fgf8 and fgf13, which could be involved in primordium morphogenesis and collective cell movement. Finally, in our survey we found two genes belonging to the Nodal pathway, squint and sox17, both highly expressed in forerunner cells (FCs) of the zebrafish gastrula [In addition, we have detected the expression of members of well known developmental and cellular signaling pathways. Among these is the Wnt pathway, which has been shown to be crucial for primordium organization . While eescribed ,42,43, welopment . The Fgfigration . Here, wgastrula . Nodal/Tsox2, sox3, oct4, pou4f2 and prox1. However, we did see overexpression of shha and b, a set of genes of the dlx family and members of the msx family, among others.Notably, the migrating primordium and the deposited proneuromasts contain precursor cells for the diverse cell types that later on differentiate to form a functional sensory organ. Therefore, it is expected that these cells will express neural progenitor cell markers, genes related to neurogenesis, and genes involved in differentiation of the mechanoreceptor hair cells. Most of the known neural progenitor cell markers expressed in the PLL primordium and neuromasts are also expressed in other tissues, including the CNS . Accordibdnf, is expressed in neuromasts and may be crucial in maintaining innervation of hair cells [The analysis of genes expressed by neurons that innervate neuromasts or by glial cells that accompany the nerve and neuromasts were beyond the scope of this study. However, they are integral components of the functioning lateral line and contribute to its development and may provide important cues for its migration and survival. For example, glial cells appear to be responsible for inhibiting neuromast formation from cells lying between neuromasts  by produir cells .In this study, we catalogue a variety of biological process and a wide number of signaling pathways involved in lateral line development. Molecules belonging to diverse cellular and developmental processes were uncovered using this approach, demonstrating its utility for the identification of new genes involved in the morphogenesis, migration and cell type specification within the migrating PLL primordium in the zebrafish. Importantly, some of the identified gene products can also open new insights into the understanding of cell migration, which may be crucial to create efficient therapies for a variety of human diseases.cldnb:gfp transgenic line was obtained from Darren Gilmour [Fish and embryos (AB and T/AB5 strains) were maintained in our facility according to standard procedures . The cld Gilmour . All emb Gilmour  and rais Gilmour . EmbryonCldnb:gfp embryos were dechorionated with Pronase  and transferred to glass dishes. We used the posterior third of the transgenic embryos in order to avoid recovering GFP-expressing cells in tissues other than the lateral line. Fish were anaesthetized and the tail was transected with a sharp scalpel adjacent to the end of the yolk extension. Tails of embryos were recovered from 36 hpf (for isolating primordium cells) or from 48 hpf (for neuromast cells) embryos, then transferred to Hank's medium containing 0.25% trypsin and 1 mM EDTA and incubated for 15 to 30 min at room temperature during which they were dissociated mechanically by pipetting them up and down every 5 minutes. The digestion was stopped by adding fetal bovine serum to 10% and cell suspensions were then filtered through 40 μm nylon mesh, washed twice with PBS, pelleted by centrifugation at 600Xg for 2 minutes and resuspended in L-15 medium (Sigma) supplemented with 10% fetal bovine serum. FACS of single cell suspensions was performed at room temperature using a FACSAria Sorter (Becton Dickinson) with a Coherent Innova 70 laser at 488 nm and 200 mW power. GFP+ and GFP- cells were sorted directly into Trizol Reagent (Invitrogen) and stored at -80°C.Total RNA was isolated from equal numbers of GFP+ and GFP- cells by extraction with Trizol Reagent, according to the manufacturer's instructions. RNA pellets were resuspended in nuclease-free water (Ambion). RNA integrity was confirmed by visualization of the ribosomal RNA on picochips for the Bioanalyzer (Agilent). Approximately 500 ng to 800 ng RNA was linearly amplified by using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) with yields ranging from 12 to 30 μg of aRNA. aRNA samples were split and labeled, half with Cy3 mono NHS ester and half with Cy5 mono NHS ester .® SYBR® Green One-Step qRT-PCR (Invitrogen) using 20 ng of RNA and 5 μM gene specific-primers in a 25 μL reaction, according to the manufacturer's instructions. PCR primers were as follows: atoh2a 5'-GCACGAGCTGTTCACCTGCTAAAAC and 5'-GACGGGATCCTCAAAGGGAAGAGQuantitative PCR was performed using SuperScript™ III Platinumβ-actin 5'-TGGCCCCTAGCACAATGAAG and 5'-GCCTCCGATCCAGACAGAGTAT;cldnb 5'-GAAGGAATTTGGATGAGCTGCGTGG and 5'-CGACAGCATGATTCCCATCAGTCCG;cd9b 5'-GTGATTGGAGGAGTCGGCATCG and 5'-CACTCGCGCACATTCACAAACG;dlx6 5'-CCAGCAGACTCAATACCTGGC and 5'-CCGCCTTGTTTCAACAGCTTCegfp 5'-GACCCTGAAGTTCATCTGCACCACC and 5'-GCGGGTCTTGTAGTTGCCGTCGTCC;epcam 5'-ATGGCCAGCCATTTGAGGTTGATG and 5'-GCCGTGCAAGAAAGAACAGAACCA.klf3 5'-CCACAGCCAAGAGAAATCGGTC and 5'-GTGTGCGTCTATGGGCTTTCAG;lect1 5'-GAGTGAAAGCTGAGGTGGCGTC and 5'-CTGGCGTCTCATTCGTGTCGC;myod 5'-ATGACACACCAAATGCTGACGCAC and 5'-TCTCTGTGGAAATTCGCTCCACGA;pecaml 5'-CGTTGGCCGGATGGTTAAATCC and 5'-GCAGCGATGGGAACTTTCACTC;ptgs2 5'-CATGTTTGCTTTCTTCGCCCAGC and 5'-GTGTTGAACCTCCAGCGTCTCTC 3;ptgds 5'-CCTGAAGAGTGATGGTTCCTGCTG and 5'-GTCAACCACGCGCATGTCATTG;ptgisl 5'-GAACAACCTCCGCCTGCTTATG and 5'-GCTTGTCAAACCGGCGAAACTC;scyba 5'-GATGAATCGCTGTAGTACGGCCGC and 5'-CCGGATCTTCGGCCCTTTTCTTG;sema3 d 5'-CAGGTGTCACTGCACAGGTGCC and 5'-GTGTCCCAACAATGGCTGGATG;similar hcd205 5'-GTGCGTCAGATCTCAGTAGTTTGA G and 5'-GCTAAAGCAGTGCACCGAGTGCTG;similar hcxcr6 5'-GAAGAATGGCTGGAACCTGCAAC and 5'-CCGACTATGCCAGTCACAACC;sox8 5'-GAACGCGTTTATGGTTTGGGCTC and 5'-CCTCCACGAACGGCCTCTTCTC;sox17 5'-CCCAGACCTGCACAATGCGG and 5'-GGGCTCCAATCGCTTGTTTCGβ-actin to obtain relative expression levels. Standard deviations were calculated from triplicate measurements.Expression levels were determined by comparison to a standard curve from total RNA isolated from whole embryos. Values from GFP- and GFP+ samples were then normalized to We used "in house" 34 K zebrafish oligo microarray chips, which was derived from three sources: Compugen , MWG  and Operon . The whole set contains replicates of several positive (known housekeeping genes) and negative control oligos (random sequences) to control for the homogeneity and specificity of the hybridization.http://www.genesifter.net/ software. Data points with average quality values below 0.7 were eliminated and the datasets were Lowess normalized. Normalized data were log-transformed and GFP+ and GFP- values were separately averaged over the eight experiments. Fold differences were calculated from log averages and Student's t-test with Benjamini and Hochberg correction to generate p-values that were used to determined statistical significance. The microarray data is available at NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE25617.Two biological replicates of GFP+ and GFP- cells were cohybridized, each with one dye swap, for a total of eight hybridizations. Hybridizations were performed overnight at 45°C in Maui Mixer FL hybridization chambers (BioMicro Systems). Microarray slide post-hybridization processing and scanning were done as previously described . Data anin situ hybridizations (ISH) were carried out essentially as described in [Whole mount ribed in , with poPathway analysis of the genes enriched in the primordium was done with MetaCore software (GeneGo). The known relationships between the human orthologs of the zebrafish genes were used to generate the interaction network. The network was then manually examined and curated. In addition, an extensive search of the "GeneGo processes" was done for genes known to be involved in cell migration/adhesion and cancer pathways. The primordium-enriched genes involved in two or more of these processes were identified.The authors declare that they have no competing interests.VEG designed and performed all the experiments. JL participated in the microarray and pathways analysis. AE carry out the microarray experiments. MB, EJV and VR participated in the design of the study and established the cell sorting conditions. MLA and SMB designed the experiments, contributed to the conclusions and drafted the manuscript. All authors read and approved the final manuscript.Supplementary Table 1. Complete list of genes enriched in microarray experiment at 36 hpf.Click here for fileSupplementary Table 2. Complete list of genes enriched in microarray experiment at 48 hpf.Click here for fileSupplementary Table 3. Known PLL primordium and neuromasts markers.Click here for fileSupplementary Figure 1. Pattern of the neuromast deposition in the embryonic PLL. The number and position of the neuromasts was analyzed in control (A), cd9bMO (B-C), and f11rMO (D-E) embryos at 48 hpf. Red arrows indicate the position of the PLL neuromasts.Click here for fileSupplementary Table 4. List of genes enriched at 36 and 48 hpf.Click here for file


Growing evidence supports the notion that impairment of mitochondrial signaling in the endothelium is an early event and a causative factor in the development of diseases such as atherosclerosis or diabetic complications. In this review, we want to outline the significance of mitochondria in both physiology and pathology of the vascular endothelium.Lining the inner surface of the circulatory system, the vascular endothelium accomplishes a vast variety of specialized functions. Even slight alterations of these functions are implicated in the development of certain cardiovascular diseases that represent major causes of morbidity and mortality in developed countries. Endothelial mitochondria are essential to the functional integrity of the endothelial cell as they integrate a wide range of cellular processes including CaThe online version of this article (doi:10.1007/s00424-012-1085-z) contains supplementary material, which is available to authorized users. As the blood vessel’s innermost layer, the vascular endothelium fulfills a great multitude of regulatory and sensory functions . ImpairmThe primary role of mitochondria is to produce and regulate the production of energy-rich molecules such as adenosine triphosphate (ATP), via aerobic respiration. At first impression, these cellular power plants might not seem to be of particular importance to the endothelial cell, a type of cell that usually covers over two-thirds of its energy demands by anaerobic glycolysis , 33, 1182+ and reactive oxygen species (ROS) within cells. In addition, mitochondria have been recognized to determine the fate of cells by controlling apoptosis, the process of programmed cell death. In this review, we intend to take a closer look at these specific functions and possible dysfunctions of mitochondria in the vascular endothelium.Mitochondria constitute multifunctional organelles that are able to specifically regulate the signaling of cellular messengers such as CaEndothelial cells line the inner surface of blood vessels and establish an active barrier between solid tissues and circulating blood. Over the past few decades, this delicate cellular monolayer has emerged as a highly specialized tissue that by far exceeds the sole function of forming a passive physical interface . EndotheEndothelial cells per se are quite heterogeneous and various phenotypes meet the particular demand of the individual tissue and/or organ , 48. Som2+ ER) does not drop below a certain level, since the activity of ER chaperones such as calreticulin and calnexin strictly depends on free Ca2+ [2+]ER causes protein misfolding, accumulation of these proteins and activation of the unfolded protein response and ER stress pathways mito) enhances mitochondrial ATP production, which is essential for Ca2+ uptake into the ER through sarco/ER Ca2+-ATPase (SERCA) mito upon stimulation with histamine [2+ between subcellular compartments, in turn, can initiate unfolded protein response [Mitochondrial fission during treatment with high istamine , which mistamine  Fig. 3)d-glucoseresponse  and apopresponse  both of response .Fig. 3Sc2+-dependent opening of mPTP participates in the death of human aortic endothelial cells under hyperglycemic conditions [An enhancement of certain apoptotic pathways is seen in cellular models of diabetic vascular disease  that mignditions .From a therapeutic point of view, the antidiabetic agent metformin is of special interest because in addition to its metabolic effects, it prevents the opening of mPTP . This miSpecific familial disorders and metabolic diseases such as type 2 diabetes can cause a mismatch between lipid supply and uptake capacity of the adipose tissue leading to increasing levels of free fatty acids, triglycerides and cholesterol in the blood plasma. Since the endothelium is not able to store large amounts of lipids, pathologically elevated blood lipids represent a tremendous metabolic challenge for endothelial mitochondria and a threat to the organelle’s function and integrity .2+ sequestration [Proteins that are upregulated when fatty acid availability exceeds mitochondrial oxidative capacity are UCP2 and 3 , 67. UCPstration , 145. Bestration . Anotherstration . In any stration , whereasstration . Though 2 [2+- and NO-mediated decrease in oxygen sensitivity of cytochrome c oxidase [2 levels for surrounding aerobic tissue that crucially depends on sufficient oxygen supply.Compared to other cell types such as neurons or the myocardium, the endothelium can tolerate long periods of ischemia . In view2 . This is oxidase  that, inHowever, in contrast to their high resistance to ischemia, endothelial cells seem to be particularly vulnerable during reperfusion, as they are the first to undergo apoptosis starting already after 5 min of reperfusion in isolated rat hearts . The impThe most widely discussed factor that is proposed to damage endothelial mitochondria exclusively upon reperfusion are ROS, which can originate from numerous sources during reperfusion. The three most studied sources comprise the enzymes xanthine oxidase, NADPH oxidase and, of course, the mitochondrial ETC , 161. Wh2+ channels. The significance of mitochondrial Ca2+ in endothelial reperfusion injury still requires further investigation. One can expect, though, that cytosolic Ca2+ oscillations occurring during reoxygenation [Repeated short episodes of ischemia, so-called ischemic preconditioning, improve cell survival after subsequent severe ischemia not only in cardiac muscle but also in the endothelium . This begenation  are trangenation  and exocgenation  that, in2+ handling, redox signaling, mechanotransduction and apoptosis, all of which are closely interrelated. Dysfunction of endothelial mitochondria is considered to be a causative factor in the pathophysiology of most cardiovascular diseases and, thus, represents a promising target for future therapeutic interventions. However, the complexity of mitochondrial signaling pathways and interorganelle crosstalk is generally underestimated and requires further studies in order to be profoundly understood.Compared to other tissues, the endothelium has only few mitochondria and their contribution to cellular energy production is rather insignificant. Yet these organelles are of central importance to endothelial function as they integrate a broad spectrum of physiological processes including CaSupplementary movie 1(MPG 2186 kb)Supplementary movie 2(MPG 3442 kb)Below is the link to the electronic supplementary material.


These findings all suggest that Rab25 is a tumour suppressor for colonic neoplasia.Recent investigations have increasingly focussed attention on the roles of intracellular vesicle trafficking in the regulation of epithelial polarity and transformation. Rab25, an epithelial-specific member of the Rab family of small GTPases, has been associated with several epithelial cancers. Whereas Rab25 overexpression is associated with ovarian cancer aggressive behaviour, Rab25 expression is decreased in human colon cancers independent of stage. Recent studies of mouse models of intestinal and colonic neoplasia have demonstrated that Rab25 deficiency markedly promotes the development of neoplasia. Some of these effects appear related to alterations in  Neverthβ receptors. In considering these rather subtle effects, it is important to note that small defects in the efficiency of dynamic trafficking over an extended period of time may lead to progressive deficits, perhaps contributing to the age-related increase in cancer incidence.Thus, although it is clear that Rab25 can impact epithelial cell transformation, the exact mechanism remains elusive. It appears that the effects of Rab25 expression depend largely on context. Thus, either overexpression or loss of Rab25 expression can lead to transformation. It seems likely that these findings result from cell-specific roles for Rab25 in the trafficking of specific cargoes. Thus, if, as is documented in MDCK cells , then ovMin mice onto the Rab25-deficient background induces a marked increase in tumours, and crossing of Smad3+/− mice onto the Rab25 knockout background yields large invasive tumours. These results indicate that Rab25 is an important tumour suppressor in the colon. Given the results in both ovarian and breast cancer studies, it is likely that Rab25 influences the trafficking and recycling of a number of key regulators of polarity and signalling involved in transformation. Thus, either loss of Rab25 or overexpression of Rab25 may lead to cell transformation based on the cell-specific alteration in surface protein delivery.Loss of Rab25 in human colon cancers is associated with poorer patient prognosis. Although Rab25-deficient mice do not develop spontaneous colon tumours, breeding of Apc


They are secreted proteins consisting of an LRR (leucine-rich repeat) domain and a so-called epilepsy-associated or EPTP (epitempin) domain. Both domains are thought to function in protein–protein interactions. The first LGI gene to be identified, LGI1, was found at a chromosomal translocation breakpoint in a glioma cell line. It was subsequently found mutated in ADLTE  epilepsy) also referred to as ADPEAF . LGI1 protein appears to act at synapses and antibodies against LGI1 may cause the autoimmune disorder limbic encephalitis. A similar function in synaptic remodelling has been suggested for LGI2, which is mutated in canine Benign Familial Juvenile Epilepsy. LGI4 is required for proliferation of glia in the peripheral nervous system and binds to a neuronal receptor, ADAM22, to foster ensheathment and myelination of axons by Schwann cells. Thus, LGI proteins play crucial roles in nervous system development and function and their study is highly important, both to understand their biological functions and for their therapeutic potential. Here, we review our current knowledge about this important family of proteins, and the progress made towards understanding their functions.The development and function of the vertebrate nervous system depend on specific interactions between different cell types. Two examples of such interactions are synaptic transmission and myelination. LGI1-4 ( Synapse formation and maturation require multiple interactions between presynaptic and postsynaptic neurons that are mediated by a diverse set of synaptic proteins  or Schwann cells in the PNS ; reviewed in Nave, . As descAs suggested by its name, leucine-rich glioma inactivated, the LGI protein family was discovered in gliomas. Loss of one copy of chromosome 10 is a common event in high-grade gliomas. In a search for genes that are mutated in these gliomas, Cowell and colleagues identified a gene in the 10q24 region that was rearranged as a result of a t balanced translocation in a T98G glioblastoma multiforme cell line  has one LGI gene] and are found in all vertebrate genomes that have been examined to date. Early vertebrate genomes underwent two rounds of whole genome duplication , two copies of lgi2 (lgi2a and lgi2b), a single copy of lgi3 and no copy of lgi4 . Following whole genome duplication events, gene families often contract, as genes with redundant functions are lost  are an order of fish that diverged from teleost fishes prior to their third genome duplication and are thus more closely related to the ancestors of the tetrapods. The coelacanth genome . The LRRs with the N- and C-terminal flanking sequences form a slightly curved structure with parallel β-strands forming the concave face of the domain  of the LGI1 EPTP domain have been published, based on the structure of the β-propeller protein WDR5  domain, resides in a glycosylation site that is conserved among all LGI members   or dominant negative (i.e. mutant LGI1 interferes with wild-type LGI1 or its binding partners). The incomplete penetrance of many ADPEAF mutations, and the instability and/or inability to be secreted of many LGI1 proteins resulting from them suggests that these mutations are haploinsufficient  ratio in hippocampal slices after incubation with LGI1; it suggests that LGI1 is an extracellular factor-controlling synaptic strength at excitatory synapses  transmembrane protein family . More thAdam23 knockout mice  forms a hetero-tetramer between two NR1 and two NR2 subunits. A hallmark of glutamatergic synapse maturation is the change in postsynaptic NMDA-R NR2 subunit composition , which is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons  have produced similar results  result from the spontaneous release of synaptic vesicles in the absence of a stimulus; changes in their frequency suggest a presynaptic defect, whereas changes in their amplitude suggest a postsynaptic defect. In brain slices from their Lgi1 mutant mice, Fukata et al. . High-level inhibition of lgi1a produces developmental abnormalities, including reductions in brain, eyes and tail, presumably due to increased CNS apoptosis; these fish also display seizure-like hyperactive swimming behaviours -generated leucine to arginine mutation (L385R) in the fourth EPTP domain of LGI1 recapitulate the susceptibility to audiogenic seizures seen in ADPEAF, and display a similar profile of anti-epileptic drugs that suppress those seizures  signalling, reducing ERK  signalling, and reducing matrix metalloproteinase expression in glioma and other cells in culture  in high-grade gliomas, and was proposed to function as a tumour suppressor gene  is a neurological autoimmune disease associated with antibodies against a variety of antigens  has been deleted appear normal  caused PNS hypomyelination without affecting central myelin  mice, in which LGI4 is not secreted  sympathetic and dorsal root ganglia (reviewed in Jessen, Over the last decade, LGI proteins have emerged as important regulators of cellular interactions in the nervous system and their mutation has been associated with diverse pathologies such as epilepsy, psychiatric disorders and hypomyelination. However, their mechanism or mechanisms of action remain a mystery. Advances in understanding one LGI family member may inform us about the functions of others. LGI proteins are secreted proteins, but because most mutations in LGI proteins affect their secretion, these mutations do not tell us much about the mechanism of action of LGI proteins. The disease-associated mutation in LGI1 that appears to affect its interactions with other proteins rather than its secretion (Striano et al., All LGI proteins appear to interact with ADAM22/23/11 receptors albeit probably with very different affinities. The high degree of identity among LGI proteins and their affinity for these ADAM receptors suggest that they act through a similar mechanism. However, it is not known whether the LGI proteins are functionally equivalent or serve distinct functions in different parts of the nervous system at different developmental stages. The one published experiment that addresses this issue demonstrated that LGI3 could not replace LGI1 in CNS synaptic development (Fukata et al., LGI proteins evolved with vertebrates, but many of the intercellular interactions in which they function, such as synapses between neurons, clearly predate them. Are the biological functions of LGI proteins required in invertebrates, and if so, what proteins perform them? If LGI proteins perform vertebrate-specific functions, could their appearance have been a crucial step in the evolution of complex vertebrate nervous systems? As we learn more about what LGI proteins do, the answers to these questions will become apparent.LGI gene family.Importantly, we anticipate that ongoing and future clinical, genetic and biochemical work directed towards an understanding of the biology and pathology of LGI proteins will ultimately lead to novel approaches in combating the devastating neurological diseases associated with mutations in the


Thyroid hormones, 3,5,3'5'-tetraiodothyronine (thyroxine) and 3,5,3'-triiodothyronine (T3) contribute to the regulation of key cellular processes, including proliferation, differentiation, apoptosis and metabolism. Thus, deregulation of thyroid hormone pathway can result in serious disturbances of cellular physiology such as those observed in tumoral transformation. The intra- and extracellular concentrations of thyroid hormones (TH) are regulated by three types of iodothyronine deiodinases . These enzymes catalyze two types of reactions: deiodination in position 5' of iodothyronines , resulting in activation of thyroid hormones, and deiodination in position 5 of iodothyronines , which results in inactivation of thyroid hormones. Changes in expression and activity of iodothyronine deiodinases result in modulations of thyroid hormone concentrations and thus influence cellular processes. The actions of thyroid hormone are mediated by thyroid hormone receptors (TRs) which influence intracellular processes via modulation of expression of target genes. In this process, known as genomic mechanism of TH action, thyroid hormone receptors act as transcription factors whose activity is modulated by the presence of ligand, T3. TH can also influence cellular processes via non-genomic pathways in which TH modulate activity of membrane or intracellular receptors and activate different intracellular signaling pathways, involving protein kinases, for instance PI3K, and Akt/PKB.In the process of carcinogenesis the signaling pathway of thyroid hormones is disturbed. This is illustrated by observations of mutations and deregulated expression of thyroid hormone receptors, altered expression and activity of iodothyronine deiodinases as well as decreased intracellular concentrations of T3 in tumours. Whether these changes play a causative role in the process of carcinogenesis is currently a matter of discussion. In vitro studies in cancer cell lines and mouse models showed that increased expression of DIO3 together with enhanced proteasomal degradation of DIO2 in basal cell carcinoma stimulates tumoral proliferation. Moreover, thyroid hormone receptor beta-1 was shown to act as a powerful suppressor of tumour invasiveness and metastasis in mouse models of breast cancer and hepatocarcinoma. These studies suggest that local tissue hypothyroidism may rather support tumoral transformation and even initiate enhanced cancerous proliferation. In contrast, studies on the effects of systemic hypothyroidism and cancer risk show opposite or conflicting results. For instance, hypothyroidism was associated with increased risk of hepatocellular carcinoma, and cancers of lung and prostate. In case of breast cancer, different studies reported that hypothyroidism was linked to reduced risk of the disease in humans while when studied in mice, hypothyroidism reduced tumour growth but enhanced metastasis. Finally, studies performed on cell line and mouse xenografts of thyroid and kidney cancers showed that inhibition of integrin-mediated thyroid hormone signaling can effectively result in inhibition of tumour growth.All these studies show that the role of thyroid hormones in carcinogenic process is complex and presumably depends on type of cancer and extra- or intracellular effects of TH signaling. The results showing benefits from the use of TH pathway genes in cancer treatment and diagnosis suggest that further and more in depth studies are needed.


Down syndrome (DS), a chromosomal condition caused by the trisomy of chromosome 21, is associated with various immunological impairments, including a high incidence of autoimmune diseases  and anatomical changes in the thymus. The autoimmune regulator (AIRE) is a transcription factor whose gene maps in 21q22.3 and controls the ectopic expression of a large set of peripheral tissue antigen genes (PTA) in medullary thymic epithelial cells (mTECs). This phenomenon, termed promiscuous gene expression (pGE), plays a key role in central tolerance as demonstrated by the inactivation of AIRE resulting in the rare recessive autoimmune polyendocrinophathy-candidiasis-ectodermal dystrophy syndrome (APECED). DS patients carry three copies of AIRE which may result in its overexpression or, more rarely, its underexpression. To investigate the possibility that DS associated autoimmunity is favored by the impairment of pGE as a consequence of changes in AIRE expression and/or anatomical disorganisation of the thymus, we investigated AIRE, PTA and cell marker gene expression in thymi of DS individuals vs controls. Gene expression was assessed on cDNA from 19 DS and 21 controls total thymus samples by qPCR using TaqMan probes. Interestingly, AIRE gene expression was significantly reduced in thymus from DS vs controls (p = 0.0003). 7 PTA genes  were also reduced, more markedly for KLK3 and SAG (P = 0.002 and 0.0004 respectively). This changes seems unlikely to result from a reduction in the number of epithelial thymic cell since the levels of keratin expression were not reduced. Allele-specific quantification of three alleles of the AIRE gene demonstrated that, in spite of its overall reduction, the three copies of AIRE genes are expressed in the thymus of DS patients, thus indicating that reduction of AIRE expression is not caused by allele silencing (imprinting). Conclusion, autoimmunity associated to DS is probably in part caused by a reduction of AIRE expression that results in an impaired pGE rather that by a reduction of thymic epithelial cells as part of anatomical disorganization.


In addition, microRNAs affect all facets of the immune/inflammation responses to infection, which may control HPV clearance. We thus hypothesized that microRNA polymorphisms modify the association between HPV16 seropositivity and OSCC risk.Both microRNAs were genotyped and HPV16 serology was determined in 325 cases and 335 matched controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using univariate and multivariable logistic regression models.Four single-nucleotide polymorphisms in miR146 rs2910164 GG genotype and HPV16 seronegativity, risk of OSCC was increased among those with CG or CC genotype and HPV16 seronegativity , GG genotype and HPV16 seropositivity , and CG or CC genotype and HPV16 seropositivity . Similar results were found for miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444. Analyses stratified by tumor sites and smoking status showed that each polymorphism significantly increased the risk of HPV16-associated squamous cell carcinoma of the oropharynx (SCCOP), and such effect modification was particularly prominent in never smokers.Overall, each polymorphism had no significant main effect on OSCC risk. Compared with the risk among individuals with both microRNA polymorphisms modify the risk of OSCC associated with HPV16 seropositivity, particularly in patients with SCCOP and never smokers. Larger studies are needed to verify our findings.Our results indicate that  Oral squamous cell carcinoma  accounts for the majority of head and neck malignant tumors and is one of the most common malignancies worldwide. It has been estimated that approximately 36,540 new cases of OSCC will be diagnosed and 7,880 individuals will die from these cancers in 2012 in the United States MicroRNAs are highly conserved, single-stranded, short, noncoding RNAs of about 22 nucleotides that regulate gene expression through completely or partially base pairing with target mRNAs at the 3′-untranslated region, resulting in mRNA cleavage or translational suppression microRNAs play key roles in a broad range of physiologic and pathologic processes microRNAs remain largely unclear, previous studies have indicated that microRNAs may participate in tumorigenesis, functioning as tumor suppressors and/or oncogenes microRNAs may be involved in regulation of the immune and inflammatory response systems microRNAs may affect susceptibility to HPV-associated OSCC.hsa-146a was over-expressed in SCCHN cell lines and tumor tissues and hsa-mir-149 was down-regulated in squamous cell carcinoma of the tongue and hsa-mir-146a polymorphism may also affect the miRNA expression miR146 G>C, miR196 C>T, and miR499 T>C may affect cancer risk and prognosis miR146 rs2910164, miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444) and risk of head and neck cancers, but no significant association between each SNP and cancer risk was found microRNAs in regulating the immune and inflammatory response systems and thereby mediating HPV infection, we hypothesized that genetic variants in these four microRNAs alter the association between HPV16 seropositivity and risk of OSCC. To test this hypothesis, we evaluated the joint effect of HPV16 serology and these four common microRNA SNPs on risk of OSCC.Previous studies have demonstrated that Patients with incident OSCC were recruited as part of an ongoing molecular epidemiology study of head and neck cancers through the Head and Neck Center at The University of Texas MD Anderson Cancer Center between December 1996 and October 2002. All patients were recruited without restrictions on age, sex, cancer stage, or histology and had newly diagnosed, histopathologically confirmed, untreated OSCC. Excluded from participation were patients with second primary tumors; primary tumors of the sinonasal tract, nasopharynx, hypopharynx, or larynx; primary tumors outside the upper aerodigestive tract; cervical metastases of unknown origin; and histopathologic diagnoses other than SCC. In addition, patients who had received a blood transfusion in the past 6 months or who were receiving immunosuppressive therapy were excluded. Among patients initially contacted for participation, approximately 95% of eligible incident cases agreed to participate in the study.A pool of cancer-free subjects was recruited from the Kelsey-Seybold Foundation, a multispecialty physician practice with multiple clinics throughout the Houston metropolitan area, and from healthy visitors who accompanied cancer patients to outpatient clinics at MD Anderson Cancer Center but were genetically unrelated to the cases. In this pool of cancer-free controls, each individual was first surveyed by means of a short questionnaire to determine his or her willingness to participate in research studies. Each eligible subject was subsequently interviewed to collect demographic and epidemiologic information, such as age, sex, ethnicity, smoking status, and alcohol consumption status. The overall response rate for the controls was approximately 78%. Excluded from participation were individuals with history of any cancer, history of immunosuppressive therapy, or blood transfusion in the past 6 months.Smoking status was categorized as “ever smokers” (those who had smoked more than 100 cigarettes in their lifetime) and “never smokers” . Drinking status was categorized as “ever drinkers”  and “never drinkers” (those who never had such a pattern of drinking). Both Institutional Review Boards of UT MD Anderson Cancer Center and Kelsey-seybold approved the study, and written informed consent was obtained from all study subjects. In this study, 335 cancer-free controls were selected from the pool of potential controls by frequency matching with the patients by age (±5 years), sex, and smoking and drinking status. To avoid confounding factors due to ethnic characteristics, all study subjects included were non-Hispanic whites, who accounted for the vast majority of the cases.HPV16 L1 virus-like particles generated from recombinant baculovirus-infected insect cells were used to test for antibody against HPV16 L1 capsid protein in the plasma of study participants by using a standard enzyme-linked immunosorbent assay, as described previously miR146 rs2910164, miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444. The microRNA SNPs, primers, and restriction enzymes  used for each SNP are shown in From each blood sample, a leukocyte pellet was obtained from the buffy coat by centrifugation of 3 mL of whole blood. The pellet was used for genomic DNA extraction with the DNA Blood Mini Kit  according to the manufacturer's instructions. A polymerase chain reaction-restriction fragment-length polymorphism assay was used to amplify the fragments that contained SNPs of microRNA genotypes, and HPV16 serology between cases and controls were evaluated using the chi-square test. To evaluate the association of HPV16 serologic status and microRNA genotypes with OSCC risk, odds ratios (ORs) and 95% confidence interval (CIs) were calculated using both univariate and multivariate logistic regression analyses. Logistic regression was also used to assess the potential interaction effects by evaluating departures from the models of additive and multiplicative interactions between selected variables. A more-than-additive interaction was suggested when OR11>OR10+OR01−1, for which OR11 = OR when both factors were present, OR10 = OR when only factor 1 was present and OR01 = OR when only factor 2 was present. A more-than-multiplicative interaction was suggested when OR11>OR10×OR01. We assessed the interaction by reporting the P values from the Wald test for testing the coefficients  were different from 0, where the interaction term consisted of the product of the two variables: microRNA polymorphism and HPV16 seropositivity. We also assessed the joint effects of HPV16 serology and microRNA genotypes on OSCC risk, and the joint effects were further stratified by tumor site and smoking status. Statistical Analysis System software  was used for all statistical analyses. All tests were two-sided, and P<0.05 was considered the cutoff for statistical significance.The distributions of selected demographic variables, tobacco smoking, alcohol consumption, P>0.05). However, we observed that HPV16 seropositivity was significantly more common in patients than in controls (P<0.001). HPV16 seropositivity was associated with risk for OSCC , particularly for SCCOP , but not for oral cavity cancers , after adjusting for age, sex, smoking status, and drinking status.The final analysis included 325 OSCC cases and 335 controls. Of the 325 cases, 188 (57.8%) had SCCOP, and 137 (42.2%) had SCC of the oral cavity. The distributions of demographic characteristics and known OSCC risk factors are summarized in microRNA SNPs among the controls were all in agreement with the Hardy-Weinberg equilibrium . Overall, we did not find any statistically significant association of any of the four microRNA SNPs with the risk of OSCC, but we did find that miR146 rs2910164 and miR499 rs3746444 had a minor effect on the risk of SCCOP , and individuals with the miR499 rs3746444 combined CT and CC genotypes had a higher risk of SCCOP than individuals with the TT genotype .The genotype distributions of the four of SCCOP Table 3.microRNA genetic variants. Specifically, compared with individuals with both miR146 rs2910164 GG genotype and HPV16 seronegativity, those with both GG genotype and HPV16 seropositivity had an increased risk of OSCC , and the risk was even higher among those with both CG or CC genotype and HPV16 seropositivity . In contrast, compared with individuals with both miR149 rs2292832 CT or TT genotype and HPV16 seronegativity, those with both CT or TT genotype and HPV16 seropositivity had an increased risk of OSCC , and the risk was even higher among those with both CC genotype and HPV16 seropositivity . Similar results were observed for the associations between miR196 rs11614913 and miR499 rs3746444 SNPs and risk of HPV16-associated OSCC .As shown in ted OSCC Table 4.microRNA SNPs on the risk of HPV16-associated OSCC were confounded by smoking status, we performed stratified analyses of joint effects of HPV16 seropositivity and microRNA variants on risk of OSCC by smoking status (In order to explore whether the effects of g status Table 6.microRNAs on risk of OSCC, but we did observe a significant association between miR146 rs2910164 and miR499 rs3746444 and a moderately increased risk of SCCOP. However, we found that the joint effect of HPV16 seropositivity and each of these microRNA SNPs increased the risk of OSCC, although we did not observe any significant interaction for such joint effect on risk of OSCC. Moreover, such effect modification was more pronounced for SCCOP as opposed to SCC of oral cavity and in never smokers than in ever smokers. Our results are in agreement with the characteristics of SCCOP associated with HPV infection, indicating that microRNA SNPs might play a role in the development of HPV16-associated SCCOP.In this study, overall, we did not observe a significant main effect of each SNP of these MicroRNAs act as omnipresent regulators of gene expression and are involved in many cellular processes, including inflammation and immune responses. Recent studies have demonstrated that microRNAs participate in mediating inflammatory and cytokine signaling as well as innate and acquired immune response to viral infection by targeting critical elements in inflammatory signaling pathways MicroRNAs also modify viral-host interactions, which play central roles in the development and progression of infection-associated tumors.microRNAs may lead to individual variations in immune function, inflammation, and apoptosis that modify viral immune escape, antiviral defense, and evasion of apoptosis, leading to modification of the risk of infection-associated cancers. Numerous studies have shown that some microRNAs are up-regulated, while other microRNAs are down-regulated in head and neck cancers microRNAs may be associated with the development, progression, and prognosis of head and neck cancers Therefore, functional genetic polymorphisms of microRNA SNPs and the risk of head and neck cancers. One study indicated that the miR196 rs11614913 variant reduced the risk of head and neck cancers miR146 rs2910164, miR149 rs2292832, and miR196 rs11614913 did not modify the risk of head and neck cancers independently of HPV infection but that miR499 rs3746444 moderately reduced the risk of head and neck cancers microRNA SNPs and HPV16 infection on OSCC risk. HPV16 infection has recently been identified as one of the primary etiologic factors for causes of SCCOP, but oral cavity and laryngeal cancer mainly result from tobacco and alcohol consumption. In our current study, although we had a smaller study size, we found that microRNA SNPs and HPV16 seropositivity may function jointly in the development of OSCC, particularly in patients with SCCOP and in never smokers.Recently, several authors have reported associations between microRNA genetic variants and HPV16 infection jointly play a role in the development of OSCC has not been fully clarified, a joint effect of microRNAs and HPV16 infection on susceptibility to OSCC is biologically plausible. HPV16 is oncogenic: it encodes viral oncoproteins E6 and E7, which inhibit p53 and Rb cell cycle tumor suppressors. MicroRNA variants might functionally affect expression of genes involved in myriad cellular processes, including inflammation and immune response pathways, subsequently controlling the host's ability to clear HPV and HPV's ability to escape immune surveillance microRNA variants might act jointly in the development of OSCC. However, further studies are required to validate the hypothesized functionality of these SNPs.Although the precise mechanism by which the common microRNA SNPs and HPV seropositivity on the risk of OSCC was much stronger in never smokers than in ever smokers. These data further support the notion that risk genotypes of the four common SNPs of microRNAs may be involved in the development of HPV-associated OSCC in non-Hispanic white never smokers. However, the modifying effect of each of microRNA SNPs on risk of OSCC associated with HPV16 was not statistically significant. This lack of significance could be either because there was no such interaction effect in these subgroups or because the small sample sizes in each substratum limited the statistical power to detect a significant interaction effect. Therefore, the significance and degree of such interaction in each subgroup needs to be further investigated in future studies with larger sample sizes.In the present study, our analysis stratified by tumor site showed that the effect modification was more pronounced for SCCOP than for oral cavity SCC, reinforcing the concept that there are differences in the etiology of cancers of the oropharynx and oral cavity. Additionally, the analysis further stratified by smoking status for each SNP showed that the joint effect of Although the present study minimized potential confounding factors, several limitations should be considered. First, a possible selection bias in patient recruitment cannot be ruled out as our study was a hospital-based case-control study and as the cases and controls were not selected from the same target population. In addition, only non-Hispanic white subjects were included in our study. It is therefore uncertain whether these results are generalizable to other ethnic populations. Second, the effects of potential demographic confounding factors were minimized by adequate frequency matching on age, sex, smoking status, and alcohol use, but stratified analyses included a limited number of individuals in each subgroup. As such, the role of these SNPs and HPV16 in OSCC will require confirmation in larger studies in different populations. Third, because individual patients may have had differences in their immune response to HPV16 infection or antibody instability, HPV16 seropositivity may not reflect the tumor's true HPV16 status, leading to possible misclassifications of some patients as HPV16 seronegative who had tumors that actually were HPV16 DNA positive. This misclassification could result in a major selection bias for the estimates of association. Finally, lack of information on changes in sexual practices in current study did not allow us to evaluate its potential influence on risk of HPV-associated OSCC, particularly SCCOP. Therefore, our future studies should include the patients with tumor HPV status and sexual behaviors to further evaluate our findings once such information become available. Nevertheless, the use of serologic status may provide a possibility to include a cancer-free control group for the present case-control study design.microRNA variants may modify the risk of HPV16-associated OSCC, particularly in patients with SCCOP and in never smokers. However, further prospective studies with larger sample sizes are needed to validate our findings.In conclusion, our results indicate that