Participants and presenters were a healthy mix of junior and established scientists (47 % of participants being graduate students and postdocs), staying true to the vision of the first meeting in 1997 and the spirit of Cold Spring Harbor Laboratory. The breadth and depth of exciting, unpublished work towards discovering fundamental principles and direct applications in agriculture is impossible to capture in a short meeting report. Therefore, here we highlight some transformative areas whose potential impact is likely to reach beyond plant biology.The 10A major theme across the sessions was the development of disruptive tools and their application to address technical challenges and to answer key biological questions. Ronan C. O'Malley  described DNA affinity purification sequencing (DAP-seq), a method he pioneered in the Ecker laboratory and is using to create an atlas of Arabidopsis transcription factor (TF) binding sequences. DAP-seq involves capturing and sequencing genomic DNA fragments using a tagged TF bound to an affinity bead. O'Malley subjected all ~1800 Arabidopsis TFs to DAP-seq and identified motifs for >500 TFs. Interestingly, 60 TFs capture most motif types because members of TF families tend to bind to similar motifs. He also reported that DNA methylation within a TF-binding motif alters binding of 76 % of TFs, with the majority (72 %) being inhibited by methylated sites. Future directions include double DAP-seq or sequential DAP-seq to identify more biologically relevant complex binding sites and to elucidate mechanisms of combinatorial control of gene expression.Oropetium thomaeum. Resurrection plants such as O. thomaeum and Selaginella lepidophylla can lose most of their cellular water content during desiccation but ‘come back to life’ upon re-watering. Hence, the genomic resources of these plants have the potential to shed light on mechanisms of stress tolerance. O. thomaeum has the smallest known genome among the grasses (~250 Mbp), and was sequenced at ~65× coverage using PacBio’s P6-C4 chemistry making it the first published genome sequenced using single-molecule sequencing. The exceptional quality of the resulting genome assembly was enabled by high-quality, long genomic DNA fragments (20–30 kb) and by the long read lengths obtained (the N50 of the read lengths was ~16 kb). Advances in sequencing technology, such as PacBio’s long-read technology, are poised to usher in a new era of platinum-standard reference genomes.Robert VanBuren  described the single-molecule genome sequencing of the desiccation-tolerant grass Philippa Borrill  described an expression visualization and integration platform (expVIP) that has been used to develop and analyze a hexaploid wheat gene-expression atlas comprising 418 diverse RNA-seq samples. This platform was crucial for integrating layers of next-generation sequencing data to reveal the genetic architecture regulating nutrient remobilization and senescence in wheat, and to prioritize targets of the senescence-controlling wheat TF NO APICAL MERISTEM b1 (NAMb1) for future study. Multiple advances such as those described above will facilitate future molecular genetic studies in crops that have large complex genomes, such as wheat and sugarcane.6-methyladenosine (m6A) modification for mRNA structural integrity, which is essential for transcriptome function and homeostasis. The loss of m6A results in a global increase of mRNA secondary structure around start and stop codons, and many m6A modified transcripts are defense related. Gregory mentioned that there are 152 additional types of RNA modifications, whose functional roles remain unknown. He described a new method his lab has developed called interaction profile sequencing (PIP-seq), which allows the identification of RNA–protein interaction sites as well as proteins interacting with specific RNA structures or modifications.Scientific progress is driven not only by disruptive technologies but also by disruptive concepts and out-of-the-box thinking. Several talks touched on this area. For example, Brian Gregory  presented work from his and other labs, which aims to determine the global landscape of RNA secondary structures and RNA–protein interactions. Besides the well-known structures of non-coding RNAs, he showed how all RNAs, including mRNAs, have secondary structures whose particular conformations might influence transcript function. In addition, he discussed the potential importance of NArabidopsis thaliana, he identified numerous elements that were enriched in intergenic and transposon sequences. Traditionally, plants are not known to harbor many long-distance enhancer elements, but Dorrity's study shows that there may be a wealth of long-distance regulatory elements in plants.Another interesting concept was that of long-distance transcriptional regulatory elements in plants, presented by Michael Dorrity . Dorrity used self-transcribing active regulatory region sequencing (STARR-seq) to rapidly identify and quantify plant enhancer elements in full-genome libraries. Using this method on A. thaliana, Drosophila melanogaster, and Homo sapiens. Initial results include an in-depth characterization of a novel regulator that controls organ size in plants. Approaches like this could tackle the problem of assigning molecular functions to the ‘dark matter’ of genomes, which remains a problem even for intensively studied model organisms.Seung Rhee  described recent work to identify novel transcriptional regulators in eukaryotes. Rhee mentioned that despite unprecedented progress in genome sequencing in the past 20 years, all sequenced genomes harbor 25–75 % of protein-encoding genes whose molecular functions remain unknown. Rhee described a computational method that does not rely on sequence homology to identify new transcriptional regulators, as well as its application to several model organisms such as Several speakers highlighted the pace with which some of that ‘dark matter’ in large crop genomes is becoming illuminated. Cristobal Uauy  demonstrated the power of a genome-enabled phenotyping and sequencing platform for hexaploid bread wheat. By combining analysis of a large TILLING population with exon-capture approaches, Uauy’s team and consortium partners aim to identify wheat homoeologs that contribute to important yield traits such as grain weight.Resistance (R) genes, which encode receptors that recognize various infectious pathogens. In the past, attempts to improve resistance relied largely on the painstaking introduction of major-effect R genes into high-yielding varieties for use in agriculture. Efficient capture of new, valuable resistance traits across crop germplasm is now achievable, as shown by Thomas Kroj  Montpellier, France) and Beat Keller . Kroj highlighted the discovery of co-acting R gene pairs in plant genomes, which have integrated additional ‘decoy’ domains to effectively betray pathogen attack. Keller described efforts to harness the natural diversity of wheat and maize R gene complements against damaging fungal diseases. This is providing fundamental insights to how R genes evolve and functionally diversify. Information from natural allelic variants of the wheat Pm3 powdery mildew R gene is enabling Keller’s team to engineer altered receptor modules and combine useful new crop disease resistance traits for robust resistance in the field.The complexity of crop genomes is, in part, due to the expansion and diversification of disease Brachypodium distachyon, which have altered responses to abiotic stresses such as heat and drought. Christopher Topp  has focused on developing imaging tools to quantify root architectural and physiological traits in maize, and to measure the degree of plasticity in root growth dynamics in response to changing nutrient availability. Topp and a team of engineers are developing an imaging facility to measure the root behavior of maize genetic variants in the field.A prominent thread in several talks was the value of high-throughput phenotyping in model and crop plants. This is enabling the characterization of developmental processes at many scales and is uncovering the genetic variation underlying potentially useful traits. Malia Gehan  described the utility of an open-source image analysis software suite called PlantCV that she and colleagues have developed, which enables the quantification of seedling growth (biomass) and photosynthetic performance over time. Using this software and a high-throughput phenotyping platform, the Mockler group is beginning to identify natural genetic variants in the model grass species Boechera stricta plants against insect herbivore damage. Although clearly measurable in a large natural B. stricta population, the fitness contribution and heritability of allelic variation at this locus is very small. This rare example of ‘real world’ ecological genetics and the effect of genotypic variation on fitness in nature emphasizes the need to measure crop productivity traits under field conditions.These and other studies described at the meeting emphasize the multidisciplinary nature of genome-enabled quantitative biology to harness new phenotypic variation and genetic epistasis for crop improvement. Important next steps will be to determine whether the variation in growth habits, biomass, or stress tolerance observed in controlled environments contributes to bioproductivity and fitness in the field. An important cautionary tale was presented by Thomas Mitchell-Olds  who described his study of allelic variation at a biochemical defense pathway locus that underlies tolerance of A. thaliana root development, crucial transcriptional feed-forward and feed-back loops remain to be discovered, as demonstrated by Jazz Dickinson  who is combining genetics, synthetic biology, and two-photon selective plane illumination microscopy (2P-SPIM) approaches to dissect spatially and temporally relevant processes in asymmetric root cell division.Descriptions of state-of-the-art, high-throughput technologies and genome biology were complemented nicely by presentations on genetic, molecular, and spatial dissections of biological processes in model and crop species. In A. thaliana. He also described variation in the network topology between annual flowering A. thaliana and perennial relatives such as Arabis alpina. Coupland identified an age-related signaling circuit that influences the ability of plants to initiate flowering. In another study, Matthias Berens (a graduate student in the Tsuda lab at MPIPZ) uncovered an effect of leaf age on hormonal decision-making in growth responses to environmental stress.George Coupland (Max Planck Institute for Plant Breeding Research (MPIPZ), Germany) outlined his group’s progress in defining the transcriptional network underlying seasonal control of flowering in A. thaliana. The interplay of hormone pathways regulates the sorting of cambial cells into high-order vascular tissues and secondary vascular expansion, as observed most dramatically in trees. Helariutta’s team is using knowledge gained from his analysis of A. thaliana to characterize the genetic basis of vascular architecture variation and adaptation in birch trees.A neat combination of genetics, synthetic biology, and imaging by Ykä Helariutta  is providing new insights into how early organizing centers control vascular development in In another potentially exciting step forward, Kun Huang  is developing in vivo sensors to track the biogenesis and movement of small RNAs in plants in response to developmental or environmental stimuli. For this, Huang described a modified ‘Spinach’ fluorescent RNA aptamer-based detection system , which works in vitro but not yet in vivo other than in Chinese Hamster Ovary cells. She described a new system using a biliverdin-binding fluorescent RNA aptamer to detect small RNAs in vivo.th anniversary of the meeting series and we anticipate a celebration of genome-enabled plant biology of the past, present, and future.Here we illustrate, using a few examples, some of the exciting new directions in plant biology presented at the meeting. These endeavors leverage today’s technological revolutions in genome sequencing and imaging and will undoubtedly impact fields beyond plant biology. We, along with David Jackson , are thrilled to organize the next meeting in 2017. It will be the 20


In response to external stimuli, cells polarize morphologically into teardrop shapes prior to moving in the direction of their blunt leading edge through lamellipodia extension and retraction of the rear tip. This textbook description of cell migration implies that the initial polarization sets the direction of cell migration. Using microfabrication techniques to control cell morphologies and the direction of migration without gradients, we demonstrate that after polarization, lamelipodia extension and attachment can feedback to change and even reverse the initial morphological polarization. Cells do indeed migrate faster in the direction of their morphologically polarization. However, feedback from subsequent lamellipodia extension and attachment can be so powerful as to induce cells to reverse and migrate against their initial polarization, albeit at a slower speed. Constitutively active mutants of RhoA show that RhoA stimulates cell motility when cells are guided either along or against their initial polarization. Cdc42 activation and inhibition, which results in loss of directional motility during chemotaxis, only reduces the speed of migration without altering the directionality of migration on the micropatterns. These results reveal significant differences between substrate directed cell migration and that induced by chemotactic gradients. Directional cell motility plays a central role in embryonic development , tissue The second step of cell migration, directional protrusion of lamellipodia at the leading edge, is driven by actin polymerization, 12 and The RhoGTPases, Rac, Rho, or Cdc42, are key players in modulating cell migration and cytoskeletal dynamics in all four steps–27. Cdc4Cells interact simultaneously with soluble signalling molecules and their substrate. While significant progress has been made in identifying the molecular components and signalling pathways involved in cell migration during chemotaxis–38, how microarrays of closely spaced teardrop-shaped adhesive islands to amplify the natural directional persistence (MANDIP) of cells, we have demonstrated that 3T3 fibroblasts and human microvascular endothelial cells can be directed to migrate continuously along preset paths without chemoattractants, external fields, or mechanical manipulation[Micropatterned cells have been observed to extend lamellipodia, filipodia, and microspikes most aggressively at sharp corners of their constraining adhesive islands. Cells cipulation, 44. CelThe ability to guide cells to migrate along and against their morphological polarization enables us to investigate if and how substrate guided directional migration differs from the sequential four-step polarize-extend-attach-retract model for chemotaxis. During chemotaxis, gradients in the binding of soluble chemoattractants lead to local activation of RhoGTPases, which regulate actin cytoskeleton organization and contractility, induces directional migration. However, in contrast to soluble chemoattractants whose binding cannot impart physical forces to cells, the binding of cell integrins to the ECM result in focal adhesion complexes that transmit both physical and chemical cues. Here we will examine if substrated directed cell movement requires the activation of specific RhoGTPases that are central to chemotaxis.2) was chosen based on the previous study that show the migration tendency is higher when cell spreading is restricted[We use MANDIP micropatterns A and B  to direcestricted. The spaestricted. On micrestricted.The seemingly minor rearrangement of the teardrop islands to form micropattern B forces the cell movement against the morphological polarization. As cells adopt the shape of the teardrop islands, they are polarized morphologically to migrate from their blunt end. However, lamellipodia extended from the blunt end rarely attach perpendicularly to a neighboring island due to the absence of permissive adhesion sites directly in front of their blunt-end. In contrast, lamellipodia extended from the tip, can attaches readily to the blunt of a neighboring island. As a result, tip-to-blunt, counter-clockwise migration along the teardrops dominates >90%, . This suMigrating fibroblasts have been shown to respond to external cues such as chemotactic gradients, wounding, and electric fields by reorienting their Golgi apparatus toward the direction of migration. The reoHere, we investigate if and how cells on micropattern B reorient their Golgi as they reverse the polarity imposed by the teardrop shape, so as to migrate in the direction of successful lamellipodia attachment from their rear tip. To do this quantitatively as the cells migrate around the micropatterns, the location of the nucleus was identified to be either in the blunt half (R1) or tip half (R2) of the teardrop shapes. When the nucleus is located in the tip half (R2), the cell is translocating and spanning between the two neighboring islands and the nucleus typically do not reside completely within the tip but suspended between the two neighboring islands .For cells migrating on micropattern A, the Golgi apparatus is localized predominantly in front of nuclei (r1) irrespective of whether the cell is transvering between the two adhesive islands  and dominant negative (DN) Rac1, RhoA, and Cdc42. Rac1 activation at the blunt leading edge of cells promotes and maintains lamellipodia protrusions–50. HoweRho activation at the rear of cells increases actomyosin based contractility, 55 and CA-Cdc42 cells aggressively extend actin-based membrane protrusions at their edges. These actin protrusions form filopodia that spread beyond the adhesive island and form attachments on neighboring island. As cells migrate from one island to another new filopodia attach and old ones detach . These fin vitro wound closure studies[CA-Cdc42 or DN-Cdc42 cells migrate on non-patterned substrates with reduced persistence. The spee studies where DNIn chemotaxis, the first step of cell migration—morphological polarization is thought to direct the subsequent steps of lamellipodia extension and attachment. Here we show that for substrate-directed cell migration, lamellipodia extension and attachment can feedback to change the morphological polarization dictated by the initial shape of the cell. This feedback arising from the continuous exploration of the cell’s local environment through membrane protrusions is likely a key mechanism for directed cell movement on non-homogeneous substrates. Unproductive protrusions that fail to attach are retracted, while protrusions that attach successfully to the substrate can reorient the morphology leading to positive feedback. The concept that cell substrate interactions can influence directional cell movement has been proposed by others, but to our knowledge, the idea that cells can move consistently against their initial polarization through substrate interactions and Golgi apparatus reorientation has neither been proposed nor observed. Here, we offer experimental evidence that such a feedback mechanism not only exists but is strong enough that lamellipodia attachment can reverse morphological polarization.Dominant negative Rac mutants exhibit inhibited chemotaxis for several cell types, and subRhoA, which regulates contractility, stimulates cell motility in chemotaxis. On our substrate-directed studies, RhoA activation has a very significant effect, with CA RhoA cells migrating almost twice as fast along the initial polarization direction. Even when migrating against their initial polarization (micropattern B), CA RhoA cells migrate at a higher speed than CA Rac1 cells highlighting the significant role of cell contractility during substrate-directed movement. In all cases, the p-values all reject the null hypothesis that the mutants have the same average hop time as the wild type fibroblast.The most striking difference we found between substrate-guided and chemotactic migration is the role of Cdc42. Cdc42 is generally accepted to be a key regulator of cell polarity during chemotaxis–68. Cdc4It has been shown that signal cross talk between different RhoGTPases and clonal variability can occur when using dominant negative or constitutively active mutants. For exaAltogether, the results presented here brings to light a new layer of complexity on the role of physical cell-substrate interactions in reshaping polarized cell morphology and directional migration even when Cdc42 is inhibited. These findings highlight the importance of understanding the interplay between physical and biochemical cues that guide cell migration during during tissue morphogeneis, wound healing, and cancer metastasis.Tissue culture dishes were purchased from Fisher Scientific  and used as received. Polydimethylsiloxane  was obtained from Dow Corning . Alexa 488-phalloidin, 4',6-diamidino-2-phenylindole (DAPI), were purchased form Molecular Probes . Phosphate buffer saline was purchased from Sigma . Opti-MEM media was purchased from invitrogen, Serum Supreme, were purchased from Cambrex biosciences .Random copolymers of OEGMA and MA  were prepared by free radical polymerization of 10 wt% methanolic solutions of the two monomers (80:20 OEGMA to MA mass ratio) at 60°C following a procedure described previously. PolymerMicropatterns of different geometric shapes, e.g., squares, circles, ellipses and rectangles were fabricated on silicon wafers using standard photolithographic techniques. From this silicon master, complementary polydimethylsiloxane (PDMS) replicas were prepared and used as stamps in subsequent microcontact printing steps to form Rac1, RhoA and Cdc42 constitutively active mutants  and dominantly negative mutants  were gifts from Prof. Yi Zheng  and were generated as described previously–76. BrieConstitutively active and dominant negative mutants of Rac1, RhoA and Cdc42 (NIH 3T3 fibroblasts) were cultured in Opti-MEM media (w/o phenol red) supplemented with 10% fetal bovine serum and 25mM hepes (Invitrogen). All mutants except CA-Rac1 were grown by selection with G418 (350 μg/ml). Cell cultures were thermostated using a stage-mounted heater equipped with a temperature controller. Patterned cells (500 cells/mL) were supplemented with media containing human PDGF (5 ng/mL) and HEPES buffer (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid; 50 mM) 30 min prior to time-lapse imaging. Phase-contrast images were recorded each hour for the time lapse figures.Only single cells confined within single islands were considered for analysis. Cells were considered to have “hopped” when their nuclei and 90% of their cell area have relocated from one island to another. The 99% confidence intervals of the average speed of motile cells were calculated based on the standard deviation of data collected from at least 100 hopping events and a minimum population of 30 motile cells.Cells were fixed with 3.7% paraformaldehyde for 10 minutes, washed in phosphate buffered saline, and then permeabilized with 0.2% Triton X100 for 5 minutes. Samples were then rinsed with PBS and incubated with Alexa 594-phalloidin, Alexa 488 conjugated anti-golgin-97 (human) and DAPI to stain for F-actin, golgi apparatus and nuclei respectively. Images of the patterned cells were acquired using a Nikon TE-2000 inverted microscope with Metamorph software The polarization of Golgi patterned on microarray patterns A and B  was quan


Incisional hernias remain a surgical challenge when balancing surgical morbidity, functional restoration, and risk of recurrence. Laparoscopic intraperitoneal onlay mesh (IPOM) placement reduces postoperative wound infections and allows fast patient recovery. Yet, current IPOM techniques do not achieve closure of the midline hernia gap, thereby increasing the risk of persistent mesh bulging with poor abdominal wall function. We propose a novel triple-step hernia repair technique that includes tension-free midline reconstruction. It is achieved through laparoscopic dorsal component separation and laparoscopic suture closure of the midline with a 1.0 polydioxanone suture sling. Combining dorsal abdominal wall component separation, a midline closure with adequate suture strength, and IPOM reinforcement merges the benefits of open and laparoscopic hernia repair. This triple-step technique allows static and functional laparoscopic abdominal wall reconstruction.The online version of this article (doi:10.1007/s00268-014-2747-0) contains supplementary material, which is available to authorized users. Laparoscopic intraperitoneal onlay mesh placement with omission of laparotomy for hernia repair is beneficial for patients’ physical function and reduces the incidence of surgical-site infections. Functional restoration of the abdominal wall is more effective, however, with open hernia repair, which allows reconstruction of the midline with approximation of the rectus abdominis muscle. Another disadvantage of not closing the hernia gap during laparoscopic intraperitoneal onlay mesh (IPOM) placement is the subsequent development of seroma of the original hernia sac or a bulging phenomenon caused by herniation of the mesh through the gap. We propose a triple-step procedure to compensate for some of the shortcomings of simple laparoscopic IPOM placement: (1) laparoscopic transversus abdominis fascia release; (2) laparoscopic use of a regular slowly absorbable polydioxanone suture (PDS) sling (1.0) for fascial closure; (3) IPOM reinforcement of the closed hernia gap.The patient is placed in supine position with both arms alongside the abdomen. The surgeon and first assistant stand on the right side of the patient with the monitor opposite on the left side. The scrub nurse stands to the right of the surgeons. A mini-laparotomy is made close to the right ventral axillary line in mid-abdomen. After capnoperitoneum is established via a 10-mm trocar, two additional 5-mm trocars are inserted close to the rib cage and in the lower right abdomen, respectively. Initially, intraabdominal pressure is adjusted to 12 mmHg. Adhesions to the abdominal wall must be taken down, including removal of relevant preperitoneal fat to delineate the abdominal fascia. Similarly, the fascial wound edge is cleared from adherent tissue to enable well-defined stitch placement. By applying external pressure, the hernia sac can be inverted into the abdomen to facilitate its laparoscopic removal by carefully avoiding injury to the adjacent skin. All resected tissues can be extracted using an endobag. Afterward, laparoscopic dorsal component separation is performed as described by Milburn et al. [The second step of the operation is now initiated. The needle of the PDS sling is inserted cephalad from the hernia gap via a 5-mm skin incision. The needle is then reversed under laparoscopic control and extracted via the same skin incision Fig. . The sliLaparoscopic IPOM placement as described elsewhere  is the tSo far, 11 patients  have undergone laparoscopic closure of the fascial gap prior to IPOM reinforcement. Among them, three patients required laparoscopic dorsal component separation to reduce fascial tension. No surgical-site infections or signs of recurrence have been noted. There have been no seromas or signs of bulging. In all cases, the abdominal shape was restored with closure of the fascial gap.We propose a new, simple laparoscopic triple-step technique that meets the requirements of incisional hernia repair concerning abdominal wall reinforcement and tension-free repair. The midline closure is established using a strong PDS sling suture regularly applied during open surgery together with dorsal component separation that consists of transversus abdominis fascia release. Final IPOM reinforcement ensures the strength of the hernia repair. Our technique also follows the recommendations for abdominal fascial closure in terms of the stitch length and the suture length/wound length ratio of at least 4 . We findSupplementary material 1 (M4 V 19700 kb)Supplementary material 2 (M4 V 15733 kb)Below is the link to the electronic supplementary material.


The identification of genes involved in the modulation of inflammatory processes has allowed the delineation of a new group of diseases called “Monogenetic Autoinflammatory Syndromes - MAISs”.At the moment, 25 syndromes and their gene-disorders are known. Some of them are well known , most of them are rare diseases .These disorders of innate immunity characterized by episodes of fever and systemic inflammatory symptoms affect for instance the serosal surfaces and bear the risk of developing reactive systemic (AA) amyloidosis due to excessive production of serum amyloid-A (SAA). SAA is deposited in various organs, particularly the kidneys, with the consequent progressive development of kidney failure[We report a now 26-year old patient, who was first admitted aged 5 months due to periodic fever. Additional patient's history included polyarthritis, uveitis and a worsening of renal function over the years due to histological signs of sarcoidosis in the kidney. Different immunosuppressive treatments with steroids, azathioprin, methotrexat and cyclosporin did not prevent renal failure requiring dialysis in 2005. In 2010 our patient underwent renal transplantation. Despite standard triple immunosuppression patient's symptoms, especially uveitis, occurred even after transplantation whenever steroids were reduced. The initial diagnosis “juvenile sarcoidosis” was corrected to “Blau Syndrome” due to mutation analysis carried out in April 2012 (mutation on NOD2 gene). Thanks to treatment with adalimumab since 2012, uveitis and polyarthritis are under control. Kidney function has remained stable with creatinin values within normal range up to this day.A 54-year old dialysis patient with atrophic kidneys (reason unknown) underwent renal transplant in October 2012. A few days after discharge a fever of unknown origin occurred with an increase of c-reactive protein (CRP), creatinin, severe abdominal pain and diarrhoea without detection of pathogens. As Familial Mediterranean Fever (FMF) was suspected patient underwent a colchicin therapy. The creatinin, CRP and SAA value decreased at normal range within 6 weeks of treatment. To confirm the diagnosed FMF, a mutation analysis was carried out. We found a heterocygous mutation in the MEFV gene, which confirms the FMF diagnosis.We report a women born in 1971 who was diagnosed with reduced kidney function in 2008. In addition the patient has been suffering from cold-dependent urticaria and joint pain without known reason since childhood. There was a rapid worsening of renal function over the next months. The kidney biopsy shows signs of amyloidosis (picture a-f). Mutation analysis in June 2013 confirms the diagnose “Familial Cold-Autoinflammatory Syndrome - FCAS”. Due to treatment with anakinra, the cold-dependenturticaria and joint pain are eliminated, SAA is normalized and serumkreatinin and proteinuria are decreased.Monogenetic Autoinflammatory Syndromes are rare causes of kidney failure due to excessive production of serum amyloid-A (SAA). The detection by means of mutation analysis allowed a disease-specific therapy with stabilization of serumcreatinin and SAA in addition to reduced disease-specific symptomes even in advanced kidney failure and after renal transplantation.Written informed consent for publication of their clinical details was obtained from the patient/parent/guardian/relative of the patient.


Restoration of tropical forest depended in large part on seed dispersal by fruit-eating animals that transported seeds into planted forest patches. We tested effectiveness of dispersal agents as revealed by established recruits of tree and shrub species that bore seeds dispersed by birds, bats, or both. We documented restoration of dispersal processes over the first 76 months of experimental restoration in southern Mexico. Mixed-model repeated-measures randomized-block ANOVAs of seedlings recruited into experimental controls and mixed-species plantings from late-secondary and mature forest indicated that bats and birds played different roles in the first years of a restoration process. Bats dispersed pioneer tree and shrub species to slowly regenerating grassy areas, while birds mediated recruitment of later-successional species into planted stands of trees and to a lesser extent into controls. Of species of pioneer trees and shrubs established in plots, seven were primarily dispersed by birds, three by bats and four by both birds and bats. Of later-successional species recruited past the seedling stage, 13 were of species primarily dispersed by birds, and six were of species dispersed by both birds and bats. No later-successional species primarily dispersed by bats established in control or planted plots. Establishment of recruited seedlings was ten-fold higher under cover of planted trees than in grassy controls. Even pre-reproductive trees drew fruit-eating birds and the seeds that they carried from nearby forest, and provided conditions for establishment of shade-tolerant tree species. Overall, after 76 months of cattle exclusion, 94% of the recruited shrubs and trees in experimental plots were of species that we did not plant. The future of tropical forests will be determined by interplay of climate change, conservation, deforestation, natural succession, and ecological restoration Here we address ecological results and consequences of dispersal processes that are relevant to maintaining heterogeneity and accelerating ecological succession in highly altered agricultural landscapes. Our approach uses the interdependence of most rainforest trees on birds and mammals that disperse seeds as a critical phase of tropical tree life cycles (4). The point is to shape foraging routes of fruit-eating animals by providing plantings that offer cover and/or food, and consequently facilitate seed dispersal into fenced habitat islands that serve as stepping stones among forest remnants for “countryside” plants and animals capable of existing in agricultural landscapes Our study considers processes of effective dispersal, which involves removal of seeds from parent trees to sites where germination, establishment and survival are possible We test the null hypothesis that fruit-eating birds and bats play comparable roles in promoting tree and shrub recruitment over the first 76 months of experimental restoration in an agricultural-rainforest mosaic in southern Veracruz, Mexico. Some fruit-eating birds and bats forage locally within pastures; others commute long distances to and from feeding areas The study site was in an agricultural mosaic in the Los Tuxtlas Region of southeastern Veracruz, ∼1 km NE of the nearest edge of the Los Tuxtlas Biological Station The12 ha site where we conducted the study was cattle pasture embedded in an agricultural mosaic of rainforest, isolated trees, and living fences. In August 2006 we established 24 fenced 30×30 m plots separated by 35 m of active cattle pasture arranged in a 3×8 grid . Eight exclosures were planted with 12 seedlings of 12 native animal-dispersed species (>10 cm high), and eight with 12 native wind-dispersed species with 12 seedlings each Criteria for designations of tree successional status and dispersal category are consistent with general practice This experiment is expected to span 30 years. Prior publications address legacy effects through month 24 after cattle exclusion P≤0.05. Statistics are accomplished with SAS and Systat 13.Primary analytical tools are mixed-model repeated-measures randomized-block ANOVAs. Pioneer and later-successional species and recruitment densities are evaluated independently. Independent variables include time , dispersal agent  and treatment (planted or unplanted), with interactions. F-statistics are considered significant when the Bonferroni adjustment is significant at −2 to 0.0023 m−2 from 16 to 76 months after cattle exclusion (P<0.0001). Significant differences existed in overall contribution of dispersal categories to pioneer-species densities (P<0.0001), but interaction of dispersal category of pioneer species densities with time was not significant (−2 in 2007 to 0.005 m−2 in 2013 (P<0.0001). Significant overall differences by dispersal category of individual pioneer recruits existed (P<0.0001), but again the interaction of dispersal category with time was not significant . At 76 months after cattle exclusion, species densities of later-successional trees and shrubs dispersed primarily by birds were five times higher than those dispersed by both bats and birds, with a significant interaction of dispersal category with time , with a significant interaction of dispersal category with time . In pastures at Los Tuxtlas, C. obtusifolia is both bird- and bat-dispersed Use of fruiting trees by bats and birds is to a degree context-specific. C. obtusifolia and Trema micrantha and sapling Bursura simaruba, appeared in the plots directly after fencing, reflecting legacies of seeds brought to figs or other fruiting trees that were cut during site preparation In the present experiment, legacy effects faded over time. A few older recruits, including reproductive Planted stands reduce both seed and establishment limitation. Birds that potentially disperse seeds are more likely to forage in clumps of shrubs or trees than in open areas, stay longer in larger than smaller stands of woody vegetation, and regurgitate or defecate more seeds in tree islands than in the open −2 month−1−2, indicating immense mortality of seeds or young seedlings. In contrast, planted plots averaged ∼ one recruit 16 m−2. These densities were well below those under large fenced figs in a pasture landscape Only a small proportion of seeds resulted in established seedlings in pasture plots. Early in the present experiment, we reported seed input of woody plants into fenced plots >90 m from forest of one seed in 10 mother than those that we planted With the exception of common pioneers, species planted in our experiment are in higher densities than occur in nature. High densities differentially affect survival of planted species, and will likely bias early recruitment in favor of species 2) stands. At least to established seedling, sapling and in some case adult stages, this is occurring, with a clear increase in establishment of later-successional species between 60 and 76 months, and little recruitment of most planted or early-recruited pioneers that are fruiting in planted plots.Scattered colonists from forests will likely have a different dynamic. Plots with 120 m of edge admit diffuse light, explaining high establishment of pioneers during the first five years of cattle exclusion Artibeus lituratus, up to 70 g) carry Dipteryx (Fabaceae) fruits weighing >20 g for hundreds of meters, but even small fruit bats  disperse a variety of tree seeds >20 mm long in extensive forest It is too early to know whether the imbalance of bird- versus bat-dispersed later-successional recruits will persist. In addition to dispersal of small-seeded pioneers, bats disperse many large-seeded, late-successional species in continuous forest in both the Old- and New-World Tropics Ocotea uxpanapana (Lauraceae) are present, as are 20 well-established  Tetrorchidium rotundatum , a widespread animal-dispersed tree that is nonetheless endangered in Mexico. A realistic goal for maintenance and recovery of substantial tropical biodiversity is to sustain and re-establish dispersal processes that preserve or create as much habitat heterogeneity and connectivity as rural economies permit.Our study has significant implications for maintaining biodiversity in human-dominated landscapes. Mixed-species plantings potentially maintain and locally increase “countryside diversity” of tropical plants and animals that are capable of persisting in or moving through human-dominated landscapes Habitat islands left as remnants or planted as stepping stones in matrices between forest remnants serve a variety of functions. Connectivity preserves landscape species richness at large scales Table S1Dispersal mode and successional status of recruited species, including references for published reports.(DOC)Click here for additional data file.Appendix S1Criteria for designating dispersal modes and successional status of plants.(DOC)Click here for additional data file.Data Spreadsheed S1Data used for analyses.(XLSX)Click here for additional data file.


Biomedical big data, as a whole, covers numerous features, while each dataset specifically delineates part of them. “Full feature spectrum” knowledge discovery across heterogeneous data sources remains a major challenge. We developed a method called bootstrapping for unified feature association measurement (BUFAM) for pairwise association analysis, and relational dependency network (RDN) modeling for global module detection on features across breast cancer cohorts. Discovered knowledge was cross-validated using data from Wake Forest Baptist Medical Center’s electronic medical records and annotated with BioCarta signaling signatures. The clinical potential of the discovered modules was exhibited by stratifying patients for drug responses. A series of discovered associations provided new insights into breast cancer, such as the effects of patient’s cultural background on preferences for surgical procedure. We also discovered two groups of highly associated features, the HER2 and the ER modules, each of which described how phenotypes were associated with molecular signatures, diagnostic features, and clinical decisions. The discovered “ER module”, which was dominated by cancer immunity, was used as an example for patient stratification and prediction of drug responses to tamoxifen and chemotherapy. BUFAM-derived RDN modeling demonstrated unique ability to discover clinically meaningful and actionable knowledge across highly heterogeneous biomedical big data sets. As a whole, biomedical big data provide multi-scale and diverse descriptions of biological or pathological events at different “throughput levels” across a range of experimental platforms and clinical systems. For example, in the TCGA dataset, breast cancer has been profiled at molecular, cellular, tissue, patient, and population levels in terms of biological traits as well as drug and treatment responses in short-term  and long-term  contexts. Types of data range from high-throughput omics assays to conventional demographic and diagnostic features. Besides the research and trial data collected from specific cohorts910111314“drinking seawater” paradox and limit full-spectrum analysis of rich features. For features available in a pan-dataset analysis, each dataset only covers a small portion of them. Such intrinsic “local deficiency” of features in each dataset and heterogeneity of feature availability across biomedical big datasets raises unique challenges and demands novel approaches for data mining. This limitation causes a “drinking seawater” paradox: the more the seawater is consumed to ease thirst, the more water in the body will be lost, and the worse the thirst will be; similarly, the more that biomedical big datasets with heterogeneous features are incorporated, the fewer features and samples are qualified for analysis, and the less knowledge can be discovered. This paradox stems from the heterogeneous coverage of features across datasets. Imagine that a virtual “data table” for knowledge discovery was established by incorporating multiple biomedical big datasets for breast cancer  or human errors . However, for “structurally missed features”, as demonstrated in Missing value imputation approaches such as k-nearest-neighbor16Another strategy is to combine local knowledge discovery in individual datasets and global summaries of the results crossing datasets2122Pairwise association analysis24uniform metric to enable unbiased analysis of pairwise feature associations across datasets, whereas RDN allowed global analysis and summarization of the knowledge discovered by BUFAM. Thus, the BUFAM-derived RDN model provides a general solution for the feature deficiency problem during the integration of biomedical big data sets of heterogeneous features. Such strategy enables efficient and effective use of heterogeneous biomedical big data for understanding diseases and optimizing clinical decisions.To address this “drinking seawater paradox”, we developed the relational dependency network (RDN) of biomedical features based on bootstrapping for unified feature association measurement (BUFAM). BUFAM provided a all datasets that share these two features. Once all associations that are directly testable are profiled, an RDN of discovered associations can be established. Network-based analysis tools can be applied to this RDN to generate a global understanding of the pairwise discovered knowledge, and to further identify associated features that do not share enough data to test direct associations. Feature heterogeneity226The design of the BUFAM-derived RDN approach is illustrated in As illustrated in The unified relationships among heterogeneous biomedical features, evaluated by BUFAM p-values . The JacOur association results were consistent with current knowledge about breast cancer. For example, estrogen receptor (ER) status, an important breast phenotype widely used for patient subtyping and treatment design, was associated with patients’ ethnicity . The ER-282 test), which was not reported before. Asian patients were less likely to choose lumpectomy (7.7%) compared with White (27.4%) and African (32.5%) American patients. However, about 50% of Hispanic patients chose lumpectomy. Hispanic and Asian patients were also less likely to choose either simple or modified radical mastectomy , compared to African American (52.5%) and White (55.8%) patients.We also observed an ethnicity-specific preference regarding choices for surgery  if batch effects overwhelmed true associations and 2) if the discovered associations were consistent with literature. Originally identified30The 21-gene signatureThe discovered and validated associations were not only fully consistent, but also consistent with published results. For example, the strong positive association between the Oncotype DX Score with higher histologic grades  is consiBUFAM provides a flexible metric platform for customized and scalable data mining. The default statistical tests , bootstrAfter performing data imputation for numeric features using the KNN algorithm, most pairs of features that demonstrated associations of various strengths before imputation were no longer distinguishable. Among 10 numeric-to-numeric associations, 9 showed apparent strong associations . Figure structurally missed features resulting from differences of feature coverage among datasets, in 80% of cases, current data imputation approaches introduced artifacts that overwhelmed the original data patterns.Using the KNN algorithm for imputation of numeric data has been one of the most reliable approaches for processing missing data. It has shown robustness against distribution, percentage34Features are not always imputable. An essential assumption for KNN imputation is that features have a strong enough association that the similarity defined by any feature is likely to be consistent. Under this assumption, if a sample is missing a feature, a set of samples with similarity to this sample in all other features  can be used to estimate the missed feature of the first sample. Although this assumption works well for microarray data1734We compared the results of our pan-dataset association analyses with meta-analyses in terms of feature coverage, patient coverage, and association results . The uniCompared with meta-analyses, the greatest strength of the BUFAM approach was the unified and robust measurement of feature associations across cohorts. Since all associations were assessed using the same strategy, the measurements were less sensitive to the cohorts used. Meanwhile, since more patients could be used for knowledge discovery , BUFAM f dataset .Cohort-specific biases, especially the sampling bias of patients, often challenge meta-analysis. BUFAM provides a fair incorporation to minimize such biases. Cohort-specific bias with respect to some features, however, may introduce artifacts and reduce statistical sensitivity of the BUFAM approach. Careful data cleaning and normalization are thus crucial for the sensitivity of BUFAM.Strong associations among features were visualized as a relational dependency network . The ER in situ immunity patterns, and the other from the tumor growth and invasion patterns.The signaling pathway annotation enrichment analysis showed that the ER module was associated with a wide range of immune pathways , includiDecisions regarding surgical procedures were strongly associated with pathologic staging, HER2 status, and histologic types. These features included pathologic stage using the TNM systemThese “clinically omitted” features reflect a gap between clinical practice and the current molecular profiling achievement. The explosive growth of omics data and rapid progress in bioinformatics approaches have generated numerous molecular signatures for subtyping patients through molecular-level evidence. Many of these signatures provide important information about mechanistic differences in disease among individuals and have demonstrated strong clinical potential. However, none of these breast cancer molecular signatures and molecular subtyping actually significantly affects clinical decisions regarding surgeries. A major reason could be that most molecular signatures were developed for identifying suitable drugs, not types of surgeries. For example, the Oncotype DX score was established to predict responses to chemotherapy and the risk of distant metastasis.Our results suggested that even for such a relatively small RDN (44 nodes), the challenges of data sparseness and heterogeneity could be addressed, and biologically and clinically meaningful knowledge could be extracted for further research and validation.Using the ER module as an example, we further illustrated how modules of heterogeneous features from disparate resources integrative contribute to better therapeutic designs. We first demonstrated that the RDN module detection allowed cross-cohort integration of features whose associations could not be directly measured. Then we showed that such integration was biologically meaningful by gene set enrichment analysis using BioCarta signaling pathway signatures. Finally, we demonstrated the clinical relevance of the detected ER module by using the associated signatures to stratify responses to tamoxifen-related therapies. We chose these therapies as the use case for the ER module because tamoxifen, an ER antagonist, is the typical therapy for patients with ER-positive breast cancer.We showed that the BUFAM approach could reliably and sensitively discover associations among features by integrating data from diverse and structurally heterogeneous datasets. However, in many cases, due to the lack of data, we could not directly measure associations between two intrinsically related features. This challenge was addressed by projecting these BUFAM-discovered pairwise associations onto a unified RDN, allowing indirect associations among features based on their connections. We used network-related approaches37The RDN module detection allowed indirectly associating such features. The TCGA cohort shares three clinical features with the other two cohorts: patient age, ER status, and PR status . These “The clinical relevance of the ER module could not be directly assessed using the associated features because none of the three cohorts we used includes all these features . Thus, wet al. reported more regulatory (FOXP3 positive) T cells in ER-negative than ER-positive patients; once the latter group showed high levels of regulatory T cells, they were at higher risk for tumor relapseet al.et al.The ER module exhibited strong associations with patient immunity patterns and responses to tamoxifen and chemotherapy, consistent with the literature. Adams and colleagueset al.et al.Chemotherapies also modulate the immune systemet al.Our results also suggest that the conflicting reports regarding ER and immunity- related drug responses might due to heterogeneity in patients. The immune active subtype patients might benefit most from natural immune surveillance, and were very sensitive to the immune suppression effects of chemotherapies. How combination therapy (tamoxifen with chemotherapies) affects patient survival is also not clear. For example, Osborne and colleagues reported antagonism between tamoxifen and melphalan or fluorouracilReliably evaluating associations among biomedical features in big datasets is challenging, largely due to the lack of reliable ground truth and often inconsistent literature reports. For this exploratory work, we developed a comprehensive strategy to check the discovery from three aspects: 1) Cross-domain validation. We used data from the WFU EMR data, an independent dataset used for clinical purposes, to examine discovered associations. Different from traditional strategies of using similar datasets in the research domain for cross-validation, the EMR dataset was more independent and thus stricter; 2) Translation-oriented validation. We examined the clinical relevance and the translational potentials of the discovered knowledge using inter-cohort cross-validation. In details, we identified patient subgroups on one cohort (TCGA) according to the discovered associations, and then applied the subtyping model to another cohort (WFCCC) to examine whether stratified patients showed different drug responses. This evaluation of effectiveness examined whether the obtained knowledge was clinically actionable. 3) Literature-based validation. We limited validation to those features that had been well studied in the literature. We used literature-based validation for Oncotype DX Score-related associations. However, we did not heavily rely on evidence from literature, since the widely existing inconsistent  reports undermine confidence in such “dry validation”. Our comprehensive validation strategy provided more reliable and practical evaluation of the biologic and clinical values of the BUFAM-based RDN approach as well as the discoveries described herein.We developed a bootstrapping-based unified feature association approach and used it to analyze three major breast cancer data repositories. We incorporated demographic, diagnostic, treatment and molecular features of patients, analyzed the associations of these features across heterogeneous datasets, visualized the discovered knowledge as a pan-BRCA (Breast Cancer) relational network, and validated these discoveries with electronic medical record data. Two sets of strongly associated features, the ER and the HER 2 modules, were discovered and annotated with the underpinning molecular signaling signatures. Four novel immunity-related breast cancer subtypes were discovered based on the ER module and demonstrated clinical potential for designing precision therapy schemes. The BUFAM-derived RDN modeling approach demonstrated the ability to detect clinically actionable associations across highly heterogeneous biomedical big data. Our approach for the first time overcomes the obstacle of big-data-associated feature heterogeneity, and allows efficient utilization of diverse datasets for unified knowledge discovery.TCGA: The Cancer Genome AtlasMDACC: MD Anderson Cancer Center11WFCCC: Wake Forest Comprehensive Cancer CenterBreast cancer patient data from four repositories were used in this study: The WakeOne EMR data of breast cancer patients were obtained with appropriate approval from the Institutional Review Board of Wake Forest University.The overall pipeline of the BUFAM-derived RDN knowledge discovery is illustrated in We developed BUFAM  to normaFormalized mathematical details are provided in Significant feature-to-feature relations, evaluated by BUFAM p-values, were extracted to establish a relational dependency network (RDN). After adjusting for multiple testing, associations of false discovery rates (FDRs) less than 0.01 were used to build the RDN model. Features were associated as modules using Girvan-Newman’s modularity clustering algorithmData were validated using the WakeOne EMR data. A total of 28 associations were validated using corresponding association methods.Numeric features were used to compare the performance of the BUFAM association before and after KNN imputationBUFAM was performed on each separately and all cohorts together. Results were compared based on three aspects of feature associations: coverage, sample size used for each, and statistical significance.Discovered feature modules were annotated using a customized gene set enrichment analysis (GSEA) analysis, with the underlying signaling pathways using 217 BioCarta54Patients were subtyped according to the discovered ER module using the TCGA cohort, and the drug responses of each subtype were compared using the WFUCCC dataset. Patients from the TCGA cohort were clustered against the ER module using the module-specific BioCarta signaling signatures and a sparse k-means approach. Four patient subtypes were discovered: Immune Inert, Neutral, Active, and Responsive subtypes. WFUCCC patients were classified into these subtypes accordingly, and their responses to four types of adjuvant treatment strategies  were evaluated by Kaplan-Meier survival analysis with log-rank test and the Cox proportional hazards model, using the distant metastasis-free survival time as the index. More details are provided in http://ctsb.is.wfubmc.edu/express/db/drugsig.htm).All data except the WakeOne EMR were available using SQL through the DrugSig Translational Data Warehouse (PostgreSQL) hosted by Center for Bioinformatics & Systems Biology, Wake Forest School of Medicine. For more details, see our website .


New evidence has highlighted that miRNA production and trafficking can be dysregulated in both autoimmmune and neurological disorders. Multiple sclerosis (MS) in particular is an autoimmune pathology leading to neurodegeneration. Profiling studies performed on cells derived from MS patients have described a dysregulated network of miRNAs in both immune and neural cells. Interestingly, new evidence has emerged showing that circulating miRNAs are also dysregulated in MS body fluids, including plasma/serum and cerebrospinal fluid. This review summarizes the current scientific theories on the function of this altered circulating miRNA network. It builds up new insights about miRNA transfer mechanisms including extracellular vesicle trafficking involved in cell-to-cell communication and the possible physiopathological functions of these transfers in MS. Finally, this review proposes that monitoring altered miRNA expression levels could serve as a potential biomarker read-out of MS subtype and severity. Multiple sclerosis (MS) is a human lymphocyte-mediated autoimmune disease affecting the central nervous system (CNS). Neurodegeneration of the CNS is associated with both neuron demyelination and inflammation in the so-called active lesions. It is common to separate relapsing–remitting MS (RRMS), in which periods of relapses (attacks) alternate with periods of remission, from primary progressive MS (PPMS) characterized by a constant worsening of CNS condition and minor remissions. In about 60% of RRMS cases, the disease secondarily turns into a progressive form (secondary progressive or SPMS). miRNAs are small non-coding RNAs of about 20 nucleotides inhibiting the expression of their mRNA targets through degradation and/or by stopping mRNA translation . In mostThe expression of miRNAs in MS was first assessed in cells derived either from MS patients’ blood samples or from active lesions , 4. InteUsing a combination of established methods of miRNA profiling, it is now possible to propose a provisional network of dysregulated miRNAs in MS. These approaches provide new insights and raise some interesting issues: how are disrupted cellular and circulating miRNA networks related together? What could be the role of circulating miRNAs in MS physiopathology? Which molecular vectors carrying circulating miRNAs would be the most relevant to study and could these vectors be miRNA-associated actors of MS pathogenesis? This review first recapitulates the up-to-date network of dysregulated circulating miRNAs in MS patients. Second, extracellular vesicles (EVs) are considered as miRNA vectors and are proposed to be involved in miRNA-mediated pathogenesis. Finally, circulating miRNAs are proposed to be convenient, reliable, and accurate biomarkers to differentiate MS subtypes and evaluate MS severity.Junker and colleagues have described the first miRNA profiling analysis performed on MS patient brain lesions in 2009 . Exponenvia the transcription of miRNA-derived cDNA (Amino Allyl MessageAmp™ II aRNA amplification kit from Life Technologies). Such differences in the procedure for miRNA profiling lead to massive variability. Researchers now aim at standardizing these techniques to comprehend profiling metadata  or plasma have been extensively used to assess miRNA expression levels , 18, 19.kip1 driving apoptosis . A significant number of miRNAs were commonly dysregulated between plasma and immune cells  and between plasma and the CNS  Figure  and Tregpoptosis . Also, mpoptosis .+ T cells (eightfold)  , and in ghtfold)  and as ightfold) , 11, 24 + T cell apoptosis  or sometimes in vivo to be recipient for EVs. In the CNS, neural cells, including glia cells, also exchange EVs  autoimmune model aiming at reproducing MS pathology features. It is now accepted that in both MS and its EAE model, pathogenesis involves lymphocytes including B cells , Th17 anThe implication of cellular miRNAs in MS and EAE pathogenesis is currently strongly investigated. In order to define the new concepts about circulating miRNA function, it is necessary to consider different elements: (i) producing and recipient cells for EVs, (ii) cellular and extracellular miRNA dysregulation, and (iii) cellular miRNAs functionally involved in MS/EAE. Some miRNA function and distribution seem relevant to suggest circulating miRNA implication in Th17-mediated, Th1-mediated, and brain-resident cell-mediated MS pathogenesis Figure .+ T cells  and quantify MS severity,the fact that collecting blood miRNAs and measuring their expression is easy, poorly invasive, and cheap,the robustness of circulating miRNAs which are highly stable, andthe accuracy using composite biomarkers rather than a single one.So, why are biomarkers for MS still arousing interest? Emerging biomarkers, such as circulating miRNAs, display several advantageous features in comparison with previous methods:The chronicity in MS pathology is the cause of repetitive relapses causing irreversible neurodegeneration. Depending on MS subtypes (PPMS or RRMS), medical care will be different. Thus, the possibility to anticipate MS evolution is crucial. Different statistical methods are used to predict the diagnostic accuracy of a biomarker: the area under the curve (AUC) Box A and theBox 1How can we estimate the diagnostic accuracy of miR-633 and miR-922 to distinguish MS from other neurological diseases (OND) ?First method: receiver operating characteristic (ROC) curves (A) plot sensitivity (y axis) versus 1 − specificity in (x axis). An experiment good predictor curve will locate between a random classifier (50% chance predicting MS or OND) and a perfect classifier (100% chance predicting MS or OND). Calculation of the area under the curve (AUC) gives the level of diagnostic accuracy, which corresponds to the probability that random MS patients have higher (if upregulated) or lower (if downregulated) miRNA levels than OND ones.Second method: it is possible to determine a sensitivity and specificity(B) score for each cut-offs (estimated threshold of miRNA level separating MS from OND), thus drawing the ROC curve. The highest sensitivity and specificity score indicates the most relevant cut-off. The example in (C) represents the result of sensitivity and sensibility (left) and the predicted results using only miR-633 (right).sibility  (left) aFenoglio and colleagues  found ouAlthough its procedure is not the most convenient, the analysis of CSF miRNAs from MS patients is relevant considering that it may better reflect the level of brain damage. The authors mentioned the promising future of miRNAs as biomarkers of MS  but aptlThere is current evidence that at least 60 circulating miRNAs would be dysregulated in MS patient’s blood and profiling results are continuously emerging. The current challenge relies on linking this network with the network of cellular dysregulated miRNAs. EVs are the most relevant miRNA carriers and could explain the relation between these two dysregulated networks. Both immune and neural cells, active actors of MS physiopathology, produce and receive EVs. This observation has to be considered besides the demonstrated implication of some cellular miRNAs, including miR-326 and miR-155, which were found to promote inflammation and demyelination.Indeed, the hypothesis of miRNA-containing EVs raises the issue of a possible cell non-autonomous amplification of inflammation and demyelination through abnormal miRNA transfer. It has to be seriously considered during a period of relapse, in which symptom worsening is sudden. Interestingly, current treatments to prevent from worsening effects of relapses were shown to reduce miRNA dysregulation in peripheral blood mononuclear cells and T cells in particular –65, but FJ acquired the data and drafted the manuscript. ND revised the manuscript and made substantial contributions to its final content and design. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lens culinaris), groundnut (Arachis hypogaea), and pigeon pea (Cajanus cajan) were found to experience lower drought-induced yield reduction compared to legumes such as cowpea (Vigna unguiculata) and green gram (Vigna radiate). Yield reduction was generally greater when legumes experienced drought during their reproductive stage compared to during their vegetative stage. Legumes grown in soil with medium texture also exhibited greater yield reduction compared to those planted on soil of either coarse or fine texture. In contrast, regions and their associated climatic factors did not significantly affect legume yield reduction. In the face of changing climate, our study provides useful information for agricultural planning and research directions for development of drought-resistant legume species to improve adaptation and resilience of agricultural systems in the drought-prone regions of the world.Food legume crops play important roles in conservation farming systems and contribute to food security in the developing world. However, in many regions of the world, their production has been adversely affected by drought. Although water scarcity is a severe abiotic constraint of legume crops productivity, it remains unclear how the effects of drought co-vary with legume species, soil texture, agroclimatic region, and drought timing. To address these uncertainties, we collected literature data between 1980 and 2014 that reported monoculture legume yield responses to drought under field conditions, and analyzed this data set using meta-analysis techniques. Our results showed that the amount of water reduction was positively related with yield reduction, but the extent of the impact varied with legume species and the phenological state during which drought occurred. Overall, lentil ( Legumes rank among humanity's most important agricultural food crops. They are grown in almost every climatic region and on a wide range of soil types. Legumes are only second to cereals in terms of contribution to food security , serve aWorld demand for legumes is expected to grow in the foreseeable future, not only in developing countries, but also in the developed nations given the trend towards healthy dieting. As the therapeutic uses of legumes are better understood  and the -1 due mainly to fluctuation in annual rainfall . It. It21]. 2 concentrations, which eventually limit photosynthesis and shoot growth. On the other hand, the second mechanism—osmotic regulation through increased solute concentration—is less energy demanding [Legume plants have at least two ways to resist drought: (i) drought avoidance via efficient stomata regulation, and (ii) drought tolerance via osmotic adjustment which usually allows root growth to proceed under drought condition –27. Leguemanding . Therefoemanding . The solemanding –35. Thisemanding . Maintaiemanding , 37, whiemanding . The use2 fixation might be inhibited by water deficit [2 fixation of some legumes. For example, nodules formed at the endodermis  such as in faba bean and groundnut, are able to resist water stress better than those that are superficially attached  such as in cowpea, black gram and green gram [2 fixation. Consistent with our findings, the legumes species that exhibit relatively high N2 fixation during drought  also tend to produce higher yields during drought compared to the species that have limited N2 fixation during drought  [2 fixation to water deficits in regions where drought is a recurring phenomenon.Legumes are unique in their capacity to resist drought because of their interaction with N-fixing  bacteria and arbuscular mycorrhiza –40. Alth deficit , numerou deficit , 43. Thi deficit  since le deficit . Varietyeen gram . While ieen gram , limitin cowpea) . Some le cowpea) . TherefoP<0.01) compared to drought that occurred during the early and late reproductive stages under the same amount of water reduction  resulted in yield reduction (43.4%) similar to the reduction observed when drought occurred throughout the growing season  in medium-textured soils compared to either fine-textured (30.9%) or coarse-textured soils  and the developing countries (0.8 tons ha-1) which are located mostly in tropical regions [When separated into tropical and non-tropical regions, our results showed that no difference in yield reduction between legumes planted in the tropics (35.4%) and in the non-tropical regions 36.6%; . However regions . Possibl regions ; 2) soil regions ; and 3)  regions . FarmersWe did not find any significant difference in crop yield between legumes grown in dryland and non-dryland regions probably because even in their natural conditions, legumes of dryland origin  usually experience terminal drought and show a yield increase if irrigation is applied during the reproductive phase . The simIn this study, through meta-analytic techniques, we investigated how the effects of drought on yield of legume crops co-vary with legume species, soil texture, agroclimatic regions, and drought timing. Many regions of the world have experienced significant shifts in the pattern and amount of rainfall, thus raising concern of a growing water scarcity problem and increasing frequency of crop failure. This study provides useful information that could inform agricultural planning and management to minimize drought-induced yield loss. Since our results showed that the effects of drought on yield reduction varied with species, soil texture, as well as drought timing, this study underscores the need to prioritize the selection and development of drought resistant legume species adapted to the drought-prone regions of the world. Since the effects of drought on legume production was found to be less affected by climatic regions  but was more related to legume species, the selection and promotion of drought-resistant legume species could provide an approach to minimize the impact of droughts. When selecting for drought-resistant species, phenological plasticity could be an important trait to consider given the irregularity in rainfall pattern and the observation that drought generally causes higher yield reduction when it occurs during the reproductive stage compared to during vegetative growth. Among the species of crop legumes, common bean could be the species that requires the most research since it ranks third among legumes in terms of production  and secoS1 File(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Splenosis is a rare condition that results from the autotransplantation of splenic parenchyma into unexpected locations such as the abdomen or subcutaneous tissue. In the presence of coexisting injury to the diaphragm intrathoracic transplantation can occur emerging as single or multiple pleural-based masses. This occurs after traumatic rupture of the spleen and is usually asymptomatic, only to be discovered incidentally on routine thoracic or abdominal imaging. To our knowledge this is the third documented case of combined intrathoracic and subcutaneous splenosis found in English literature. This occurred in a 71-year-old male involved in a motor vehicle accident at age 19 requiring urgent splenectomy. He has a significant cigarette smoking history and was referred to our hospital for further evaluation of an abnormality seen on shoulder X-ray. Splenosis is a rare condition that results from the autotransplantation of splenic parenchyma into unexpected locations such as the abdomen or subcutaneous tissue, usually after traumatic splenic rupture . In presWe report a case of combined intrathoracic and subcutaneous splenosis in a 71-year-old male involved in a motor vehicle accident at age 19 requiring urgent exploratory laparotomy and splenectomy. He had a significant smoking history and was referred to our hospital for further evaluation of an incidental abnormality seen on shoulder X-ray.A 71-year-old Hispanic male was referred to our institution for the evaluation of an incidental 4 cm left peripheral mid-lung opacity on shoulder X-ray  as well At the time of evaluation, he did not have any respiratory symptoms. His physical examination was unremarkable with the expected healed surgical scar at the left lateral abdomen. A computed tomography (CT) scan of the chest was ordered to better characterize the incidental lesion. This study revealed multiple pleural-based noncalcified masses, the largest measuring up to 7.2 centimeters, mild centrilobular and paraseptal emphysema, and two subcutaneous masses at the site of his 51-year-old healed abdominal incision Figures  and 4. TF-fluoro-2-deoxygluocose (FDG) positron emission tomography/CT (PET/CT) revealed the previously detected pleural masses to be only mildly FDG-avid . There wBecause of his risk factors for primary lung malignancy he underwent left sided video-assisted thoracoscopic surgery (VATS) and biopsy. Surgical specimens demonstrated pleural-based masses which were grossly friable and maroon-tanned. Histologically, two well-delineated areas compatible with white and red pulp were noted . OccasioSplenosis is a unique, acquired condition first described by von Kuttner in 1910 . It resuIn the presence of coexisting diaphragm abnormalities or injury intrathoracic implantation may occur leading to one or more pleural-based splenic nodules . This waSubcutaneous splenosis is exceedingly rare with less than 20 reported cases, 16 of which were recently reviewed by Papakonstantinou et al. They found that the majority of cases occur after gunshot wounds or, as with our patient, occur at the site of abdominal surgical scars [Splenosis is generally an asymptomatic condition but there have been thoracic cases presenting with pleurisy  and hemo99mTechnetium (Tc) labeled heat-damaged erythrocyte scanning is the preferred method because it is significantly more specific than 99mTechnetium sulfur colloid, indium 111-labeled platelet, or 99mwhite blood cell scanning due to diminished liver uptake [In patients with history highly suggestive of splenosis and without risk factors or symptoms indicative of malignancy diagnosis can be obtained nonsurgically using radionucleotide scintigraphy or Magnetic Resonance Imaging (MRI). r uptake , 17, 18.99mheat-damaged erythrocyte radionucleotide scanning. In general, MRI has significantly better resolution when compared to other imaging modalities and for this reason may be superior in the diagnosis of splenosis [An alternative noninvasive technique is Ferumoxide MRI . This meplenosis .In cases similar to our own where there is suspicion, malignancy risk factors, or inconclusive imaging studies a tissue biopsy should be obtained for definitive diagnosis. CT-guided fine needle Aspiration (FNA) could be employed when nodules are anatomically amenable. Unfortunately, without the histologic architecture provided by a resection, a small needle biopsy or FNA would not be able to differentiate the splenic elements from chronic inflammation, an intraparenchymal lymph node or a low grade lymphoma. However, the most important contributory information would be the absence of a carcinoma, mesothelioma, or metastatic disease. Ultimately, for thoracic lesions video-assisted thoracoscopic surgery (VATS) with biopsy can be used to obtain a tissue diagnosis. In very rare symptomatic cases this approach is diagnostic as well as therapeutic. Fine needle aspiration or surgical biopsies allow cytopathological analysis which may potentially rule out primary pulmonary malignancies, pleural malignancies, pleural-based metastatic disease, lymphomas, and other varieties of cancer. Histologically ectopic splenic tissue is similar to normal spleen with encapsulated parenchyma consisting of red and white pulp with lymphoid follicles. Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae [Because splenosis is generally asymptomatic, surgical excision of ectopic splenic nodules is often unnecessary and may subject patients to unnecessary surgical risks –23. Morefluenzae . Despitefluenzae . Anecdotfluenzae . Neverthfluenzae . Considefluenzae  it is stfluenzae .99mTc labeled heat-damaged erythrocyte scintigraphy or Ferumoxide MRI. Should risk factors for malignancy be present or the aforementioned studies be inconclusive a CT-guided FNA or VATS with biopsy can be performed to rule out malignancy and obtain a definitive diagnosis. Even though the immunologic functionality of ectopic splenic tissue is debated surgical excision is not recommended unless the patient is symptomatic.To conclude, we present the third documented case of combined thoracic and subcutaneous splenosis , 30. Thi


Dental adhesive systems used for bonding dental resins to enamel and dentin have evolved through several "generations,"with changes in chemistry, mechanism, number of bottles, application technique, and clinical effectiveness. The trend in thelatest generation of dental bonding systems is to reduce the number of components and clinical placement steps. Theintroduction of i bond, a single-bottle dental adhesive system, is the latest of the new generation materials, and combinesetchant, adhesive, and desensitizer in one component. This paper describes different dentin bonding agents, its evolution,mechanism of action and different commercially available dentin bonding agents and their role in the retention of pit andfissure sealant. Dentin/enamel adhesives allow bonding of resin-basedcomposites and compomers to primary and permanent teeth.Adhesives have been developed with reported dentin bondstrengths exceeding that of enamel.1The preventive function of pit and fissure sealant isachieved by the adherence of the material to the acid etchedenamel surface thus physically occluding the pits and fissurefrom the rest of the oral environment. The clinical successof adhesives allows for more conservative preparation whenusing composite restorative materials.The use of the bonding agent may alter the rheology ofthe material allowing it to flow better into the fissures andacid etched surface. The use of phosphate containingbonding agent may result in chemical bonding to the calciumof the enamel surface as well as micromechanical retentionfrom the sealant.2Dentin bonding systems consist of bifunctionalmolecules: (1) A methacrylate group that bonds to therestorative resin by chemical interaction and (2) a functionalgroup that is able to penetrate wet dentin surface. The useof dentin bonding agents between the tooth and fissuresealant can be beneficial for reducing microleakage whenthere is contamination of the enamel.Retention rates are of interest as sealant effectiveness isdirectly related to its retention and completely sealed fissuresshould not develop caries. Studies have shown improvedresults when an intermediate bonding layer is appliedbetween enamel and sealant which increases, bond strength,reduce microleakage and enhances flow of resins intofissures.First generation bonding agents: The foundation of modernadhesive dentistry was laid in 1995, Buonocore discoveredthat acrylic resin could be bonded to human enamel thatwas conditioned with 85% phosphoric acid for 30 seconds.Buonocore found that acid-etching enamel caused a largeincrease in resin enamel bond strength (ca. 20-25 MPa).But they tried those same procedures on dentin, they weredisappointed to find that the resin-dentin bond strengths toacid-etched dentin was very low (5-10 MPa). He predictedthat this bonding technique could be used in various dentalprocedures, including class III and class V restorations andpit and fissure sealants. They included NPG-GMA.N-phenylglycine glycidyl methacrylate), the polyurethanes,and the cyanoacrylates. SS White’s Cervidentwhich had very poor clinical results when used to restorecervical erosion lesions without mechanical retention. or HEMA .Phosphate ester dentin bonding agents: Representativebrands: Bondlite (SDS/Kerr), Creation bond (Den-Mat),Prisma universal bond (Caulk), and Scotchbond (3M).Polyurethane dentin bonding agents: Representativebrand: Dentin-Adhesit (Ivoclar Vivadent).A third generation of bonding agents was introduced in themid to late 1980’s and these either modified or removed thesmear layer to permit resin penetration in to the underlyingdentin.Examples are: Scotchbond 2 (3M ESPE), Gluma (Bayer),Tenure , Syntac classic (Ivoclar Vivadent) andXR bonding system (Kerr)4Fourth generation dentinal adhesive were developed in theearly to mid 1990’s and represent a significant advancementin dentistry. They are based on the ‘total-etch’ conceptwere both enamel and dentin are simultaneously etched with37% phosphoric acid for 15 seconds. Following this thetooth is washed and gently dried to leave the dentin surfacemoist so as to prevent the exposed collagen network. Nexta primer of hydrophilic monomers is applied. Due to theirvolatile nature, the solvent displaces water from collagennetwork and allows penetration of the primers. Finally theadhesive resin is applied which may be Bis GMA, UDMAwith TEGDMA and HEMA. The adhesive resincopolymerizes with the primed dentin and also compositeresin applied over it.Introduction of this total etch concept was based on thework of Fusayama and others in Japan. Bertolotti and Kancaproposed a technique that involved phosphoric acid – etchingof dentin as well as enamel followed by the application ofrelatively hydrophilic resins. Products such as All- Bond 2, Optibond FL (Kerr), Perma Quick (Ultradentproducts) and Scotch bond multipurpose (3M ESPE).5With the development of fourth generation dentin bondingagents, the concept of hybrid layer formation was put forthas the mechanism of bonding to dentin. This concept wasproposed by Nobo Nakabayashi in 1982. This layer is formedby the inter diffusion of the low viscosity monomers intothe exposed collagen network and the intertubular dentin toform a micromechanical bond with dentin. The hybrid layeris very strong and tough when properly formed, and affordsenormous micromechanical retention for resin composites.6Another major advance in dentin bonding technique thatoccurred in the early 1990s was the "wet" (or moist)bonding technique developed by Dr. John Kanca, whodiscovered that if one left some residual water in acid-etcheddentin, bond strengths could be doubled.7Examples: Scotchbond multipurpose, All bond 2.Composition: Scotchbond multipurpose.4All bond 2.4Fifth generation bonding agents: The fifth-generationsystems introduced during the mid 1990s combined theprimer and adhesive while maintaining high bond strengths.It is simplified version of the fourth generation adhesive. Inthis primer and adhesive are present in the same bottle ratherthan two different bottles. Though they require fewer stepsin achieve dentin bonding, these agents are inferior to fourthgeneration bonding agents in terms of their bond strength.Examples are single bond (3M), one-step (Bisco), Glumacomfort bond (Heraeus Kulzer), Opibond solo (kerr). Thenew self-etching primers can utilize the smear layer as alegitimate bonding substrateWhen single bottle primer/adhesives are applied, thesolvent may diffuse into the water, instead of vice versaforcing adhesive monomers to undergo phase changes,forming blisters, resin globules, etc.A great advantage is that self etching primers aredesigned to be used on dry dentin. Although one should notover desiccate dentin, the dentin surface can be briefly driedfollowing cavity preparation because the dentin ismineralized. This avoids all issues of how moist or wet thedentin should be prior to bonding. It is far easier to produceuniform dryness than it is uniform wetness. Anotheradvantage of these self etching primer systems is that theydo not etch very far into the dentin beneath smear layers.This avoids removal of smear plugs in the tubules and seemsto be responsible for the lack of postoperative sensitivityassociated with these technique-insensitive adhesivesystems.8Even though the hybrid layer is thin, resin dentin bondstrengths are very high. The first marketed modern selfetching primer (ca. 1989-1991) was Scotch Prep.Composition: Single bond, one step, Gluma comfort bond.4Sixth generation bonding agents: The sixth-generation dentaladhesive systems introduced in the late 1990s and early2000s eliminated the separate acid-etching step byincorporating an acidic primer that was placed on the enameland dentin after tooth preparation and isolation from saliva.This dentinal adhesive, include self etching primer whereprimer and etchant are in one bottle and the adhesive resinis in another bottle. They show good bond strength to dentinbut not to enamel. Examples are-Prompt-L-Pop (3M),Clearfil SE Bond (Kuraray) Xeno (Dentsply) It is importantto note that although self-etching.Primer products bond well to roughened or preparedenamel, they form a significantly weaker bond to unpreparedenamel if the enamel is not first etched with a standardphosphoric acid etchant. The bond strength to dentin andenamel is lower than the fourth and fifth generation systems.Composition: Clearfill SE bond.4Seventh generation bonding agents: Seventh-generationsystems were introduced in late 2002 and it combinedetchant, primer, and adhesive in a single bottle, eliminatingan additional mixing and/or placement step over the sixthgenerationsystems. i-bond is a seventh-generation, singlecomponent,no-mix, one-step application dental adhesivewith an etchant, adhesive, desensitizer, and photoinitiator.This i-bond (Heraeus kulzer) was said to perform etching,disinfecting, priming and bonding in a single step. Thebenefits using this bonding agent increases patient comfort,reduces chairside time, decreases contamination, increasesefficacy, which would promising in pediatric patients inpreventing pit and fissure caries. It contains UDMA, 4-META in an acetone/water solvent and Gluma desensitizer,4-META monomer is responsible for conditioning due toits acid function. UDMA is used as cross linking and filmforming monomer to add the required stability to the adhesivelayer.9Examples: G-bond- GC corp, i-bond- Heraeus Kulzer.Composition;10A modification of the sealant application technique wasproposed by Hitt and Feigal (1992), with the use of a dentinbonding agent layer between the etched enamel and thesealant. Some studies confirmed the benefits of theapplication of bonding agents under sealants in etchedenamel that was contaminated by saliva . Such studies showed reduction ofmicroleakage and increase in the retention rate of the sealant.A low-viscosity hydrophilic material bonding layer, as partof or under the actual sealant, is recommended for longtermretention and effectiveness. Feigal, Hitt and Splieth(1993) believe that using bonding agent is useful to increasefissure sealant retention on teeth contaminated with saliva.The results of the clinical study by Boksman et al (1993),reported that the use of bonding does not increaseretention.13In vitro and invivo studies report that use of a bonding agent will improvethe bond strength and minimize microleakage.15Symons et al (1996) reported that sealant penetration ofenamel fissures was enhanced by the use of the All- bond 2(Bisco) and Scotchbond multipurpose systems.In another study, Feigal and Hebling in (2000) assessedthe effect of bonding as an intermediate layer onmicroleakage reduction on saliva contaminated enamel andthey also showed that using bonding agent as an intermediatelayer reduces microleakage on saliva-contaminated enamel.11The use of a hydrophilic adhesive prior to sealantplacement improves retention of the sealant and decreasesmicroleakage, the use of self-etching adhesive systems maybe a valid and promising alternative to acid etching withphosphoric acid. These adhesives have recently beenintroduced to simplify the bonding procedures and reducethe technique sensitivity by eliminating the etching, rinsingand drying steps of the bonding protocol.16Pashley et al in (2001) found no correlation between thebonds to enamel of these adhesive systems and their pH. So far, the literature does notprovide a straight forward answer whether mild self-etchadhesives bonded to enamel can withstand the mechanicaland chemical challenges of the oral cavity.17The dental literature supports the use of tooth bondingadhesives when used according to the manufacturer’sinstruction unique for each product, as being effective inprimary and permanent teeth in enhancing retention ofrestorations, minimizing microleakage, and reducingsensitivity.18In another research, Pinar et al (2004) assessed theclinical performance of sealants with and without a bondingagent and showed that the placement of bonding under thefissure sealant did not affect the clinical success of thesealant. It has been reported that the functional monomersin self-etching adhesives can chemically interact withhydroxyapatite11Venker D J et al 2004) reported that some clinical andlaboratorial studies have shown that the association of selfetchingadhesive systems to pit-and-fissure sealants is lesseffective than the use of etch-and-rinse adhesive systems.04 reportThe addition of one- bottle system with primer andadhesive components together may enhance the initial bondto enamel and also improve the initial bond between thebonding agent and the sealant. The fifth generation singlebottle system that were tested improved sealant success,showing significant hazard ratios of 0.53 for occlusalsealants and 0.35 for buccal/lingual sealants.21A study by Arzu Pinar et al (2005) observed that the useof bonding agent as an intermediary layer between enameland sealant did not affect sealant success during a 24-monthperiod.21Complete penetration of the etchant into the fissuresshould occur for a durable retention of the sealant to enamel.In a previous study by Bottenberg et al (1996) none of thetested commercially available etchants was able to penetratefarther than 17% of the total fissure depth in a fissuremodel.23The self-etching adhesive systems (Adper single bondand Clearfil S3-bond) used in the study Jaciara MirandaGomes-Silva et al (2008) is considered as a weak selfetchingprimer (pH 2.5) and its hydrophilic acid functionalmonomer (10-MDP) has an intense chemical interactionwith the hydroxyapatite. Clinically acceptable retention rateshave been reported and may thus be the result of a two-foldmechanism: increased micromechanical retention in additionto the chemical interaction.23Torres et al (2005) reported that the application of abonding agent (Prime and Bond) layer underneath the resinpit-and-fissure sealant placement resulted in significantlyhigher Shear bond strength to the saliva-contaminated andnoncontaminated groups. The authors also found thatindividual or simultaneous curing of the bonding agent andthe sealant had no influence on bond strength to contaminatedenamel. The different results found in the study by JaciaraMiranda et al (2008) may be attributed to the fact that adpersingle bond 2 etch-and-rinse adhesive system contains wateras a co-solvent, which gives a lower volatility to this materialcompared to other adhesive systems that contain onlyacetone as a solvent.23In a study by Boksman L (1993) on 2 years clinical evaluationof retention of pit and fissures placed with and without useof bonding agents showed that, the use of Scotch bond 2(third generation) and prisma universal bond (secondgeneration) did not increase the long-term retention rate. At6 months evaluation the sealant placed with use of bondingagent showed higher retention rate than its counterpartplaced without a bonding agents. But at 2 years level theuse of bonding agent prior to sealant application did notincrease the retention rate.13Third-generation dentin bonding systems: These systemsalter or remove the smear layer prior to bonding and producebond strengths ranging from 16 to 2612-14 MPa. Some ofthe products produce bond strengths approaching thoseformed to enamel. Clinical retention rates of 100% at 2years have been reported. 24One set of criteria used to distinguish fourth generationproducts from earlier ones has been: Ability to bond asstrongly to dentin as to enamel, ability to bond strongly tomoist dentin, and technique insensitivity. An additionalcriterion may be the ability to bond, many different types ofsubstrates . Being able to bond to these substrates,generally means that, the dentin bonding agents enhancesthe strength of the bond of composite resin to them. Theability of dentin bonding agents to bond resin to metal hasbeen demonstrated and commented upon by severalresearchers.25In a research conducted by RJ Feigal (2000) on improvedsealant retention on bonding agents, showed that the fourthgeneration dentin bonding agents ,Tenure primer and Scotchbond multipurpose primer, was not significantly effectivein sealant retention.13Fifth-generation products are essentially distinguishedby being one-step or one-bottle products.In general, these products have limitations. Many requireat least as much time to apply or even more time than threecomponentproducts and they lack many of the componentsnecessary to perform multisubstrate bonding. It also appearsimportant to apply multiple coats of these agents so thatthere is an adequately thick resin layer on top of the hybridlayer.26A study on retention of pit and fissure sealant with orwithout bonding agents by Arzu Pinar et al (2005) showsthat, the clinically acceptable marginal integrity rates forsealants with a bonding agents after 3,6,12,24 months were93%, 93%, 83% and 79% respectively. Similar way marginaldiscoloration were studied and no color change was seenwere 96%, 93%, 81% and 75% respectively. Anatomic formof sealant were showed that after 6 months it is about 98%,after 12 months 83%, and after 24 months it was 79%. Heconcluded that the use of bonding agents as an intermediarylayer between enamel and sealant did not affect sealantsuccess during 24 months period.9In a study by Baca P, et al (2007) on retention of threefissure sealant and a dentin bonding agents in cariesprevention showed that the marginal leakage and bond toenamel of Optibond Solo (fifth generation), a hydrophilicone-bottle filled dentin adhesive, is not affected by humidconditions. Indeed, an increase in bond strength to enamelwas reported when bonds were performed under wetconditions. Optibond Solo can achieve similar retention tofissure sealants at 12 months but appears to be moresensitive to the application technique.27A clinical study on pit and fissure sealants with andwithout a seventh generation bonding agents (2008) showedthat there was no significant effective in retention of pit andfissure sealants when compared to the conventionalphosphoric acid etching technique.2829Based on the results observed in several studies, the use ofbonding agent as an intermediary layer between enamel andsealant did not affect sealant success. In situations in whichcontrol of saliva and isolation is impossible the use of bondingfor increasing the quality of fissure sealant therapy is useful.Studies on latest generation bonding agent i- bond bondingagent showed the retention rate of sealant showed the sameretention rate of sealant without using the bonding agents.So the bonding agent i-bond was not effective in retentionof pit and fissure sealant when compared to the conventionalphosphoric acid etching technique.